Method for detaching antigenicity-enhanced cell nucleus from immobilized cell or FFPE tissue section, and antigen activator and kit used for method
阅读说明：本技术 从固定化细胞或ffpe组织切片脱离增强抗原性的细胞核的方法以及用于该方法的抗原活化剂及试剂盒 (Method for detaching antigenicity-enhanced cell nucleus from immobilized cell or FFPE tissue section, and antigen activator and kit used for method ) 是由 佐藤七月 黑岩美佳 中辻匡俊 石原英幹 于 2019-02-28 设计创作，主要内容包括：本发明提供用于使用Ki-67抗体检测含Ki-67蛋白质阳性细胞核的细胞的数的预处理方法、该检测方法、该检测方法中使用的试剂盒、及使用该方法的治疗方案选定。探讨由不旨在Ki-67抗原的活化的酶的活化,用不识别MIB-1的表位的酶(稀有切点酶)预处理试样,则含细胞角蛋白的其他抗原的抗原性也增强的同时,增强Ki-67抗原的抗原活化。结果,从FFPE切片增强抗原性的状态下使细胞核脱离,以存在于该核的Ki-67蛋白质作为目标,通过使荧光标记的抗体反应,完成客观性、再现性、普遍性更高的Ki-67阳性细胞的定量方法。(The present invention provides a pretreatment method for detecting the number of cells containing a Ki-67 protein-positive nucleus using a Ki-67 antibody, the detection method, a kit used in the detection method, and selection of a treatment regimen using the method. To investigate the activation of an enzyme not intended for the activation of the Ki-67 antigen, when a sample was pretreated with an enzyme that does not recognize an epitope of MIB-1 (rare-cutting enzyme), the antigenicity of another antigen containing cytokeratin was also increased, and the antigen activation of the Ki-67 antigen was also increased. As a result, the cell nucleus was detached from the FFPE section with the antigenicity enhanced, and the Ki-67 protein present in the nucleus was targeted, and a fluorescence-labeled antibody was reacted to complete a method for quantifying Ki-67-positive cells with higher objectivity, reproducibility, and universality.)
1.A method of detecting cells containing Ki-67 positive nuclei in an immobilized cell population using an anti-Ki-67 antibody comprising:
(1) a step of activating the antigen by pretreating the immobilized cell population with a hydrolase that does not recognize the peptide cleaving SEQ ID NO. 2, and a step of subsequently treating the antigen with a protease
(2) Process for immunostaining using anti-Ki-67 antibody, and
(3) and detecting the stained Ki-67-positive cells or Ki-67-positive cell nuclei.
2. The method of claim 1, wherein the antibody is at least one selected from the group consisting of MIB-1, DAKO-PC, Ki-S5, and a-0047.
3. The method of claim 1 or 2, wherein the enzyme is at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline-terminated peptidase and hyaluronidase.
4. The method of claim 1, wherein
The enzyme is thrombin and/or hyaluronidase,
the antibody is MIB-1.
5. The method of any one of claims 1-4, wherein the immobilized cell population is immobilized with an immobilizing agent selected from the group consisting of formalin, glutaraldehyde, alcohol, acetone, and combinations thereof.
6. The method of any one of claims 1 to 5, wherein step (1) is preceded by activation by a heat treatment.
7. The method of any one of claims 1 to 6, further comprising:
the cell nucleus is specifically stained and,
detecting stained nuclei.
8. The method of any one of claims 1 to 7, further comprising:
immunostaining for cytokeratins using anti-cytokeratin antibodies,
and detecting the stained positive cells.
9. The method of any one of claims 1 to 8, further comprising:
immunostaining for ER and/or PgR using anti-Estrogen Receptor (ER) antibodies and/or anti-progesterone receptor (PgR) antibodies,
detecting stained positive cells or positive nuclei.
10. The method of any one of claims 1 to 9 further comprising detecting a cell or nucleus in which the HER2 gene is amplified using a probe that hybridizes to the HER2 gene.
11. The method of any one of claims 1-10, wherein the immobilized cell population is contained in a tissue section.
12. The method of claim 11, wherein
The tissue slice is wrapped with a wrapping agent,
the method comprises a step of removing the coating agent and hydrophilizing the tissue section before the step of activating the antigen with the hydrolase.
13. The method according to claim 11 or claim 12, wherein a step of disrupting the cell to extract a nucleus is included between the steps (1) and (2).
14. The method of claim 13, wherein cell nuclei are extracted by disrupting the cells by shear stress.
15. The method of claim 13 or 14, wherein the cell nuclei are extracted in a buffer containing a surfactant.
16. The method of claim 15, wherein the surfactant is at least one selected from CHAPS, NP-40, and Triton-X100.
17. The method of any one of claims 1 to 16, wherein the number of stained Ki-67 positive cells or Ki-67 positive nuclei is counted.
18. The method of any one of claims 1 to 17, wherein
Fluorescence staining the Ki-67 positive cells or the Ki-67 positive cell nuclei,
counting was performed using flow cytometry.
19. The method of any one of claims 1-18, wherein the immobilized cells or the tissue slices are patient derived.
20. The method of any one of claims 1-18, wherein the immobilized cells or the tissue section are of tumor tissue origin in a patient.
21. The method of claim 20, wherein the immobilized cells or the tumor tissue are breast cancer cells or breast cancer tissue.
22. The method of claim 20 or 21, for aiding diagnosis of cancer.
23. The method of claim 20 or 21 for use in aiding diagnosis of prognosis of cancer treatment.
24. A method for determining a treatment regimen for cancer by calculating the proportion of Ki-67 positive cells within a cell population by the method of claim 20 or 21,
in case the ratio is above the cut-off value, a combination of endocrine therapy and chemotherapy is selected;
in case the ratio is below the cut-off value, the individual endocrine therapy is selected.
25. A method of treating cancer by calculating the proportion of Ki-67 positive cells within a cell population by the method of claim 20 or 21,
in the case where the ratio is above the cut-off value, a combination of endocrine therapy and chemotherapy is selected to be administered to the patient;
in the case where the ratio is below the cutoff value, endocrine therapy alone is selected for administration to the patient.
26. Method for activating Ki-67, wherein the immobilized tumor cell population or tumor tissue is pre-treated with a hydrolase which does not recognize the peptide cleaving SEQ id No. 2.
27. An antigen activator for use in a cell or tissue sample in which Ki-67 is detected by immunostaining, which comprises a hydrolase that does not recognize a peptide that cleaves SEQ ID NO. 2.
28. The antigen activator of claim 27, wherein the enzyme is at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline-terminated peptidase, and hyaluronidase.
29. The antigen activator of claim 28, wherein the enzyme is thrombin and/or hyaluronidase.
30. An antigen activator used for a cell or tissue sample in which Ki-67 and cytokeratin are detected simultaneously by immunostaining, wherein the antigen activator contains a hydrolase that does not recognize a peptide that cleaves SEQ ID NO. 2.
31. The antigen activator according to any one of claims 27 to 30, which is further used in a cell or tissue sample to be detected by immunostaining ER and/or PgR.
32. The antigen activator according to any one of claims 27 to 31, which is further used in a sample for detecting a cell that amplifies HER2 gene.
33. A kit for detecting Ki-67 positive cells in immobilized cells comprising:
a hydrolase which does not recognize a peptide cleaving SEQ ID NO 2, and
anti-Ki-67 antibodies.
34. The kit of claim 33, wherein the hydrolase is at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline-terminated peptidase and hyaluronidase.
35. The kit of claim 34, wherein the hydrolase is thrombin and/or hyaluronidase.
36. The kit of any one of claims 33 to 35 wherein the anti-Ki-67 antibody is at least one selected from MIB-1, DAKO-PC, Ki-S5 and a-0047.
37. The kit of any one of claims 33 to 36, further comprising, in combination with Ki-67, a ligand for detecting other markers used to aid in the diagnosis of cancer or to aid in the prognosis of a treatment for diagnosing cancer.
38. The kit of claim 37, wherein the ligand is at least one selected from the group consisting of an anti-cytokeratin antibody, an anti-ER antibody, an anti-PgR antibody, and a probe that hybridizes to the HER2 gene.
39. The kit according to any one of claims 33 to 38, further comprising a compound for nuclear staining.
40. The kit of any one of claims 33 to 39, further comprising a buffer for dispersing the cells, the buffer comprising a surfactant.
41. The kit according to any one of claims 33 to 40, further comprising an antigen activator for heat treatment.
42. A kit as claimed in any one of claims 33 to 41 for use in assisting the diagnosis of cancer.
43. A kit as claimed in any one of claims 33 to 41 for use in aiding diagnosis of prognosis of a cancer treatment.
44. A kit as claimed in any one of claims 33 to 41 for use in determining a treatment regimen for cancer.
45. A kit, wherein the determination of the treatment regimen is performed by calculating the proportion of Ki-67 positive cells within the cell population,
in case the ratio is above the cut-off value, a combination of endocrine therapy and chemotherapy is selected;
in case the ratio is below the cut-off value, the individual endocrine therapy is selected.
46. A kit for extracting nuclei by disrupting immobilized cells by shear stress, which comprises a buffer for cell dispersion containing a surfactant.
47. A method for extracting a nucleus by disrupting immobilized cells by shear stress generated by water flow, ultrasonic waves or the like, wherein the disruption of the cells is performed by dispersing the immobilized cells in a buffer containing a surfactant.
[ technical field ] A method for producing a semiconductor device
The present invention relates to a pretreatment method for detecting a cell containing a Ki-67 protein-positive nucleus using a Ki-67 antibody, a detection method of the cell, a kit used in the pretreatment method or the detection method, and treatment regimen selection using the detection method.
[ background of the invention ]
Formalin fixation of surgically removed tissue is the most common method in the world for preserving cancer tissue samples, and is the standard pathological technique. The most common method for preserving tissues is to immerse the whole tissue in formalin aqueous solution for a long period of time (8 to 48 hours), and then embed the whole fixed tissue in paraffin for long-term preservation at room temperature. As described above, the molecular analysis method for analyzing formalin-fixed cancer tissue is most used for analyzing the tissue of a cancer patient, for example.
An immunohistostaining method, which is one of diagnostic techniques using formalin-fixed paraffin-embedded (FFPE) tissue, is a method of attaching a slide glass by thinly cutting tissue, visualizing an antigen-antibody reaction between a biological substance present on the surface of a pathological section tissue or cell and a substance that recognizes the biological substance, and determining the presence of a target biological substance on the surface of the pathological section tissue or cell.
For example, human epithelial growth factor receptor (HER2), Estrogen Receptor (ER), and progesterone receptor (PgR) expressed in breast cancer tissues are clinically used because they are related to the effects of therapeutic methods, and are called effect predictors (non-patent document 1).
In one aspect, the presence or absence of the factor and the prognostic factor are referred to as prognostic factors. Ki-67 is also now known as a prognostic factor. In the detection of the effect-predicting factor, immunohistochemical methods (IHC methods) of major tumor tissue samples are often used. Either a method in which both the staining intensity of tumor cells and the ratio of stained cells are considered (All red Score, etc.) or a method in which the staining intensity is not evaluated and the ratio of only stained tumor cells is used for determination (J-Score, etc.).
In the case of HER2, it is generally examined by the IHC method, and the results are negative when the number of amplification products is 0 or 1+, positive when the number of amplification products is 3+, and negative when the number of amplification products is 2+, the presence or absence of amplification is examined by the FISH method (Fluorescence in situ hybridization).
In the case of ER, 3 to 8 positive cells were found in All red Score, while 10% was often set as a cutoff value when judged by the ratio of stained cells, there was also a suggestion that ER was found to be positive when 1% was present.
In the case of Ki-67, there are a method of calculating the positive ratio using the whole tissue, a method of selecting and calculating a region called a hot spot of a positive cell population, and the like.
In any case, if a certain cutoff value is set, the effect prediction factors become effective treatment protocols when they are judged to be positive.
Breast cancer is classified into various subtypes according to the expression of the effect prediction factor and the prognostic factor.
(1) Luminela (type): ER PgR positive, HER2 negative, and Ki-67 low value (< 14-20%)
(2) Luminelb (like) type (HER2 negative): ER PgR positive, HER2 negative and Ki-67 high value (14-20%)
(3) Luminelb (like) type (HER2 positive): ER PgR positive, HER2 positive
(4) Non-lumineal types: ER PgR negative and HER2 positive
(5) Triple negative type: ER negative, PgR negative, HER2 negative
These classifications also affect the selection of treatment regimens, and for example, when judged as a low Ki-67 luminela (like) type, endocrine therapy alone is mainly selected, while when judged as a high Ki-67 luminelb (like) type (HER2 negative), a combination of endocrine therapy and chemotherapy is mainly selected (non-patent
However, in the immunohistostaining method, the above-mentioned judgment is carried out by visual judgment with a microscope, and since counting is carried out manually, even an experienced pathologist requires time and labor.
Ki-67(MKI67) is a nuclear protein and is a commonly used marker for cell proliferation. Molecules expressed in nucleosomes of proliferating cells and chromosomes in the nucleus division phase are expressed at all stages of the cell cycle, and the expression level changes from the G1 late stage to the S stage, and increases in the S stage, resulting in the maximum expression level in the M stage. Thus, cells with Ki-67 expression were seen to be no longer indicative of entry into the cell cycle. However, reagents and methods for diagnosing cancer using Ki-67 as one of biomarkers are known (
In an attempt to recover nuclei from FFPE tissue sections, which is a method capable of storing tissues isolated from living bodies for a long period of time, using an enzyme, immunofluorescent staining was performed and FCM was used for detection, but since many sites of proteins are cleaved by enzymes capable of isolating nuclei from tissues, there is a high risk that antigen recognition sites are cleaved. According to non-patent document 4, for example, in an antibody targeting the same Ki-67 protein, it is reported that although in the clone name: s5, and the following clones with different antigen recognition sites: detection in MIB-1 is not possible.
Furthermore, formalin-fixed tissue or thrombin: in the case of fibronectin (plasma) fixed tissue, there is a possibility that the MIB-1 antibody may have a false positive or false negative result (non-patent document 9)
However, since the IHC method using MIB-1 has been used in clinical sites for a long time, clones that cannot use empirically selected antibodies are a great loss.
Methods of extracting proteins from FFPE tissue sections have also been investigated, but not for detecting the presence of antigen positive nuclei after treatment. In addition, although kits for extracting nucleic acids have been put to practical use, they have been limited to research reagents and have not reached the practical stage.
[ Prior art documents ]
[ patent document ]
Patent document 1: japanese patent application laid-open No. Hei 11-509930
Patent document 2: japanese patent application laid-open No. 2003-506716
Patent document 3: japanese unexamined patent application publication No. 2005-315862
Patent document 4: japanese Kohyo publication No. 2007-524103
Patent document 5: japanese laid-open patent publication No. 2009-527740
Patent document 6: japanese laid-open patent publication No. 2009-63508
Patent document 7: japanese laid-open patent publication No. 2009-122115
Patent document 8: japanese laid-open patent publication No. 2009-537822
[ non-patent literature ]
Non-patent document 1: breast cancer treatment guideline based on
Non-patent document 2: breast Cancer Research,2010,12: R56http:// break-Cancer-Research com/content/12/4/R56
Non-patent document 3: breast cancer society guidelines (Japan Breast cancer society) http:// jbcs. gr.jp/guidline/guideliline/g 6/g 61900-
Non-patent document 4: cytometric. 1997
Non-patent document 5: dowsett M, et al.Association of Ki67 in Breast cancer: (ii) registrations from the International Ki67 in Breast Cancer working group. Jnat Cancer Inst.2011Nov 16; 103(22):1656-64.
Non-patent document 6: angela Toss, et al, molecular characterization and target characterization adaptive aptamers in Breast Cancer. Breast Cancer Res.
Non-patent document 7: leers MP, et al, Multi-parameter flow cytometry with detection of the Ki67-Ag in paraffin embedded mammarycamomas.Cytometry.1997 Mar 1; 27(3):283-9.
Non-patent document 8: polley MY, et al.an International Ki67 reproducibility, J Natl Cancer Inst.2013 Dec 18; 105(24):1897-906.
Non-patent document 9: gorman, et al, Complex of Breast cardio genomic organisers on Cell Blocks incorporated by variance Methods: cellient, Formalin and thrombin. 56:289-296.
Non-patent document 10: breast cancer treatment guideline based on
Non-patent document 11: goldhirsch1, et al, personalized the treatment of the geometr with early street cancer: highlightings of the St Gallen International expert Consenssus on the Primary Therapy of Early Breast Cancer 2013 Annals of Oncology 24: 2206-2223,2013.
Non-patent document 12: goldhirsch, et al, strategies for subtype-evaluating with the diversity of the Breast cancer: highlightings of the St Gallen International expert Consenssus on the Primary Therapy of Early Breast Cancer 2011 Annals of Oncology 22: 1736-1747,2011.
[ SUMMARY OF THE INVENTION ]
[ problem to be solved by the invention ]
As described above, since the immunohistostaining method was performed in the visual examination with a microscope and counting was also performed manually, the same pathological tissue section was often used and judged differently by a pathologist (see comparative example 2). Since the ratio (%) of the presence of Ki-67-positive cells (cutoff value) affects the selection of a treatment protocol, a more objective, reproducible, and generally high method for quantifying the ratio of the presence of Ki-67-positive cells is required.
As described above, it is preferable to prepare and store a tissue sample containing a pathological tissue section as a standard pathological technique at the time of cancer excision or biopsy, and a method for quantifying the stored sample can be used.
In general, when FFPE tissue is analyzed at a single cell level, it is necessary to cleave extracellular substrates responsible for intercellular attachment and activate antigens. However, digestion using these enzymes may cleave the recognition site of the target antigen, limiting the antibodies that can be used. Therefore, in addition to the existing methods described above, a method of dispersing cells or separating cells from the nucleus becomes necessary.
[ MEANS FOR SOLVING PROBLEMS ] to solve the problems
As a result of intensive studies, the present inventors have studied a pretreatment method using an enzyme not intended for cell dispersion or detachment of cell nucleus, and succeeded in activating Ki-67 antigen while increasing the antigenicity of other cytokeratin-containing antigens when a tissue or cell sample is pretreated with an enzyme not recognizing the epitope of MIB-1 (rare-cutting enzyme). As a result, the cell nucleus was detached from the FFPE section with the antigenicity enhanced, and the Ki-67 protein present in the nucleus was targeted and reacted with a specific antibody, thereby completing a more objective, reproducible, and highly universal method for detecting Ki-67-positive cells.
Accordingly, the present invention is as follows [1] to [45 ].
[1] A method of detecting cells containing Ki-67 positive nuclei in an immobilized cell population using an anti-Ki-67 antibody comprising:
(1) a step of activating the antigen by pretreating the immobilized cell population with a hydrolase that does not recognize the peptide cleaving SEQ ID NO. 2, and a step of subsequently treating the antigen with a protease
(2) Staining procedure using anti-Ki-67 antibody, and
(3) detecting a stained Ki-67-positive cell or Ki-67-positive cell nucleus;
[2] [1] the method according to which the antibody is at least one selected from the group consisting of MIB-1, DAKO-PC, Ki-S5 and A-0047;
[3] the method according to [1] or [2], wherein the enzyme is at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline-terminated peptidase and hyaluronidase;
[4] [1] the method according to any one of the above methods, wherein the enzyme is thrombin and/or hyaluronidase, and the antibody is MIB-1;
[5] the method according to any one of [1] to [4], wherein the immobilized cell group is immobilized with an immobilizing agent selected from formalin, glutaraldehyde, alcohol, acetone, and a combination thereof;
[6] the method according to any one of [1] to [5], wherein the step (1) is preceded by activation by heat treatment;
[7] the method according to any one of [1] to [6], further comprising specifically staining cell nuclei, detecting stained cell nuclei (for example, counting);
[8] the method according to any one of [1] to [7], further comprising staining (e.g., fluorescent staining) cytokeratin with an anti-cytokeratin antibody, and detecting stained positive cells;
[9] the method of any one of [1] to [8], further comprising staining (e.g., fluorescent staining) the anti-Estrogen Receptor (ER) and/or the anti-progesterone receptor (PgR) with an anti-ER antibody and/or an anti-progesterone receptor (PgR) antibody, detecting stained positive cells or positive cell nuclei (e.g., counts);
[10] the method according to any one of [1] to [9], further comprising detecting a cell or a nucleus in which HER2 gene is amplified using a probe that hybridizes to HER2 gene.
[11] The method according to any one of [1] to [10], wherein the immobilized cell population is contained in a tissue section;
[12] [11] the method according to any one of the above aspects, wherein the tissue section is coated with a coating agent, and the method comprises a step of removing the coating agent and hydrophilizing the tissue section before the step of activating the antigen with the hydrolase;
[13] the method according to [11] or [12], wherein the method comprises a step of disrupting the cell to extract a cell nucleus between the steps (1) and (2);
[14] [13] the method wherein cell nuclei are extracted by disrupting the above cells with shear stress generated by water flow, ultrasonic waves or the like;
[15] the method of [13] or [14], wherein the above-mentioned cell nucleus is extracted in a buffer containing a surfactant;
[16] [15] the method according to any one of the above, wherein the surfactant is at least one selected from the group consisting of CHAPS, NP-40, and Triton-X100;
[17] the method according to any one of [1] to [16], wherein the number of the stained Ki-67-positive cells or Ki-67-positive nuclei is counted;
[18] [17] the method according to (1), wherein the Ki-67-positive cells or the Ki-67-positive cell nuclei are stained with fluorescence and counted by flow cytometry;
[19] the method according to any one of [1] to [18], wherein the immobilized cell or the tissue section is derived from a patient;
[20] the method according to any one of [1] to [18], wherein the immobilized cell or the tissue section is derived from a tumor tissue of a patient;
[21] [20] the method according to [20], wherein the immobilized cell or the tumor tissue is a breast cancer cell or a breast cancer tissue;
[22] the method of [20] or [21], which is used for aiding in the diagnosis of cancer;
[23] the method of [20] or [21], which is used for aiding diagnosis of prognosis of cancer treatment;
[24] a method for determining a treatment regimen for cancer by calculating the ratio of Ki-67 positive cells within a cell population by the method of [20] or [21], selecting a combination of endocrine therapy and chemotherapy in the case where the ratio is above a cutoff value, and selecting endocrine therapy alone in the case where the ratio is below the cutoff value;
[25] a method for treating cancer, wherein the ratio of Ki-67 positive cells within a cell population is calculated by the method described in [20] or [21], a combination of endocrine therapy and chemotherapy is selected in the case where the ratio is above a cut-off value, and endocrine therapy alone is selected and administered to a patient in the case where the ratio is below the cut-off value.
[26] A method for activating Ki-67, wherein the immobilized tumor cell population or tumor tissue is pre-treated with a hydrolase that does not recognize the peptide that cleaves SEQ ID No. 2;
[27] an antigen activator for use in a cell or tissue sample in which Ki-67 is detected by immunostaining, the antigen activator containing a hydrolase that does not recognize a peptide that cleaves SEQ ID NO. 2;
[28] [27] the antigen activator, wherein the enzyme is at least one selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline-terminated peptidase and hyaluronidase;
[29] [28] the antigen activator, wherein the enzyme is thrombin and/or hyaluronidase.
[30] An antigen activator used in a cell or tissue sample in which Ki-67 and cytokeratin are detected simultaneously by immunostaining, the antigen activator containing a hydrolase that does not recognize a peptide that cleaves SEQ ID NO. 2;
[31] the antigen-activating agent according to any one of [27] to [30], which is further used in a cell or tissue sample for detecting ER and/or PgR by immunostaining;
[32] the antigen-activating agent according to any one of [27] to [31], which is further used in a sample for detecting a cell that amplifies HER2 gene;
[33] a kit for detecting Ki-67 positive cells in immobilized cells comprising:
a hydrolase which does not recognize a peptide cleaving
anti-Ki-67 antibodies;
[34] [32] the kit according to any one of the above aspects, wherein the hydrolase is at least one enzyme selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline-terminated peptidase and hyaluronidase;
[35] [34] the kit according to any one of the above aspects, wherein the hydrolase is thrombin and/or hyaluronidase;
[36] [33] to [35] wherein the anti-Ki-67 antibody is at least one selected from the group consisting of MIB-1, DAKO-PC, Ki-S5 and A-0047;
[37] [33] to [36], which comprises a ligand for detecting another marker used for the auxiliary diagnosis of cancer or for the auxiliary diagnosis of prognosis of cancer treatment, in combination with Ki-67;
[38] [37] the kit according to any one of the above aspects, wherein the ligand is at least one selected from the group consisting of an anti-cytokeratin antibody, an anti-ER antibody, an anti-PgR antibody, and a probe that hybridizes to HER2 gene;
[39] the kit according to any one of [33] to [38], further comprising a compound for nuclear staining;
[40] the kit according to any one of [33] to [39], further comprising a buffer for dispersing the cells, the buffer containing a surfactant;
[41] the kit according to any one of [33] to [40], further comprising an antigen activator for heat treatment;
[42] the kit according to any one of [33] to [41], which is used for assisting in the diagnosis of cancer;
[43] the kit according to any one of [33] to [41], which is used for aiding in the diagnosis of prognosis of cancer treatment;
[44] the kit according to any one of [33] to [41], which is used for determining a treatment regimen for cancer;
[45] a kit, wherein the above-mentioned determination of the treatment protocol is performed by calculating the ratio of Ki-67 positive cells within the cell population, selecting a combination of endocrine therapy and chemotherapy in case the ratio is above a cut-off value, selecting endocrine therapy alone in case the ratio is below a cut-off value;
[46] a kit for extracting cell nuclei by disrupting immobilized cells with shear stress generated by water flow, ultrasonic waves, or the like, which comprises a buffer for cell dispersion containing a surfactant;
[47] a method for extracting a nucleus by disrupting immobilized cells by shear stress generated by water flow, ultrasonic waves, or the like, in which the cells are disrupted by dispersing the immobilized cells in a buffer containing a surfactant; and
[48] a method for detecting a cell in which an antigen of interest is present in a nucleus among a fixed cell population using an antibody to the antigen, comprising:
(1) a step of activating the antigen by pretreating the immobilized cell population with a hydrolase that does not cleave the antigen recognition site of the antibody, and a step of subsequently treating the cell population with a second hydrolase
(2) A step of staining the cells with the antibody, and
(3) and detecting the stained antigen-positive cells or antigen-positive cell nuclei.
[ Effect of the invention ]
The present invention establishes a method for detecting (quantifying) Ki-67 positive cells with high objectivity, reproducibility and universality.
By using the procedures (pretreatment reagents and procedures) provided by the present invention, detachment of nuclei that enhance antigenicity of the antigen of interest from FFPE tissue sections is achieved. The scattered nuclei thus recovered can be resolved as single nuclei. For example, proteins, nucleic acids, and the like on or in the nuclear membrane are to be detected, and can be detected by staining with a dye (fluorescent substance, chemical emitting substance, enzyme, and the like) or by using an antibody modified with such a dye. In particular, the fraction of positive nuclei (Ki-67 positivity or the like) of the target protein in the enucleated nuclei can be rapidly calculated by flow cytometry analysis.
The cutoff value of Ki-67 varies from facility to facility, because the process of visual judgment and counting by a pathologist is necessary. However, by using the procedures (pretreatment reagents and procedures) provided by the present invention, a world standard of cutoff values for pathological diagnosis with little deviation can be provided.
Furthermore, the cutoff values involved can be used to provide patients with diagnostic protocols with high QOL.
[ brief description of the drawings ]
FIG. 1 shows an image of a recovered product obtained by subjecting an FFPE tissue section of a breast cancer tissue to deparaffinization/hydrophilization and activation with a heat-treated antigen, disrupting cells using a water flow shearing apparatus, and then subjecting the resulting tissue section to immunofluorescence staining and microscopic observation. The nuclei stained with DAPI are shown on the left, and the DAPI-stained nuclei and fluorescently labeled cytokeratins (when present) are shown on the right, and it is understood from both that the nuclei are recovered while maintaining their shape.
Fig. 2 is a scattergram showing each of formalin-fixed tumor cell lines (MB231, T47D, SKBR) as activated with an antigen by heat treatment, immunofluorescent-stained, analyzed by a flow cytometer, and analyzed for the obtained data with forward scattering as the horizontal axis and side scattering as the vertical axis.
Fig. 3 is a histogram in which the area surrounded by the frame in fig. 2 is gated as the area of the cell nucleus, and the fluorescence intensity of DAPI is shown on the horizontal axis and the number of cells (nuclei) is shown on the vertical axis for the data of the gated fraction.
Fig. 4 is a histogram in which the fluorescence intensity of gated fraction data selected as the nucleus region is plotted on the horizontal axis and the number of cells (nuclei) on the vertical axis, after formalin-fixed breast cancer cells are subjected to antigen activation by heat treatment, immunofluorescent staining with an anti-cytokeratin antibody and an anti-Ki-67 antibody or their isotype control antibodies, and then analyzed by flow cytometry.
Fig. 5 is a histogram in which fluorescence intensity is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis for data of gated fractions selected as the cell nucleus region, after performing antigen activation by heat treatment on each formalin-fixed tumor cell line (MB231, T47D, SKBR), and then immunofluorescent staining with an anti-Ki-67 antibody or its isotype control antibody with or without antigen activation by thrombin, and analyzing the data by flow cytometry.
FIG. 6 is a graph showing the change in the Ki-67 positivity rate in each tumor cell line with and without thrombin treatment.
Fig. 7 is a histogram showing fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis for data of gated fractions selected as the nucleus region, after performing antigen activation by heat treatment on the formalin-fixed T47D cell line, followed by treatment with a hyaluronidase reagent, or immunofluorescent staining with an anti-Ki-67 antibody or its isotype control antibody with or without the treatment, and then analyzing the data by flow cytometry.
Fig. 8 shows a histogram in which the fluorescence intensity of gated fraction data selected as the cell nucleus region is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis, wherein the data is analyzed by a flow cytometer, and the data is plotted on the horizontal axis for fluorescence intensity.
Fig. 9A is a histogram showing data of gated fractions selected as a cell nucleus region, with fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis, in which the FFPE tissue section of breast cancer tissue is deparaffinized/hydrophilized, activated with heat-treated antigen, and cells are disrupted with water flow with or without antigen activation with thrombin, then immunofluorescent staining is performed with an anti-cytokeratin antibody and an anti-Ki-67 antibody or their isotype control antibodies, followed by analysis with a flow cytometer. Thus, the increase of cytokeratin and Ki-67 signals was observed in the thrombin-treated FFPE tissue section specimen.
FIG. 9B is a graph showing the change in the positive rates of cytokeratin and Ki-67 in the presence or absence of thrombin-treated FFPE tissue sections.
FIG. 10 is a graph showing the change in cytokeratin and Ki-67 positivity from the thickness of the thin sections.
FIG. 11 is a graph showing the correlation between the Ki-67 positivity by the method of the present invention and the positivity (average value) by the IHC method.
Fig. 12A is a histogram showing fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis for data of gated fractions selected as the nuclear region, after performing antigen activation by heat treatment on each of formalin-fixed tumor cell lines (MB231, T47D, SKBR), performing antigen activation with any of 5 digestive enzymes (thrombin, trypsin, proteinase K, dispase, proline-telopeptidase), immunofluorescent staining with an anti-Ki-67 antibody or its isotype control antibody, and analyzing with a flow cytometer.
Fig. 12B is a histogram showing data of gating fractions selected as cell nucleus regions, with fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis, by performing antigen activation by heat treatment on each of formalin-fixed tumor cell lines (MB231, T47D, SKBR), performing antigen activation with any of 5 digestive enzymes (thrombin, trypsin, proteinase K, dispase, proline-terminated peptidase), performing immunofluorescence staining with an anti-cytokeratin antibody or an isotype control antibody thereof, and analyzing with a flow cytometer.
Fig. 13A shows a histogram in which the fluorescence intensity of the gated fraction selected as the cell nucleus region is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis, using the fluorescence intensity as the horizontal axis, for the data of the gated fraction.
Fig. 13B shows a histogram in which the fluorescence intensity of the gated fraction selected as the cell nucleus region is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis, wherein the data is represented by a histogram in which the fluorescence intensity is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis.
FIG. 14 is a histogram showing fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis of data of a gated fraction selected as the nucleus region, in which data of the FFPE tissue section of a breast cancer tissue is deparaffinized/hydrophilized, activated by heat-treated antigen, activated by thrombin, disrupted by water flow, and then subjected to immunofluorescence staining using any of 2 anti-Ki-67 antibodies (MIB-1 clone and S5 clone) having different recognition sites and an anti-cytokeratin antibody or isotype control antibody thereof, followed by flow cytometry.
FIG. 15 is a histogram showing fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis for data of gated fractions selected as the nucleus region, after deparaffinization/hydrophilization of FFPE tissue sections of breast cancer tissues according to a known method, antigen activation with trypsin, immunofluorescence staining with any of 2 anti-Ki-67 antibodies (MIB-1 clone and S5 clone) having different recognition sites and an anti-cytokeratin antibody or isotype control antibody thereof, followed by analysis with a flow cytometer.
Fig. 16 is a histogram showing fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis for data of gated fractions selected as the nuclear region, by deparaffinizing/hydrophilizing FFPE tissue sections in which ER and PgR are positive or negative, performing antigen activation by heat treatment, then performing antigen activation with thrombin, further disrupting cells by water flow, then performing immunofluorescence staining with an anti-ER antibody and an anti-PgR antibody or their isotype control antibodies, and then analyzing by a flow cytometer.
Fig. 17 is a histogram showing data of gated fractions selected as cell nucleus regions, in which the fluorescence intensity is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis, using different heat-treatment antigen activators for the formalin-fixed breast cancer cell line MDA-MB-231, and then activated with thrombin, and immunofluorescent staining is performed with an anti-Ki-67 antibody and an anti-cytokeratin antibody or isotype control antibodies thereof.
FIG. 18 is a histogram showing data of gated fractions selected as a cell nucleus region, with fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis, in which the data are deparaffinized/hydrophilized, heat-treated antigen-activated, antigen-activated with thrombin, and subjected to immunofluorescence staining with an anti-cytokeratin antibody and an anti-Ki-67 antibody or their isotype control antibodies, after applying any of 4 different disruption methods (combination of masher, mortar, ultrasonication, waterflow disruption and ultrasonication) to FFPE tissue sections of breast cancer tissue, followed by flow cytometry.
FIG. 19 is a scattergram showing 2 breast cancer cell lines SKBr3 and MDA-MB-231 fixed in formalin and a lymphoblastic cell line Jurkat, which were subjected to an antigen activation treatment by heat treatment, activated with a thrombin antigen, disrupted by ultrasonic waves, stained for cell nuclei, analyzed by a flow cytometer, and analyzed for data obtained by the analysis, with the horizontal axis representing the forward scattering and the vertical axis representing the side scattering.
Fig. 20 is a scattergram showing the results of a predetermined pretreatment, thrombin treatment, cell disruption by a water stream, cell dispersion in a buffer containing various surfactants, cell disruption by ultrasonic waves, cell nucleus staining, flow cytometry analysis, and analysis of the obtained data with the horizontal axis of forward scattering and the vertical axis of side scattering.
FIG. 21 is a graph relating Ki-67 positivity in FFPE tissue sections of breast cancer tissue by IHC method calculated by different pathologists.
Fig. 22A shows histograms in which the fluorescence intensity is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis for data of gated fractions selected as the nuclear region, after performing antigen activation by heat treatment on each of the formalin-fixed tumor cell lines (MB231, T47D, SKBR), performing antigen activation with any of 3 digestive enzymes (thrombin, proteinase K, dispase), performing immunofluorescence staining with the anti-Ki-67 antibody S5 clone (Ki-S5) or its isotype control antibody, and analyzing with a flow cytometer.
Fig. 22B is a histogram in which the fluorescence intensity of data of gated fractions selected as the cell nucleus region is plotted on the horizontal axis and the number of cells (nuclei) is plotted on the vertical axis, wherein the data of each tumor cell line (MB231, T47D, SKBR) fixed in formalin is subjected to antigen activation by heat treatment, is subjected to antigen activation by any of 3 digestive enzymes (thrombin, proteinase K, dispase), is then subjected to immunofluorescence staining with an anti-cytokeratin antibody or an isotype control antibody thereof, is analyzed by a flow cytometer, and the data of the gated fractions are plotted on the horizontal axis and the vertical axis.
Fig. 23 is a histogram showing the fluorescence intensity as the horizontal axis and the number of cells (nuclei) as the vertical axis of data of gated fractions selected as the nucleus region, in which the data are analyzed by a flow cytometer after deparaffinization/hydrophilization of FFPE tissue sections of breast cancer tissues, activation with heat-treated antigens, activation with thrombin, disruption of cells by water flow, further disruption of cells by ultrasonic waves, immunofluorescent staining with an anti-cytokeratin antibody and an anti-Ki-67 antibody or their isotype control antibodies, and the subsequent analysis. The graph shows the positive rate when the threshold for positive nuclear determination was varied.
Fig. 24 is an image obtained by subjecting an FFPE tissue section known to be HER2 positive and negative to deparaffinization/hydrophilization, activation with an antigen by heat treatment, antigen activation with thrombin, cell disruption with water flow, cell disruption with ultrasonic waves, fluorescence staining of the HER2 gene by the FISH method using a DNA probe labeled with fluorescence, and observation with a fluorescence microscope.
[ DEFINITIONS ]
"immobilization of cells" means that the form of cells or the structure of a tissue, which are crosslinked by molecules or insolubilized by proteins, is stabilized by immersing a sample in a fixative solution. For the immobilization, a known method may be used, and for example, formalin solution, paraformaldehyde solution, glutaraldehyde solution, osmium tetroxide solution, acetic acid alcohol, methanol, ethanol, acetone, or the like may be used. Alternatively, the immobilization may be carried out by a freezing method.
The "Ki-67 antigen (MKI 67: Marker Of promotion Ki-67)" is a kind Of nuclear protein, and in the case Of HUMAN, is represented by the amino acid sequence (3256 amino acids) Of SEQ ID NO:1 (UniProtKB-P46013(KI67_ HUMAN)). Ki-67 is the name of an antibody found as an autoantibody in the blood of an original leukemia patient, and in the present application, "Ki-67" refers to the "Ki-67 antigen" protein unless otherwise specified. Thus, an "anti-Ki-67 antibody" refers to an "antibody" that recognizes the Ki-67 antigen.
The "antibody" may be a completely long (IgG, IgA, IgM, IgD, IgE) immunoglobulin or a fragment containing an antigen-binding recognition region thereof (so-called fragment antibody (Fab, Fab ', F (ab') 2, etc.)). The "antibody" may be derived from a mammal such as a human, a mouse, a rat, a goat, a horse, or a camel, or may be derived from a fish (including shark) or a bird (chicken).
"MIB-1" or "MIB-1 antibody" is one of the clones that monoclonal the Ki-67 antibody. Not particularly limited, and is commercially available from Dako corporation or Immunotech corporation. Ki-67 recognizes and binds to an epitope consisting of PKEKAQALEDLAGFKELFQT (SEQ ID NO: 2).
The "anti-Ki-67 antibody" is an antibody that recognizes and binds to Ki-67, and other than MIB-1, antibodies belonging to the MIB (registered trademark) family such as MIB-2, MIB-5, MIB-7, MIB-21 and MIB-24, DAKO-PC, Ki-S5 and A0047 can be used as "anti-Ki-67 antibody", and antibodies having the same properties as those used in the commercial production or clinical examination can be used as "anti-Ki-67 antibody". The anti-Ki-67 antibody can be prepared by a known method using Ki-67 antigen or a part thereof as an antigen. The "anti-Ki-67 antibody" used in the present invention preferably contains an antibody recognizing the above epitope.
"Thrombin (factor IIa)" is an enzyme involved in the coagulation of blood (serine protease) (EC number: EC3.4.21.5).
"hydrolase that does not recognize a peptide that cleaves SEQ ID NO. 2" means a hydrolase that does not recognize an epitope that cleaves MIB-1 antibody.
The protease having such properties can be known from a known database (http:// web. expasy. org/peptide _ cutter /) (Table 1). Whether or not an enzyme has such a property can be analyzed by using another antibody recognizing MIB-1 or the same epitope to confirm whether or not the enzyme cleaves the epitope.
The enzymes involved are preferably recognized and cleaved collagens of the extracellular matrix for the purpose of intracellular infiltration with antibodies and/or for the purpose of nuclear extraction. Examples of the protease include, but are not limited to, thrombin, Arg-C (clostripain) peptidase (EC 3.4.22.8), proline-terminated peptidase (EC 3.4.21.26), and the like.
[ TABLE 1]
Collagen alpha-1 (I): UniProtKB-P02452(COLA1_ HUMAN)
Ki-67：SEQ ID NO:1
For the purpose of the present invention, an enzyme that hydrolyzes a component in an extracellular substrate other than proteins such as hyaluronidase is also preferable. Hyaluronidase (EC 3.2.1.35) is a hydrolase that breaks down the β -1-4 bond of hyaluronic acid, without cleaving the peptide of SEQ ID NO: 2. The infiltration of the antibody into the cell is promoted by this property of hyaluronidase. Examples of such enzymes include hyaluronidase, glycosidase (EC 3.2.1), and N-glucanase (EC 3.5.1.52), with hyaluronidase being preferred.
In addition, although the protease that does not recognize the cleavage of the peptide of SEQ ID NO. 2 is common to hyaluronidase at the site that does not cleave the peptide of SEQ ID NO. 2, on the other hand, since both are enzymes having completely different actions, it is expected that the antigen-inactivating activity will be further increased by combining both. In particular, a combination of thrombin and hyaluronidase is preferred.
"activation with an enzyme antigen" is a process of cleaving an antigen or an extracellular substrate by enzyme treatment to amplify the reactivity of an antibody, and can be performed by immersing a sample in a reagent for dissolving an enzyme in a buffer solution adjusted in pH. The reaction is stopped after a predetermined time by heating at a temperature suitable for the enzyme reaction. During the termination of the reaction, removal of a reagent, temperature change, addition of a chelating agent, and the like are used.
The "staining with an antibody" may be performed by directly binding each antibody to a labeling substance such as a fluorescent compound, an enzyme, or a chemiluminescent substance (direct method), or by binding the labeled antibody to an antigen, or may be performed by indirectly labeling an antigen by binding a second antibody, which is specific to each first antibody and binds to the labeling substance, to a non-labeled first antibody (indirect method).
The "fluorescent compound" is not particularly limited, and examples thereof include a cyanine dye such as Cy3, and a fluorescent substance such as Fluorescein Isothiocyanate (FITC), allophycocyanin, and rhodamine. Each antibody (or the second antibody thereof) is preferably labeled with a fluorescent dye that emits light at a different fluorescent wavelength (for example, a fluorescent substance of Alexa Fluor (registered trademark) series).
The "labeling enzyme" is not particularly limited, and examples thereof include alkaline phosphatase and horseradish peroxidase.
The "chemiluminescent substance" is not particularly limited, and examples thereof include LUMINOR, AMPPD (registered trademark), CSPD (registered trademark), and CDP-Star (registered trademark).
In one embodiment, when there are a plurality of antigens to be detected/quantified in addition to Ki-67 in the cell population in the tissue section,
(1) for the same (FFPE) tissue section, each ligand (for example, an anti-cytokeratin antibody, an anti-ER antibody, an anti-PgR antibody, a nucleic acid hybridizing with HER2 gene, or a second antibody or nucleic acid binding thereto) may also be labeled with a label that makes recognition of other ligands such as a fluorescent dye of different fluorescent wavelength possible, and detected/quantified, or
(2) Multiple (FFPE) tissue sections assumed to be homogeneous may be prepared from the same tissue mass, and each target substance may be subjected to detection/quantitative staining (e.g., immunofluorescent staining, FISH).
The term "specifically stain the cell nucleus" refers to labeling with a compound that specifically stains the cell nucleus in the immobilized cell, and is not limited to this, but is preferably a compound for staining nucleic acid that is not permeable to the cell, and examples thereof include fluorescent dyes such as DAPI and Propidium Iodide (PI).
Cells or nuclei stained by fluorescence can be counted by Cytometry (Cytometry). Cytometry refers to a cell assay that quantitatively determines a large number (thousands to millions) of cells in 1 each within a short time (seconds to minutes). In Cytometry, there are Flow Cytometry (Flow Cytometry) in which cells in suspension are introduced into cells using 1 sensor region each to stop the Flow of the cells, scattered light, fluorescence, and the like are measured at high speed, and Imaging Cytometry (Imaging Cytometry) in which a cell mass attached to a multi-well plate or a slide glass is scanned with laser light to obtain a fluorescence image, scattered light, a transmission light image, and the like, and information is extracted for each 1 cell by cell image processing.
The "step of extracting cell nucleus" refers to extraction from a fixed cell (tissue) cell nucleus in a state where the structure is maintained. The cell disruption can be performed by a means for generating shear stress to the cells, and the method is not limited to these, and can be performed by a known method such as water jet disruption, a masher, a mortar, ultrasonic disruption, a net, a French press, homogenizer treatment, and glass bead treatment.
Preferably, the present invention is a method for further enhancing a staining signal without disappearing antigenicity from FFPE tissue and without limiting cell nucleus detachment using an antibody or a detection method, comprising the steps of:
(1) paraffin removal/hydrophilization of thin-cut FFPE slices
(2) Activation of antigens by heat treatment
(3) Activation of antigens by enzymes
(4) Extracting cell nucleus by breaking tissue and cell
In the present invention, the thickness of the FFPE tissue slice used for thinning is preferably not more than 60 μm, and the detection of a desired marker can be performed as the thickness of the tissue at the time of thinning for the purpose of enhancing the efficiency of the pretreatment step, and is preferably 20 μm, but not limited thereto. The number of thin slices can be increased by a plurality of numbers depending on the size of the specimen and the number of detached nuclei necessary for subsequent studies.
"embedding" means that the tissue piece (block) is set to a constant and uniform hardness, the middle cavity portion in the tissue is sealed, the tissue piece (block) has a strength that does not peel off at the time of thin cutting, and the "embedding agent" is infiltrated into the tissue piece (block) for the purpose of increasing the storage stability. The "embedding agent" is not particularly limited, and examples thereof include paraffin, paraffin derivatives, collodion, carbowax, agarose, non-heparin-treated serum, collagen, cellulose derivatives, chitin derivatives, chitosan derivatives, and mixtures thereof.
The term "deintercalation/hydrophilization" as used herein means the removal of an embedding agent (e.g., paraffin) used for embedding and the replacement of an organic solvent used for the removal with an aqueous solvent. The section is immersed in an organic solvent such as xylene to remove the embedding agent, and then ethanol solutions having a plurality of concentrations with a concentration gradient are sequentially immersed in the section from a high concentration to a low concentration in place of the organic solvent. Examples of the ethanol concentration gradient include, but are not limited to, 100%, 95%, 90%, 70%, and 50%.
The "activation by heat-treated antigen" in the present invention refers to a step of removing the antigen recognition site by heat treatment when it is formed by crosslinking at the time of fixation to cover the antigen recognition site. In the heat treatment, an antigen activator for heat treatment containing a citric acid buffer, a surfactant, a chelating agent, a reducing agent, or the like is used. The antigen activator for heat treatment is not particularly limited, and commercially available Histo VT One (NACALALI TESQUEE), antigen activation solution pH9(Nichirei Biosciences), ImmunoSaver (Nissan EM), and the like can be used.
The term "disruption by water flow" means that cells and tissues can be disrupted by shearing force of water flow, and the cells and tissues are disrupted by water flow generated by rotating a blade at 10,000rpm every 1 minute under ice-cooling using, for example, a water flow shearing apparatus (RP-10) of Sysmex corporation.
"disruption by ultrasound" means that cells and tissues can be disrupted by the shearing force of ultrasound, using, for example, an ultrasonic disruption apparatus (VCX130PB) from SONICS & MATERIALS corporation, which is exposed to ultrasound at an output intensity of 20% for 30 seconds.
The step of disrupting using ultrasonic waves is advantageous in disrupting lymphocytes, not only cell walls of lymphocytes, but also nuclei of lymphocytes, since lymphocytes are disrupted preferentially over other blood cells. Therefore, when it is desired to preferentially break lymphocytes, for example, when the presence of lymphocytes is an obstacle to analysis of other cells, it is preferable to include a breaking step by ultrasonic waves.
In the method for detaching nuclei of the present invention, 2 or more of the above-mentioned methods may be combined, and a combination of water shearing and ultrasonic disruption is also preferable.
In the step of extracting cell nuclei, the tissue or cells may be immersed or dispersed in a commonly used buffer solution, and for example, tris (hydroxymethyl) aminomethane buffer solution, phosphate buffer solution, carbonate buffer solution, glycine buffer solution, acetic acid buffer solution, tartaric acid buffer solution, citric acid buffer solution, triethanolamine buffer solution, boric acid buffer solution, Good buffer solution, or the like may be used. Of course, since the surfactant has an action of promoting detachment of the cell nucleus, it is preferable to extract the cell nucleus by using a buffer to which the surfactant is added. The surfactant is not particularly limited as long as it does not affect the cell nucleus, and examples thereof include anionic surfactants (carboxylic acid type, sulfonic acid type, sulfate ester type, phosphate ester type, etc.), cationic surfactants (quaternary ammonium salt type, alkylamine salt type, type having a pyridine ring, etc.), amphoteric surfactants (betaine type, sulfobetaine type, amine oxide type, alkylimidazole type, amino acid type, etc.), and nonionic surfactants (ester type, ether type, ester ether type, alkanolamide type, alkylglycoside type, etc.). Preferred examples of the nonionic surfactant include TritonX-100, TritonX-405, NP-40, Briji-35, Briji-58, Tween-20, Tween-80, BPSH-25, Octyl Glucoside, and Octylthio Glucoside.
Malignant tumors are tumors that infiltrate into surrounding tissues or cause metastasis among cell groups (including tumors, benign tumors, and malignant tumors) that grow in an involuntary manner by genetic variation, and the terms of malignant tumors (malignantumors) are classified into the following (1) to (3) in pathology:
(1) carcinoma (Carcinoma): a malignant tumor of epithelial tissue origin,
(2) sarcoma (Sarcoma): a malignant tumor of non-epithelial tissue origin,
(3) further: leukemia and the like,
in the present application, "cancer" refers to cancerous tumors. There are no particular restrictions on the cancer cells for head and neck cancer (palate cancer, (upper, middle, lower) pharynx cancer, larynx cancer, tongue cancer, thyroid cancer), chest cancer (breast cancer, lung cancer (non-small cell lung cancer, small cell lung cancer)), digestive cancer (esophagus cancer, stomach cancer, duodenum cancer, large intestine cancer (colon cancer, rectal cancer), liver cancer (hepatocellular cancer, cholangiocellular cancer), gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer, cancer of the urinary organs (kidney cancer, ureter cancer, bladder cancer, prostate cancer, penis cancer, seminal (testicular) cancer), genital cancer (uterine cancer (cervix cancer, uterine corpus cancer), ovarian cancer, vulvar cancer, vaginal cancer), skin cancer (basal cell cancer, squamous cell cancer), and the like.
Ki-67 may be used alone or in combination with other markers to assist in the diagnosis of cancer, or to assist in the prognosis of a treatment for diagnosing cancer.
Cytokeratin is an example of a marker for epithelial cells used to distinguish carcinoma from sarcoma. Cytokeratin is a major skeletal protein of epithelial cells, and about 20 to 30 subtypes (
The Estrogen Receptor (Estrogen Receptor, ER) is one of the molecules belonging to the steroid Receptor superfamily, also known as follicle hormone Receptor. There are 2 isoforms in the ER, each called era and ER β. These were generated from independent genes (ESR1, ESR 2). ESR1 was present at 6q25.1 and ESR2 was present at 14q 21-22.
The Progesterone receptor (Progesterone receptor (PR or PgR) is a nuclear protein belonging to
About 2 out of 3 primary breast cancers are hormone receptor positive breast cancers, and it is known that ER positive breast cancers tend to increase in japanese women. Since PgR is expressed by the estrogen-ER complex, the presence or absence of PgR becomes the benchmark for the functional normal activity or absence of estrogen and ER. The expression of these hormone receptors is now detected by immunohistochemical methods using pathological tissue specimens.
HER2 is a glycoprotein of approximately 185kDa present on the cell surface and is a receptor-type tyrosine kinase. Has a structure similar to that of the epithelial growth factor receptor (EGFR, alias ERBB1), also known as EGFR2, ERBB2, CD340, or NEU. The genes encoding HER2 proteins are HER2/neu and erbB-2, and are present on the long arm of chromosome 17. In addition, HER2 is a protein belonging to the human epidermal growth factor receptor (HER/EGFR/ERBB) family (EGFR family).
The HER2 protein is involved in regulation of cell proliferation and differentiation in normal cells, and amplification or gene mutation of the HER2 gene and control of cell proliferation-differentiation cannot occur for some reason, and thus cells become malignant. The HER2 gene may also be an oncogene, and gene amplification is seen in many types of cancers.
The samples obtained in the above-described steps of antigen activation by an enzyme and further extraction of cell nuclei can also be used in the analysis of these markers in combination with Ki-67 for the purpose of assisting diagnosis. In particular, an enzymatic treatment combining thrombin and hyaluronidase is preferred when the analysis of these markers is also performed. Of course, the detection of the HER2 gene can also be performed using a nucleic acid extracted by a known method after nuclear detachment.
A "patient" can be a mammal, can be a "human" or a "non-human mammal.
"tissue section" refers to a section of isolated tissue from a human or non-human animal. May be a "tissue slice" taken in a biopsy. The "tissue section" may be isolated, and then stored in a frozen state.
The "biopsy" refers to a procedure of taking a part of a pathological tissue with a scalpel, a needle, or the like for observation with a microscope or the like at the time of diagnosis. In the case of breast cancer, biopsy is performed from the patient's breast by "resection" (total removal of a lump of tissue); "open biopsy" (excise a portion); "core biopsy" (removal of a portion of tissue with a thick needle); or "Fine Needle Aspiration (FNA) biopsy" (removal of tissue or body fluid with a fine needle).
The "antigen activator by a lytic enzyme" and the "antigen activator by heat treatment" may be present in the form of a solution in which the components are dissolved, or may be in the form of a dried solid. In the case where either or both of these components are in a solid state, the "kit" may contain a "solvent" for dissolving these solid components.
"antigen activator used in a specimen in which Ki-67 is detected by immunostaining" means an enzyme activator used in a specimen in which cells or cell nuclei are detected using an anti-Ki-67 antibody, typically, from an immobilized cell population or tissue.
"prognosis of cancer therapy" refers to the confirmation of prediction of the therapeutic effect of chemotherapy (anticancer agent therapy), endocrine therapy, surgery (tumor extirpation), radiation therapy, and the like on a patient.
The "kit for detecting Ki-67 positive cells in an immobilized cell population" contains a hydrolase which does not recognize the peptide cleaving SEQ ID NO. 2 and an anti-Ki-67 antibody. The hydrolase is preferably at least one enzyme selected from the group consisting of thrombin, Arg-C (clostripain) peptidase, proline-terminated peptidase and hyaluronidase, and more preferably thrombin and/or hyaluronidase. The anti-Ki-67 antibody is preferably at least one selected from the group consisting of MIB-1, DAKO-PC, Ki-S5 and A-0047. The anti-Ki-67 antibody may be labeled directly with a fluorescent dye, an enzyme, a chemiluminescent substance, a radioactive element, or the like, or the kit may contain a 2 nd antibody that binds to the anti-Ki-67 antibody, the 2 nd antibody being labeled with a fluorescent dye, an enzyme, a chemiluminescent substance, a radioactive element, or the like. The kit may contain, in combination with Ki-67, ligands (e.g., antibodies or probes, etc.) for detecting other markers used to aid in the diagnosis of cancer or to aid in the prognosis of a treatment for diagnosing cancer. For example, an anti-cytokeratin antibody, an anti-ER antibody, an anti-PgR antibody, a probe that hybridizes to HER2 gene (typically, under stringent conditions) (these or a second ligand (such as an antibody or nucleic acid) thereof may also be labeled), and a compound for staining nucleic acid (for example, DAPI or Propidium Iodide (PI)) may be mentioned.
The kit may further contain a buffer for immersing or dispersing the tissue or cells used in the "procedure for extracting nuclei", and preferably, the buffer contains a surfactant. As the surfactant, CHAPS, NP-40, and Triton-X100 are preferable, and NP-40 and Triton-X100 are more preferable. Thus, according to the invention of the present application, there is provided a kit for extracting cell nuclei from cells (usually, immobilized cells) by shear stress generated with water flow, ultrasonic waves or the like, comprising a buffer for cell dispersion containing a surfactant.
The "cutoff value" (division point or morbid state identification value) refers to a numerical value for discriminating positive and negative of the test result. In the case of Ki-67, values of 14-20% are now recommended for classification of LuminalA (type-like) and LuminalB (type-like) types.
The term "anticancer agent" refers to a drug for treating or preventing cancer. Without being limited thereto, they are classified into molecular target drugs, alkylating agents, metabolic antagonists, plant alkaloids, anticancer and antibiotic agents, platinum agents, hormonal agents, biological response modifiers and the like.
"endocrine therapy" refers to a treatment for inactivating a female hormone (estrogen) that promotes the proliferation of hormone-dependent breast cancer, and includes, but is not limited to, hormone agents that administer antiestrogens, Lh-RH agonist preparations, aromatase inhibitors, progesterone preparations, and the like.
"chemotherapy" refers to the treatment of cancer with chemicals that exert an anti-cancer effect. The present invention is not limited to this, and the present invention preferably includes single administration or combined administration of an anthracycline anticancer agent (daunorubicin, doxorubicin, epirubicin, idarubicin, etc.), a taxane anticancer agent (paclitaxel, docetaxel, etc.), a platinum (platinum) agent (cisplatin, oxaliplatin, etc.), etc., which are inhibitors of DNA synthesis and replication, which are inhibitors of cell proliferation, and the like, and preferably includes combined administration of an anthracycline anticancer agent and a taxane anticancer agent. In the present specification, the term "hormone agent" is used to include "endocrine therapy".
[ examples ] A method for producing a compound
The present invention will be described in further detail with reference to the following examples, comparative examples and reference examples, but the present invention is not limited to these examples.
< reference example 1: observation of nucleus detachment by Water flow shearing
[1] materials and methods ]
FFPE tissue sections of breast cancer tissues were deparaffinized/hydrophilized, activated with heat-treated antigens, dispersed using a water flow shearing apparatus, and then subjected to immunofluorescent staining and microscopic observation of the recovered products.
[ 1-1.FFPE tissue blocks ]
FFPE tissue blocks of breast cancer tissue purchased from Proteogenex corporation were used.
[ 1-2 ] preparation of FFPE chips ]
A slide microscope slide microtome (manufactured by Thermofish) was used to thin-cut the FFPE tissue block to prepare a section having a thickness of 20 μm.
[ 1-3. De-Paraffin/hydrophilization ]
A sufficient amount of xylene was added to the thin-cut FFPE slices, and after standing for 10 minutes, the xylene was removed. This step was performed again to completely remove the paraffin wax. Subsequently, the sheet was immersed in 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and deionized water for 3 minutes in this order to hydrophilize the sheet.
[ 1-4 ] activation by Heat-treated antigen ]
Histo VT One manufactured by NACAALAI TESSQUE was added thereto diluted 10-fold with pure water, and the mixture was heated at 98 ℃ for 20 minutes with a hot block. After heating, the mixture was allowed to stand at room temperature for 20 minutes, and then the antigen activating solution was removed.
[ 1-6 ] Water stream disruption ]
The tissue was disrupted in TBS1mL at 10,000rpm for 1 minute under ice-cooling using a water jet cutter (RP-10) from Sysmex.
[ 1-7 ] immunofluorescent staining ]
4% BSA/TBS to which 10% normal goat serum (and light) was added to a microtube containing disrupted material disrupted by a water flow, and the mixture was allowed to stand at room temperature for 30 minutes and then blocked. For immunofluorescence staining, pan-Cytokeratin antibody (Abcam, rabbit polyclonal antibody: Anti-wind monoclonal antibody (AB9377-500)) was used as the 1 st antibody, and goat Anti-rabbit secondary antibody Alexa488 was used as the 2 nd antibody. The 1 st antibody reaction time was 50 minutes, the 2 nd antibody reaction time was 30 minutes, and the dye dapinosution (and light) for staining cell nuclei was added when the 2 nd antibody reaction time elapsed. The reaction was carried out at room temperature, and after addition of the 2 nd antibody, the reaction was carried out in the dark. In the antibody dilution using 0.5% BSA/TBS, each step in 1 times 0.5% BSA/TBS washing operation. The slide was loaded with 5 μ l of LDAPI embedding medium (and light) and the sample after the 2 nd antibody reaction was loaded thereon. Thereafter, the cover glass was covered and left to stand for 5 minutes, and then, the periphery of the cover glass was sealed with a nail polish by vertically pressing from above. When observed, the mixture was kept at 4 ℃ in the dark.
[ 1-8 ] fluorescence microscope ]
For microscopic observation, an EVOS integrated microscope, a fluorescence microscope (Thermoscientific), was used. DAPI Light Cube (Ex 357nm, Em 447nm) and GFP Light Cube (Ex 470nm/Em 510nm) were used for observation. The eyepiece is used at 10 times and the objective lens is used at 40 times.
[ 2. results ]
The nuclei observed with a fluorescence microscope are shown in FIG. 1. As shown in the left panel, the nucleic acid exists in a circular shape, and as shown in the right panel, cytokeratin exists around the nucleic acid. Thus, it was confirmed that the nuclei and the skeleton were not broken and remained circular, and were collected as single nuclei without aggregation.
< reference example 2: identification of cytokeratin and Ki-67-derived signals in formalin-fixed breast cancer cells
[1] materials and methods ]
Formalin-fixed breast cancer cells were activated with heat-treated antigen, immunofluorescent-stained, and signals of cytokeratin and Ki-67 were confirmed by flow cytometry.
[ 1-1. cell ]
3 breast cancer cell lines, T47D, MDA-MB-231 (shown as MB231 or 231 in the figure), and SKBr3 (shown as SKBR in the figure) were used in the ATCC (American Type Culture Collection).
[ 1-2 ] cell culture and formalin fixation ]
T47D cell line was cultured in RPMI-1640 medium, MDA-MB-231 cell line in Leibovitz's L-15 medium and SKBr3 cells in McCoy's 5A medium supplemented with 10% Fetal Bovine Serum (FBS). After the cells were sufficiently proliferated, the culture broth was aspirated, and after washing with PBS, TrypLE Express (thermolfisher) was added. After recovery of the cells, the cells were centrifuged and washed with PBS. Furthermore, the cells were suspended in PBS sufficiently and dispensed at
[ 1-4 ] activation by Heat-treated antigen ]
The same treatment as in reference example 1 was carried out.
[ 1-7 ] immunofluorescent staining ]
4% BSA/TBS to which 10% normal goat serum (and light) was added to a microtube containing the cells activated with the heat-treated antigen, and the microtube was left standing at room temperature for 30 minutes and then blocked. Immunofluorescence staining a mixture of mouse and rabbit antibodies was used for 2-fold staining. The
[ 1-8 ] flow cytometry assay ]
A Falcon (registered trademark) cell filter 5mL tube was passed through a 35 μm (380 mesh) (for flow cytometry) filter, and then transferred to a predetermined container, and measured by flow cytometry (Sysmex: Space). The measurements were carried out according to the machine manual.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
For analysis of the obtained measurement data, FLOWJO v10 software manufactured by FLOWJO LLC was used. Data analysis was performed in the following procedure according to the specification attached to the software. On a scattergram with the horizontal axis of the forward scatter and the vertical axis of the side scatter, the main regions shown in fig. 2 are selected. Next, fluorescence intensity of DAPI was plotted on the horizontal axis, the number of cells (nuclei) was plotted on the vertical axis, and the main region was used as a nucleus gate (fig. 3). The positive rates of cytokeratin and Ki-67 were calculated as the ratio of the number of cytokeratin-positive nuclei and the number of Ki-67-positive nuclei in the whole cell nucleus. The threshold for positive nuclei was used as a 95 percent value for isotype control.
[ 2. results ]
Fig. 4 shows a graph obtained by superimposing the histograms of the fluorescence intensities detected with the anti-cytokeratin antibody and the anti-Ki-67 antibody (black solid line) and the histogram of the fluorescence intensities detected with the isotype control antibody (gray dashed line). In any cell line, the black peak shows higher fluorescence intensity than gray. From these results, the presence of cytokeratin and Ki-67 in the breast cancer cells was confirmed by formalin fixation.
< example 1: enhancement of Ki-67 Signal in formalin-fixed cells activated by antigen with Thrombin
[1] materials and methods ]
The signal intensity of the presence or absence of thrombin treatment was compared using formalin-fixed breast cancer cells.
[ 1-1. cell ]
3 breast cancer cell lines used in reference example 2 were used.
[ 1-2 ] cell culture and formalin fixation ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-4 ] activation by Heat-treated antigen ]
The same treatment as in reference example 1 was carried out.
[ 1-5 ] activation by antigen of enzyme ]
Thrombin reagent (25mM Tris-HCl pH7.4, 150mM NaCl, 1000KU/L thrombin, 10mM CaCl) was added to the cells after activation by the heat-treated antigen2) The mixture was heated at 37 ℃ for 20 minutes using a hot block.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2. Among them, Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) was used as the 1 st antibody, and goat anti-mouse secondary antibody Alexa647 from Thermofish was used as the 2 nd antibody.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of Ki-67 Positive Rate
The procedure was carried out in the same manner as in reference example 2.
[ 2. results ]
FIG. 5 shows a graph in which the fluorescence intensities detected with the anti-Ki-67 antibody (black solid line) and the isotype control antibody (gray dashed line) are superimposed, and FIG. 6 and Table 2 show the change in the Ki-67 positivity. In any cell line, both the activation of the antigen by thrombin and the increase in fluorescence intensity were carried out, with an increase in the Ki-67 positive rate being seen.
[ TABLE 2]
< example 2: enhancement of Ki-67 signaling in formalin-fixed cells activated by antigen with hyaluronidase >
[1] materials and methods ]
The signal intensity of the presence or absence of hyaluronidase treatment was compared using formalin-fixed breast cancer cells.
[ 1-1. cell ]
The T47D cell line used in reference example 2 was used.
[ 1-2 ] cell culture and formalin fixation ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-4 ] activation by Heat-treated antigen ]
The same treatment as in reference example 1 was carried out.
[ 1-5 ] activation by antigen of enzyme ]
The hyaluronidase reagent (25mM Tris-HCl pH7.4, 150mM NaCl, 4000-.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2. Among them, Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) was used as the 1 st antibody, and goat anti-mouse secondary antibody Alexa647 from Thermofish was used as the 2 nd antibody.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of Ki-67 Positive Rate
The procedure was carried out in the same manner as in reference example 2.
[ 2. results ]
FIG. 7 shows a graph in which the fluorescence intensities detected with the anti-Ki-67 antibody (black solid line) and the isotype control antibody (gray dashed line) were superimposed. The fluorescence intensity increased by hyaluronidase treatment, with which an increase in the Ki-67 positivity was seen.
< reference example 3: identification of cytokeratins and Ki-67-derived signals in FFPE tissue sections
[1] materials and methods ]
Signals of cytokeratin and Ki-67 in FFPE tissue sections of breast cancer tissue were confirmed by flow cytometry.
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
The FFPE tissue block was thinly sliced with a microtome for slide microscope manufactured by Thermofish Co., Ltd to prepare 2 slices having a thickness of 20 μm, which were used for the study.
[ 1-3. De-Paraffin/hydrophilization ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-4 ] activation by Heat-treated antigen ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-6 ] Water stream disruption ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
For analysis of the obtained measurement data, FLOWJO v10 software manufactured by FLOWJO LLC was used. The data analysis is performed in the following order. First, a region in which the fluorescence intensity of DAPI is plotted on the horizontal axis, the number of cells (nuclei) is plotted on the vertical axis, and the fluorescence amount on the horizontal axis is 5 to 150 is plotted as the nuclear gating. The positive rates of cytokeratin and Ki-67 were calculated as the ratio of the number of cytokeratin-positive nuclei and the number of Ki-67-positive nuclei in the whole cell nucleus. The threshold for positive nuclei was used as a 95 percent value for isotype control.
[ 2. results ]
Fig. 8 shows a graph obtained by superimposing the histograms of the fluorescence intensities detected with the anti-cytokeratin antibody and the anti-Ki-67 antibody (black solid line) and the histogram of the fluorescence intensities detected with their isotype control antibodies (gray dashed line). The black peak shows higher fluorescence intensity than gray. From these findings, cytokeratin and Ki-67 were detected in the FFPE tissue sections.
< example 3: enhancement of cytokeratin and Ki-67 signals in FFPE tissue sections activated by antigen with thrombin
[1] materials and methods ]
The signal intensity of thrombin treatment was compared using FFPE tissue sections of breast cancer tissue.
[ 1-1.FFPE tissue blocks ]
2 FFPE tissue blocks obtained from 2 human breast cancer patients with different Ki-67 positivity rates were used by HC method purchased from IProteoenex.
[ 1-2 ] preparation of FFPE chips ]
The FFPE tissue block was thinly sliced with a microtome for slide microscope manufactured by Thermofish Co., Ltd to prepare 2 slices having a thickness of 20 μm, which were used for the study.
[ 1-3. De-Paraffin/hydrophilization ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-4 ] activation by Heat-treated antigen ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-5 ] activation by antigen of enzyme ]
The procedure was carried out in the same manner as in example 1.
[ 1-6 ] Water stream disruption ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
The procedure was carried out in the same manner as in reference example 3.
[ 2. results ]
Fig. 9A shows a graph in which the histograms of fluorescence intensities detected with the anti-cytokeratin antibody and the anti-Ki-67 antibody (black solid line) and the histogram of fluorescence intensity detected with their isotype control antibody (gray dashed line) are superimposed. On any of the FFPE sections, thrombin treatment and increase in the fluorescence intensity of cytokeratin, Ki-67, were carried out, with an accompanying increase in the positive rate (FIG. 9B).
< example 4: effect of the thickness of thinly sliced sections on the intensity of cytokeratin and Ki-67 signals
[1] materials and methods ]
It was confirmed whether the thickness of the section from FFPE affected the signal intensity of cytokeratin and Ki-67.
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
20
[ 1-3. De-Paraffin/hydrophilization ]
The same procedure as in reference example 1 was repeated.
[ 1-4 ] activation by Heat-treated antigen ]
The same procedure as in reference example 1 was repeated.
[ 1-5 ] activation by enzyme antigen ]
The procedure was as in example 1.
[ 1-6 ] Water stream disruption ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
The procedure was carried out in the same manner as in reference example 3.
[ 2. results ]
The thickness of the thin sections and the change in the positive rates for cytokeratin and Ki-67 are shown in FIG. 10.
Although the detection efficiency was the best among 3 of 20 μm, Cytokeratin (CK) and Ki-67(Ki67) could be detected at any thickness of 20-60 μm.
< example 5: comparison of Ki-67 Positive Rate and Positive Rate by IHC method Using flow cytometer
[1] materials and methods ]
The FFPE tissue blocks were used to compare the Ki-67 positivity by flow cytometry to the positivity by IHC method.
[ 1-1.FFPE tissue blocks ]
19 FFPE tissue blocks obtained from 19 patients with different Ki-67 positivity rates were used by IHC method purchased from Proteogenex corporation.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The same procedure as in reference example 1 was repeated.
[ 1-4 ] activation by Heat-treated antigen ]
The same procedure as in reference example 1 was repeated.
[ 1-5 ] activation by antigen of enzyme ]
The procedure was as in example 1.
[ 1-6 ] Water stream disruption ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2. Among them, Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used as the 1 st antibody, and goat anti-mouse secondary antibody Alexa647 manufactured by Thermofisiher and goat anti-rabbit secondary antibody Alexa488 manufactured by Abcam were used as the 2 nd antibody.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of Ki-67 Positive Rate Using flow cytometer ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-10 ] calculated from the Ki-67 positivity in IHC method ]
The Ki-67 positive rate by IHC method was calculated by the hotspot method using Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) as the 1 st antibody. Further, the average of 2 pathologists was considered as the variation among patients examined under the microscope.
[ 2. results ]
The correlation with the IHC method in the Ki-67 positive rate is shown in FIG. 11.
Correlation was confirmed by using IHC method as X and this method as Y. In addition, to use the cut-off value for positive nuclei as a 95 percent value for isotype control, the intercept was set at 10. As shown in fig. 11, good results of Y being 0.5632X +10 and a correlation coefficient 0.7697 were obtained. This indicates that this method can detect Ki-67 in FFPE tissue sections.
< example 6: detection of Ki-67 and cytokeratin upon antigen activation with various digestive enzymes
[1] materials and methods ]
Using formalin-fixed breast cancer cells, Ki-67 and cytokeratin signal intensities were compared for antigen activation with 5 different digestive enzymes.
[ 1-1. cell ]
3 breast cancer cell lines used in reference example 2 were used.
[ 1-2 ] cell culture and formalin fixation ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-4 ] activation by Heat-treated antigen ]
The same treatment as in reference example 1 was carried out.
[ 1-5A. antigen activation by enzymes (Thrombin) ]
The procedure was as in example 1.
1-5B. antigen activation by enzymes (Trypsin) ]
To the cells subjected to activation with the heat-treated antigen, 250. mu.L of trypsin reagent (25mM TBS pH7.4, 1MCaCl2, 1mg/mL trypsin) was added and heated at 37 ℃ for 20 minutes. After enzyme treatment, the enzyme was removed by washing with TBS.
[ 1-5C. antigen activation by enzymes (proteinase K) ]
600mAnson U/mL proteinase K (Boehringer) was diluted 40-fold (v/v) with 25mM TBS pH7.4 to prepare a proteinase K solution. To the cells subjected to activation with the heat-treated antigen, 250. mu.L of proteinase K solution was added, and the mixture was heated at 37 ℃ for 20 minutes. After enzyme treatment, the enzyme was removed by washing with TBS.
[ 1-5D. activation by antigen of enzyme (dispase) ]
The dispersing enzyme (and light) was dissolved in 25mM TBS pH7.4 to prepare a dispersing enzyme solution (3,000 PU/mL). To the antigen-activated cells, 250. mu.L of the dispase solution was added, and the mixture was heated at 37 ℃ for 20 minutes. After completion of the reaction, 2. mu.L of 1M EDTA solution (and light) was added thereto and mixed by inversion, followed by washing with TBS to remove the enzyme.
[ 1-5E. antigen activation by enzymes (proline-terminated peptidases) ]
Proline-terminated peptidase (Toyobo Co.) was dissolved in 25mM TBS pH7.4 to prepare a proline-terminated peptidase solution (10U/mL). To the cells subjected to activation with the heat-treated antigen, 250. mu.L of proline-terminated peptidase solution was added, and the mixture was heated at 37 ℃ for 20 minutes. After the reaction was completed, the enzyme was removed by washing with TBS.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2. Among them, Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used as the 1 st antibody, and goat anti-mouse secondary antibody Alexa647 manufactured by Thermofisiher and goat anti-rabbit secondary antibody Alexa488 manufactured by Abcam were used as the 2 nd antibody.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
The procedure was carried out in the same manner as in reference example 2.
[ 2. results ]
Fig. 12A and B show graphs in which the histograms of fluorescence intensities detected with the anti-Ki-67 antibody or the anti-cytokeratin antibody (black solid line) and the histogram of fluorescence intensities detected with their isotype control antibody (gray dashed line) were superimposed. Cytokeratins were detected using any proteolytic enzyme (FIG. 12B). In contrast, when 3 kinds of proteolytic enzymes, i.e., trypsin, dispase and proteinase K, capable of recognizing the amino acid sequence cleaved with SEQ ID NO. 2 were used, the signal was decreased, and the positive rate of Ki-67 was significantly decreased. On the other hand, it was revealed that thrombin and proline-terminated peptidase which did not recognize the cleavage of the amino acid sequence of SEQ ID NO. 2 had an increased positive rate of Ki-67, and the antigenicity of Ki-67 antigen was enhanced (FIG. 12A).
< example 7: detection of Ki-67 and cytokeratin in FFPE sections of breast cancer tissue using various digestive enzymes
[1] materials and methods ]
FFPE sections of breast cancer tissue were activated with 5 different digestive enzyme antigens and signal intensities of Ki-67 and cytokeratin were compared.
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The same procedure as in reference example 1 was repeated.
[ 1-4 ] activation by Heat-treated antigen ]
The same procedure as in reference example 1 was repeated.
[ 1-5A. antigen activation by enzymes (Thrombin) ]
The procedure was carried out in the same manner as in example 1.
1-5B. antigen activation by enzymes (Trypsin) ]
The procedure was carried out in the same manner as in example 6.
[ 1-5C. antigen activation by enzymes (proteinase K) ]
The procedure was carried out in the same manner as in example 6.
[ 1-5D. activation by antigen of enzyme (dispase) ]
The procedure was carried out in the same manner as in example 6.
[ 1-5E. antigen activation by enzymes (proline-terminated peptidases) ]
The procedure was carried out in the same manner as in example 6.
[ 1-6 ] Water stream disruption ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2. Among them, Ki-67 antibody (Dako, clone: MIB-1, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used as the 1 st antibody, and goat anti-mouse secondary antibody Alexa647 manufactured by Thermofisiher and goat anti-rabbit secondary antibody Alexa488 manufactured by Abcam were used as the 2 nd antibody.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
The procedure was carried out in the same manner as in reference example 3.
[ 2. results ]
Fig. 13A and B show graphs in which the histograms of fluorescence intensities detected with the anti-Ki-67 antibody or the anti-cytokeratin antibody (black solid line) and the histogram of fluorescence intensities detected with their isotype controls (gray dashed line) were superimposed. Cytokeratins were detected using any proteolytic enzyme (FIG. 13B). In contrast, in the same manner as in example 6, when trypsin, dispase, or proteinase K enzyme that recognizes the peptide cleaving SEQ ID NO:2 was used, the fluorescence intensity of the Ki-67 antibody (black solid line) was greatly reduced (FIG. 13A).
< example 8: detection of Ki-67 Using antibodies with different recognition sites >
[1] materials and methods ]
Signals of cytokeratin and Ki-67 in FFPE tissue sections of breast cancer tissue were confirmed by flow cytometry. The Ki-67 antibody used was the MIB-1 clone, and the S5 clone was used as an antibody different from the recognition site.
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The same procedure as in reference example 1 was repeated.
[ 1-4 ] activation by Heat-treated antigen ]
The same procedure as in reference example 1 was repeated.
[ 1-5 ] antigen activation treatment by enzyme ]
The procedure was as in example 1.
[ 1-6 ] Water stream disruption ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2. Among them, as the 1 st antibody, Ki67MIB-1 antibody (Dako, mouse monoclonal antibody) or Ki 67S 5 antibody (Millipore, mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used, and as the 2 nd antibody, goat anti-mouse second antibody Alexa647 (Thermofish Co.) and goat anti-rabbit second antibody Alexa488 (Abcam Co.) were used.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
The procedure was carried out in the same manner as in reference example 3.
[ 2. results ]
FIG. 14 shows a graph in which the histogram of fluorescence intensity detected with the anti-Ki-67 antibody or the anti-cytokeratin antibody (black solid line) and the histogram of fluorescence intensity detected with their isotype control (gray) are superimposed (upper panel: Ki-67; lower panel: cytokeratin). The peak of the Ki-67 antibody showed higher fluorescence intensity than the isotype control. This suggests that the detection of cytokeratins and Ki-67 in FFPE tissue sections was confirmed not only by the MIB-1 clone of Ki-67 but also by the S5 clone, that Ki-67 could be detected in the specimen.
< comparative example 1: detection of Ki-67 signals in FFPE tissue sections by known methods
[1] materials and methods ]
Detection of cytokeratin and Ki-67 signals in FFPR tissue sections of breast cancer tissue was carried out by a known prior art method (non-patent document 4: Cytometry 27: 283-289).
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The same procedure as in reference example 1 was repeated.
1-5 antigen activation by enzymes (trypsin): conventional methods ]
Trypsin reagent (25mM PBS pH7.4, 1mg/ml CaCl) was added to hydrophilized tissue sections21mg/mL trypsin) was added thereto, and the mixture was heated at 37 ℃ for 70 minutes. After the enzyme treatment, the enzyme was removed by washing with PBS.
[ 1-7 ] immunofluorescent staining ]
4% BSA/TBS to which 10% normal goat serum (and light) was added to a microtube containing a sample activated with an enzyme antigen, and the microtube was left standing at room temperature for 30 minutes and then blocked. For immunofluorescence staining, as the 1 st antibody, Ki67MIB-1 antibody (Dako, mouse monoclonal antibody) or Ki 67S 5 antibody (Millipore, mouse monoclonal antibody), and cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used, and as the 2 nd antibody, goat anti-mouse secondary antibody Alexa647 by Thermofisher, and goat anti-rabbit secondary antibody Alexa488 by Abcam were used. The 1 st antibody reaction time was overnight at 4 ℃.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 using a flow cytometer
The procedure was carried out in the same manner as in reference example 3.
[ 2. results ]
As shown in FIG. 15 (upper panel: Ki-67; lower panel: cytokeratin), it was detected when the S5 clone of Ki-67 was used in the conventional method described in non-patent document 4, but the Ki-67-derived fluorescence peak was greatly reduced in the sample using the MIB-1 clone antibody, and Ki-67 could not be detected.
< example 9: detection of ER and PgR in FFPE sections
[1] materials and methods ]
FFPE tissue sections in which the positivity and negativity of ER and PgR became known were used, antigen activation was performed with thrombin, nuclei were extracted by water current disruption, and signal confirmation of ER and PgR was performed using a flow cytometer.
[ 1-1.FFPE tissue blocks ]
ER-positive specimen (All red Score ER7) and PgR-positive specimen (All red Score PgR8) purchased from Proteogenex were used as positive specimens for ER and PgR, and ER and PgR-negative specimen (All red Score ER0 and PgR0) were used as negative specimens for ER and PgR.
[ 1-2 ] preparation of FFPE chips ]
Prepared in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The same procedure as in reference example 1 was repeated.
[ 1-4 ] activation by Heat-treated antigen ]
The same procedure as in reference example 1 was repeated.
[ 1-5 ] activation by antigen of enzyme ]
After activation by heat-treated antigen, ER-positive subjects undergo antigen activation with thrombin. In addition, PgR-positive and ER and PgR-negative subjects are antigen-activated with thrombin or proline-terminated peptidase. The antigen activation with thrombin was performed in the same manner as in example 1, and the antigen activation with proline-terminated peptidase was performed in the same manner as in example 6.
[ 1-6 ] Water stream disruption ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-7 ] immunofluorescent staining ]
4% BSA/TBS supplemented with 10% normal goat serum (and light) was added to a microtube containing a water-flow-disrupted sample, and the mixture was allowed to stand at room temperature for 30 minutes and then blocked. Immunofluorescent staining was carried out using an ER antibody (Abcam, clone: SP1, rabbit monoclonal antibody) or a PgR antibody (Thermofisser, clone: SP2, rabbit monoclonal antibody) as the 1 st antibody and a goat anti-rabbit mouse secondary antibody Alexa488, manufactured by Abcam, as the 2 nd antibody. The 1 st antibody was reacted at room temperature for 45 minutes. The 2 nd antibody was reacted at room temperature for 40 minutes, and DAPI Solution (and light), a dye for staining cell nuclei, was added 20 minutes after the addition of the 2 nd antibody.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9. calculation of ER and PgR Positive Rate ]
For analysis of the obtained measurement data, FLOWJO v10 software manufactured by FLOWJO LLC was used. The fluorescence amount of DAPI is taken as horizontal axis, the number of cells (nuclei) is taken as vertical axis, and the region with fluorescence amount of 5-150 on horizontal axis is taken as nucleus gate. The positive rates of ER and PgR were calculated as the ratio of the number of ER-positive nuclei and the number of PgR-positive nuclei in the whole cell nucleus. The threshold for positive nuclei was used as a 90 percent value for isotype control.
[ 2. results ]
Fig. 16 shows a graph in which a histogram of fluorescence intensity detected with the anti-ER antibody or the anti-PgR antibody (black solid line) and a histogram of fluorescence intensity detected with their isotype control antibodies (gray broken line) are superimposed. Even when ER-positive and PgR-positive FFPE tissue sections were treated with an enzyme that does not recognize the peptide cleaving SEQ ID No. 2, the black peaks showed higher fluorescence intensity than gray, and ER and PgR were detected together. On the one hand, no ER and PgR were detected on ER and PgR negative FFPE tissue sections.
< example 10: comparison of Ki-67 and cytokeratin signals in immobilized breast cancer cells using different antigen activators for thermal treatment
[1] materials and methods ]
The formalin-fixed breast cancer cell line MDA-MB-231 was subjected to antigen activation by heat treatment using 3 antigen activators, and after thrombin treatment, immunofluorescence staining was performed, and cytokeratin and Ki-67 were detected by flow cytometry.
[ 1-1. cell ]
A breast cancer cell line MDA-MB-231 from ATCC (American Type Culture Collection) was used.
[ 1-2 ] cell culture and formalin fixation ]
The procedure was carried out in the same manner as in reference example 2.
Activation by heat-treated antigens (Histo VT ONE) ]
The same treatment as in reference example 1 was carried out.
Activation by Heat-treated antigen (antigen activation solution pH9) ]
Antigen activation solution pH9(Nichirei Biosciences) was used. The procedure and conditions of the treatment were the same as in reference example 1.
Activation by heat-treated antigen (Immunosaver) ]1-4C
Antigen-activating solution ImmunoSaver (Nissan EM) was used. The procedure and conditions of the treatment were the same as in reference example 1.
[ 1-5 ] activation by antigen of enzyme ]
The procedure was as in example 1.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of Positive rates of cytokeratin and Ki-67 ]
The procedure was carried out in the same manner as in reference example 3.
[ 2. results ]
FIG. 17 shows a graph in which the histogram of fluorescence intensity detected with the anti-Ki-67 antibody or the anti-cytokeratin antibody (black solid line) and the histogram of fluorescence intensity detected with their isotype control antibody (gray dashed line) are superimposed (upper panel: Ki-67; lower panel: cytokeratin). In any of the activating solutions, the black peak showed higher fluorescence intensity than gray. From these findings, it was confirmed that the detection of cytokeratin and Ki-67 in formalin-fixed cells was useful even when the activating solution was changed.
< example 11: effect of various disruption methods in the step of extracting cell nucleus on detection of cytokeratin and Ki-67 in FFPE tissue sections
[1] materials and methods ]
Cell nuclei were extracted using FFPE tissue sections of breast cancer tissue using 3 disruption methods and a combination of disruption methods, and signal intensities of cytokeratin and Ki-67 were compared using a flow cytometer.
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-4 ] activation by Heat-treated antigen ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-5 ] activation by antigen of enzyme ]
The procedure was as in example 1.
[ 1-6A. masher ]
The enzyme-treated tissue was mashed 20 times in TBS1mL under ice-cooling using a biological masher (registered trademark) II of Nippi corporation.
[ 1-6B. mortar ]
The enzyme treated tissue was triturated in TBS1mL using a mortar and pestle.
[ 1-6C. ultrasonic crumbling ]
The enzyme-treated tissue was disrupted with TBS1mL at an output intensity of 40% for 20 seconds under ice-cooling using an ultrasonic disruption apparatus (VC X130PB) manufactured by SONICS & MATERIALS.
[ 1-6D. combination of Water stream disruption and ultrasonic disruption ]
The enzyme-treated tissue was disrupted at 10,000rpm in TBS1mL for 1 minute under ice-cooling using a water jet cutter (RP-10) from Sysmex. The tissue was disrupted by a water jet with an output intensity of 20% at TBS1mL for 30 seconds under ice cooling using an ultrasonic disruption apparatus (VC X130PB) manufactured by SONICS & MATERIALS.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
For analysis of the obtained measurement data, FLOWJO v10 software manufactured by FLOWJO LLC was used. The fluorescence amount of DAPI is taken as horizontal axis, the number of cells (nuclei) is taken as vertical axis, and the region with fluorescence amount of 5-150 on horizontal axis is taken as nucleus gate. The positive rates for cytokeratin and Ki-67 were calculated as the ratio of the number of cytokeratin-positive nuclei to the number of Ki-67-positive nuclei in the whole cell nucleus. The threshold for positive nuclei was used as a 90 percent value for isotype control.
[ 2. results ]
Fig. 18 shows a graph in which the histogram of fluorescence intensity detected with the anti-cytokeratin antibody or the anti-Ki-67 antibody (black solid line) and the histogram of fluorescence intensity detected with their isotype control antibody (gray dashed line) are superimposed. In any of the disruption methods, the peak of the black solid line shows higher fluorescence intensity than the gray broken line. From these findings, cytokeratin and Ki-67 in FFPE tissue sections could be detected using various disruption methods.
< example 12: preferential disruption of lymphocytes by ultrasound disruption
[1] materials and methods ]
Using formalin-fixed breast cancer cell lines and lymphoblastic cell lines, it was confirmed whether or not ultrasonication affected the acquisition of cell nuclei.
[ 1-1. cell ]
Breast cancer cell lines SKBr3 and MDA-MB-231 and lymphoblastic cell line Jurkat were used from ATCC (American Type Culture Collection).
[ 1-2 ] cell culture and formalin fixation ]
[ 1-4 ] activation by Heat-treated antigen ]
The same procedure as in reference example 1 was repeated.
[ 1-5 ] antigen activation treatment by enzyme ]
The procedure was as in example 1.
[ 1-6 ] ultrasonic crumbling ]
The cells after enzyme treatment were disrupted with TBS1mL at an output intensity of 20% for 30 seconds under ice-cooling using an ultrasonic disruption apparatus (VC X130PB) from SONICS & MATERIALS.
[ 1-7 ] Nuclear staining ]
DAPI Solution (and light), a dye that stains the nucleus, was added and the reaction was shaded for 20 minutes.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 2. results ]
Fig. 19 shows a scattergram of each cell line with forward scattering as the horizontal axis and side scattering as the vertical axis. In the breast cancer cells SKBr3 and MDA-MB-231, the presence or absence of ultrasonication confirmed that the positions of the major nuclear regions were not changed. On the one hand, in the lymphocyte Jurkat, a major nuclear region disappears by the ultrasonic disruption. This suggests that epithelial cells are not disrupted by ultrasonication, but that lymphoblastoid cells are preferentially disrupted by ultrasonication.
< example 13: increase in number of analysis nuclei by addition of surfactant >
[1] materials and methods ]
The FFPE tissue sections of breast cancer tissues were used, and a surfactant was added during ultrasonication to confirm whether or not the cells had changed in nucleus.
[ 1-1.FFPE tissue blocks ]
2 FFPE tissue blocks obtained from 2 human breast cancer patients with different Ki-67 positivity rates were used by the IHC method purchased from Proteogenex.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-4 ] activation by Heat-treated antigen ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-5 ] antigen activation treatment by enzyme ]
The procedure was as in example 1.
[ 1-6 ] ultrasonic crumbling ]
Tissues and cells activated with the enzyme antigen were disrupted with TBS, TBS containing 1% CHAPS (and light), TBS containing 1% NP-40 (and light), or TBS1mL containing 1% Triton-X100 (and light) in TBS, under ice cooling using an ultrasonic disruption apparatus (VC X130PB) of SONICS & MATERIALS for 30 seconds at an output intensity of 20%.
[ 1-7 ] Nuclear staining ]
DAPI Solution (and light), a dye that stains the nucleus, was added and the reaction was shaded for 20 minutes.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 2. results ]
FIG. 20 shows a scattergram having the horizontal axis of the forward scattering and the vertical axis of the side scattering, when the cells are sonicated in a buffer solution containing no surfactant and in a buffer solution containing various surfactants, and Table 3 shows the total number of analyzed nuclei in the flow cytometry measurement. An increase in the total number of nuclei analyzed was observed by the addition of the surfactant.
[ TABLE 3]
Subject A
Subject B
TBS (surfactant not added)
131027
40642
+ 1% CHAPS
144233
72561
+ 1% NP-40
289745
91164
+ 1% Triton-X100
216606
97388
Comparative example 2: determination of Ki-67 Positive cells by different pathologists >
[1] materials and methods ]
The correlation of Ki-67 positivity in 2 IHC-method FFPE tissue sections was confirmed.
[ 1-1.FFPE tissue blocks ]
38 FFPE tissue blocks obtained from 38 patients with different Ki-67 positivity rates were used by the IHC method purchased from Proteogenex.
[ 1-10 ] calculated from the Ki-67 positivity in IHC method ]
The same procedure as in example 5 was repeated.
[ 2. results ]
The results are shown in FIG. 21. It was found that the Ki-67 positive rates were different from each other in the pathologist even in the same samples, and the deviation was large.
< reference example 4: detection of anti-Ki-67 antibodies with different recognition sites after antigen activation with various digestive enzymes
[1] materials and methods ]
After antigen activation using formalin-fixed breast cancer cells, any of 3 digestive enzymes (thrombin, dispase, proteinase K), followed by fluorescent immunostaining, Ki-67 and cytokeratin were detected using a flow cytometer.
[ 1-1. cell ]
3 breast cancer cell lines used in reference example 2 were used.
[ 1-2 ] cell culture and formalin fixation ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-4 ] activation by Heat-treated antigen ]
The same treatment as in reference example 1 was carried out.
[ 1-5A. antigen activation by enzymes (Thrombin) ]
The procedure was as in example 1.
[ 1-5B. activation by antigen of enzyme (dispase) ]
The same procedure as in example 6 was repeated.
[ 1-5C. antigen activation by enzymes (proteinase K) ]
The same procedure as in example 6 was repeated.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2. Among them, Ki-67 antibody (Millipore, clone: S5(Ki-S5), mouse monoclonal antibody) and pan-cytokeratin antibody (Abcam, rabbit polyclonal antibody) were used as the 1 st antibody, and goat anti-mouse secondary antibody Alexa647 (Thermofisiher Co.) and goat anti-rabbit secondary antibody Alexa488 (Abcam Co.) were used as the 2 nd antibody.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of Ki-67 Positive Rate
The procedure was carried out in the same manner as in reference example 3.
[ 2. results ]
FIGS. 22A and B show graphs obtained by superimposing the histograms of fluorescence intensities detected with the anti-Ki-67 antibody (Ki-S5) or the anti-cytokeratin antibody (black solid line) and the histogram of fluorescence intensities detected with their isotype controls (gray dashed line). The peaks of the black solid line show higher fluorescence intensity than the gray broken line when antigen activation is performed with any enzyme. From these results, it was confirmed that the antigen recognition sites of the anti-Ki-67 antibody (Ki-S5) and the anti-cytokeratin antibody, Ki-67 and cytokeratin detection were maintained in the antigen activation with any enzyme.
< example 14: discussion of threshold values for determination of cytokeratins and Ki-67 Positive nuclei in FFPE tissue sections
[1] materials and methods ]
Cytokeratin and Ki-67-derived signals in the FFPE tissue sections were detected by a flow cytometer, and the positivity when the threshold of positive nuclei was changed was calculated.
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-4 ] activation by Heat-treated antigen ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-5 ] activation by antigen of enzyme ]
The procedure was carried out in the same manner as in example 1.
[ 1-6 ] combination of Water stream disruption and ultrasonic disruption ]
The enzyme-treated tissue was disrupted at 10,000rpm in TBS1mL for 1 minute under ice-cooling using a water jet cutter (RP-10) from Sysmex. The tissue was disrupted by a water jet with an output intensity of 20% at TBS1mL for 30 seconds under ice cooling using an ultrasonic disruption apparatus (VC X130PB) manufactured by SONICS & MATERIALS.
[ 1-7 ] immunofluorescent staining ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-8 ] flow cytometry assay ]
The procedure was carried out in the same manner as in reference example 2.
[ 1-9 ] calculation of the Positive rates of cytokeratin and Ki-67 ]
For analysis of the obtained measurement data, FLOWJO v10 software manufactured by FLOWJO LLC was used. The fluorescence amount of DAPI is taken as horizontal axis, the number of cells (nuclei) is taken as vertical axis, and the region with fluorescence amount of 5-150 on horizontal axis is taken as nucleus gate. The positive rates for cytokeratin and Ki-67 were calculated as the ratio of the number of cytokeratin-positive nuclei to the number of Ki-67-positive nuclei in the whole cell nucleus. The threshold values for positive nuclei were used as 60, 70, 80, 90 and 95 percent values for isotype controls. The positive rate was calculated from the value detected by the flow cytometer (100-threshold).
[ 2. results ]
Fig. 23 shows a graph in which the threshold was changed so that the histograms of the fluorescence intensities detected with the anti-cytokeratin antibody and the anti-Ki-67 antibody (black solid line) and the histogram of the fluorescence intensity detected with their isotype control antibody (gray dashed line) coincide. The positive rate was calculated using any threshold. Thus, the positive rates for cytokeratin and Ki-67 were calculated without depending on the threshold values.
< example 15: detection of HER2 in FFPE tissue sections
[1] materials and methods ]
The amplification of HER2 was confirmed by FISH method by extracting nuclei from FFPE tissue sections in which HER2 was positive (specimen A:
[ 1-1.FFPE tissue blocks ]
The FFPE tissue block used in reference example 1 was used.
[ 1-2 ] preparation of FFPE chips ]
The procedure was carried out in the same manner as in reference example 3.
[ 1-3. De-Paraffin/hydrophilization ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-4 ] activation by Heat-treated antigen ]
The procedure was carried out in the same manner as in reference example 1.
[ 1-5 ] activation by antigen of enzyme ]
The procedure was carried out in the same manner as in example 1.
[ 1-6 ] combination of Water stream disruption and ultrasonic disruption ]
The enzyme-treated tissue was disrupted at 10,000rpm in TBS1mL for 1 minute under ice-cooling using a water jet cutter (RP-10) from Sysmex. The tissue was disrupted by a water jet with an output intensity of 20% at TBS1mL for 30 seconds under ice cooling using an ultrasonic disruption apparatus (VC X130PB) manufactured by SONICS & MATERIALS.
[ 1-7. detection of HER2 by FISH ]
The tissue-disrupted sample was mounted on a glass slide, dried, and then immersed in 10% neutral buffered formalin at room temperature for 10 minutes. Slides were washed with TBS and air dried and then probed with PathVysis (R) HER-2DNA probe kit (Abbot) (protocol FISH staining according to kit attached protocol).
[ 1-8 ] fluorescence microscope ]
In the microscopic observation, an integral fluorescence microscope BZ-X710(KEYENCE) was used. DAPI filters (Ex 360nm, Em 460nm) and TRITC filters (Ex 545nm, Em 605nm) were used for observation. The objective lens is used 20 times.
[ 2. results ]
The nuclei observed with a fluorescence microscope are shown in FIG. 24. HER2 derived fluorescent signals could be detected in HER2 positive FFPE tissue sections, and in one aspect, confirmed to be undetectable in HER2 negative FFPE tissue sections.
[ possibility of Industrial utilization ]
The present invention establishes a method for detecting (quantifying) Ki-67 positive cells with high objectivity, reproducibility and universality. Furthermore, by quantifying ER-positive cells, PgR-positive cells, and Her 2-positive cells, classification of endogenous subtypes of cancer is possible.
Detachment of nuclei that enhance antigenicity of the antigen of interest from FFPE tissue sections is achieved by using the protocol provided by the present invention (including a pretreatment process that includes antigen activation by a specified enzyme). The scattered nuclei thus recovered can be resolved as single nuclei. For example, proteins, nucleic acids, and the like on the nuclear membrane or in the nucleus are analyzed, and these objects can be detected by observing the form of staining with a dye (fluorescence), or using a ligand such as an antibody or nucleic acid labeled with an enzyme or a fluorescent dye. The method, antigen activator and kit of the present invention can be used for morphological observation with a microscope, analysis by IHC, EIA, CLEIA, digital PCR, or cytometric analysis using an imaging cytometer or a flow cytometer. In particular, according to a preferred embodiment of the present invention, the method can be used for calculating the ratio of positive nuclei (particularly, the Ki-67 positivity) of a target protein in the enucleated nuclei analyzed by flow cytometry.
By using the protocol provided by the present invention (including a pretreatment process involving activation by an antigen of a specified enzyme), an index of pathological diagnosis with less deviation can be provided.
Further, the patient can be provided with an optimal diagnosis plan by using the index with less variation. Anticancer agent treatment also imposes a large burden on patients themselves, and it is also important to determine the treatment regimen of patients before the start of treatment in order to maintain the qol (qualityofhife) of patients. In the case of the invention of the present application, it can be applied not only to a treatment protocol for chemotherapy before surgery (preoperative anticancer agent treatment) but also to a treatment protocol for (prognosis) after tumor extirpation in surgery.
Sequence listing
<110>NITTO BOSEKI CO LTD
<120> method for detaching antigenicity-enhanced cell nucleus from immobilized cell or FFPE tissue section, and antigen activator and kit for the same
<130>A00243WO01
<150>JP2018-035696
<151>2018-02-28
<160>2
<170>PatentIn version 3.5
<210>1
<211>3256
<212>PRT
<213> Intelligent (Homo sapiens)
<400>1
Met Trp Pro Thr Arg Arg Leu Val Thr Ile Lys Arg Ser Gly Val Asp
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Ile Glu Cys Asp Ile Arg Ile Gln Leu Pro Val Val Ser Lys Gln His
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Cys Lys Ile Glu Ile His Glu Gln Glu Ala Ile Leu His Asn Phe Ser
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Ser Thr Asn Pro Thr Gln Val Asn Gly Ser Val Ile Asp Glu Pro Val
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Arg Leu Lys His Gly Asp Val Ile Thr Ile Ile Asp Arg Ser Phe Arg
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Tyr Glu Asn Glu Ser Leu Gln Asn Gly Arg Lys Ser Thr Glu Phe Pro
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Arg Lys Ile Arg Glu Gln Glu Pro Ala Arg Arg Val Ser Arg Ser Ser
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Phe Ser Ser Asp Pro Asp Glu Lys Ala Gln Asp Ser Lys Ala Tyr Ser
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Gln Cys Leu Asp Asn Ser Lys Lys Asn Glu Ser Pro Phe Trp Lys Leu
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Val Leu Gln Tyr Cys Arg Lys Ser Gly Leu Gln Thr Asp Tyr Ala Thr
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Glu Pro Ala Ser Pro Glu Gln Glu Leu Asp Gln Asn Lys Gly Lys Gly
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Phe Pro Leu Tyr Glu Pro Ala Lys Met Lys Thr Pro Val Gln Tyr Ser
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Gln Gln Gln Asn Ser Pro Gln Lys His Lys Asn Lys Asp Leu Tyr Thr
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Leu Pro Thr Glu Thr Glu Ile His Asn Glu Pro Phe Leu Thr Leu Trp
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Leu Ser Ser Val Asp Ile Asn Asn Phe Gly Asp Ser Ile Asn Glu Ser
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Glu Gly Ile Pro Leu Lys Arg Arg Arg Val Ser Phe Gly Gly His Leu
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Val Ile Ser Pro Pro Ala Pro Ser Pro Arg Lys Thr Pro Val Ala Ser
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Asp Gln Arg Arg Arg Ser Cys Lys Thr Ala Pro Ala Ser Ser Ser Lys
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Ser Gln Thr Glu Val Pro Lys Arg Gly Gly Arg Lys Ser Gly Asn Leu
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Pro Ser Lys Arg Val Ser Ile Ser Arg Ser Gln His Asp Ile Leu Gln
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Met Ile Cys Ser Lys Arg Arg Ser Gly Ala Ser Glu Ala Asn Leu Ile
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Gln Thr Lys Val Ile Lys His Gly Pro Gln Arg Ser Met Asn Lys Arg
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Gln Arg Arg Pro Ala Thr Pro Lys Lys Pro Val Gly Glu Val His Ser
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Gln Phe Ser Thr Gly His Ala Asn Ser Pro Cys Thr Ile Ile Ile Gly
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Lys Ala His Thr Glu Lys Val His Val Pro Ala Arg Pro Tyr Arg Val
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Ser Gly Ile Ala Glu Met Phe Lys Thr Pro Val Lys Glu Gln Pro Gln
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Gly Lys Gln Phe Gln Gly Thr Asp Ser Gly Glu Glu Pro Leu Leu Pro
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Thr Ser Glu Ser Phe Gly Gly Asn Val Phe Phe Ser Ala Gln Asn Ala
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Ala Lys Gln Pro Ser Asp Lys Cys Ser Ala Ser Pro Pro Leu Arg Arg
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Pro Ser Lys Thr Val Ser Thr Ala Asn Arg Ser Gly Arg Ser Thr Glu
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Phe Arg Asn Ile Gln Lys Leu Pro Val Glu Ser Lys Ser Glu Glu Thr
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Asn Thr Glu Ile Val Glu Cys Ile Leu Lys Arg Gly Gln Lys Ala Thr
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Leu Leu Gln Gln Arg Arg Glu Gly Glu Met Lys Glu Ile Glu Arg Pro
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Ser Leu Gln Pro Glu Pro Ile Asn Thr Pro Thr His Thr Lys Gln
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Gln Leu Lys Ala Ser Leu Gly Lys Val Gly Val Lys Glu Glu Leu
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Lys Glu Ser Pro Lys Gln Ile Leu Asp Pro Ala Ala Arg Val Thr
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Ala Phe Met Gly Thr Pro Val Gln Lys Leu Asp Leu Ala Gly Thr
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Leu Pro Gly Ser Lys Arg Gln Leu Gln Thr Pro Lys Glu Lys Ala
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Gln Ala Leu Glu Asp Leu Ala Gly Phe Lys Glu Leu Phe Gln Thr
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Pro Gly His Thr Glu Glu Leu Val Ala Ala Gly Lys Thr Thr Lys
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Ile Pro Cys Asp Ser Pro Gln Ser Asp Pro Val Asp Thr Pro Thr
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Ser Thr Lys Gln Arg Pro Lys Arg Ser Ile Arg Lys Ala Asp Val
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Glu Gly Glu Leu Leu Ala Cys Arg Asn Leu Met Pro Ser Ala Gly
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Lys Ala Met His Thr Pro Lys Pro Ser Val Gly Glu Glu Lys Asp
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Ile Ile Ile Phe Val Gly Thr Pro Val Gln Lys Leu Asp Leu Thr
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Glu Asn Leu Thr Gly Ser Lys Arg Arg Pro Gln Thr Pro Lys Glu
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Glu Ala Gln Ala Leu Glu Asp Leu Thr Gly Phe Lys Glu Leu Phe
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Gln Thr Pro Gly His Thr Glu Glu Ala Val Ala Ala Gly Lys Thr
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Thr Lys Met Pro Cys Glu Ser Ser Pro Pro Glu Ser Ala Asp Thr
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Asp Val Gln Lys Glu Leu Ser Ala Leu Lys Lys Leu Thr Gln Thr
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Lys Ser Ile Asn Ala Phe Arg Glu Thr Ala Lys Gln Lys Leu Asp
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Pro Ala Ala Ser Val Thr Gly Ser Lys Arg His Pro Lys Thr Lys
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Glu Lys Ala Gln Pro Leu Glu Asp Leu Ala Gly Leu Lys Glu Leu
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Phe Gln Thr Pro Val Cys Thr Asp Lys Pro Thr Thr His Glu Lys
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Thr Thr Lys Ile Ala Cys Arg Ser Gln Pro Asp Pro Val Asp Thr
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Pro Thr Ser Ser Lys Pro Gln Ser Lys Arg Ser Leu Arg Lys Val
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Asp Val Glu Glu Glu Phe Phe Ala Leu Arg Lys Arg Thr Pro Ser
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Ala Gly Lys Ala Met His Thr Pro Lys Pro Ala Val Ser Gly Glu
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Lys Asn Ile Tyr Ala Phe Met Gly Thr Pro Val Gln Lys Leu Asp
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Leu Thr Glu Asn Leu Thr Gly Ser Lys Arg Arg Leu Gln Thr Pro
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Lys Glu Lys Ala Gln Ala Leu Glu Asp Leu Ala Gly Phe Lys Glu
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Leu Phe Gln Thr Arg Gly His Thr Glu Glu Ser Met Thr Asn Asp
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Lys Thr Ala Lys Val Ala Cys Lys Ser Ser Gln Pro Asp Pro Asp
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Lys Asn Pro Ala Ser Ser Lys Arg Arg Leu Lys Thr Ser Leu Gly
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Lys Val Gly Val Lys Glu Glu Leu Leu Ala Val Gly Lys Leu Thr
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Gln Thr Ser Gly Glu Thr Thr His Thr His Thr Glu Pro Thr Gly
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Asp Gly Lys Ser Met Lys Ala Phe Met Glu Ser Pro Lys Gln Ile
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Leu Asp Ser Ala Ala Ser Leu Thr Gly Ser Lys Arg Gln Leu Arg
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Thr Pro Lys Gly Lys Ser Glu Val Pro Glu Asp Leu Ala Gly Phe
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Ile Glu Leu Phe Gln Thr Pro Ser His Thr Lys Glu Ser Met Thr
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Asn Glu Lys Thr Thr Lys Val Ser Tyr Arg Ala Ser Gln Pro Asp
1730 1735 1740
Leu Val Asp Thr Pro Thr Ser Ser Lys Pro Gln Pro Lys Arg Ser
1745 1750 1755
Leu Arg Lys Ala Asp Thr Glu Glu Glu Phe Leu Ala Phe Arg Lys
1760 1765 1770
Gln Thr Pro Ser Ala Gly Lys Ala Met His Thr Pro Lys Pro Ala
1775 1780 1785
Val Gly Glu Glu Lys Asp Ile Asn Thr Phe Leu Gly Thr Pro Val
1790 1795 1800
Gln Lys Leu Asp Gln Pro Gly Asn Leu Pro Gly Ser Asn Arg Arg
1805 1810 1815
Leu Gln Thr Arg Lys Glu Lys Ala Gln Ala Leu Glu Glu Leu Thr
1820 1825 1830
Gly Phe Arg Glu Leu Phe Gln Thr Pro Cys Thr Asp Asn Pro Thr
1835 1840 1845
Thr Asp Glu Lys Thr Thr Lys Lys Ile Leu Cys Lys Ser Pro Gln
1850 1855 1860
Ser Asp Pro Ala Asp Thr Pro Thr Asn Thr Lys Gln Arg Pro Lys
1865 1870 1875
Arg Ser Leu Lys Lys Ala Asp Val Glu Glu Glu Phe Leu Ala Phe
1880 1885 1890
Arg Lys Leu Thr Pro Ser Ala Gly Lys Ala Met His Thr Pro Lys
1895 1900 1905
Ala Ala Val Gly Glu Glu Lys Asp Ile Asn Thr Phe Val Gly Thr
1910 1915 1920
Pro Val Glu Lys Leu Asp Leu Leu Gly Asn Leu Pro Gly Ser Lys
1925 1930 1935
Arg Arg Pro Gln Thr Pro Lys Glu Lys Ala Lys Ala Leu Glu Asp
1940 1945 1950
Leu Ala Gly Phe Lys Glu Leu Phe Gln Thr Pro Gly His Thr Glu
1955 1960 1965
Glu Ser Met Thr Asp Asp Lys Ile Thr Glu Val Ser Cys Lys Ser
1970 1975 1980
Pro Gln Pro Asp Pro Val Lys Thr Pro Thr Ser Ser Lys Gln Arg
1985 1990 1995
Leu Lys Ile Ser Leu Gly Lys Val Gly Val Lys Glu Glu Val Leu
2000 2005 2010
Pro Val Gly Lys Leu Thr Gln Thr Ser Gly Lys Thr Thr Gln Thr
2015 2020 2025
His Arg Glu Thr Ala Gly Asp Gly Lys Ser Ile Lys Ala Phe Lys
2030 2035 2040
Glu Ser Ala Lys Gln Met Leu Asp Pro Ala Asn Tyr Gly Thr Gly
20452050 2055
Met Glu Arg Trp Pro Arg Thr Pro Lys Glu Glu Ala Gln Ser Leu
2060 2065 2070
Glu Asp Leu Ala Gly Phe Lys Glu Leu Phe Gln Thr Pro Asp His
2075 2080 2085
Thr Glu Glu Ser Thr Thr Asp Asp Lys Thr Thr Lys Ile Ala Cys
2090 2095 2100
Lys Ser Pro Pro Pro Glu Ser Met Asp Thr Pro Thr Ser Thr Arg
2105 2110 2115
Arg Arg Pro Lys Thr Pro Leu Gly Lys Arg Asp Ile Val Glu Glu
2120 2125 2130
Leu Ser Ala Leu Lys Gln Leu Thr Gln Thr Thr His Thr Asp Lys
2135 2140 2145
Val Pro Gly Asp Glu Asp Lys Gly Ile Asn Val Phe Arg Glu Thr
2150 2155 2160
Ala Lys Gln Lys Leu Asp Pro Ala Ala Ser Val Thr Gly Ser Lys
2165 2170 2175
Arg Gln Pro Arg Thr Pro Lys Gly Lys Ala Gln Pro Leu Glu Asp
2180 2185 2190
Leu Ala Gly Leu Lys Glu Leu Phe Gln Thr Pro Ile Cys Thr Asp
2195 2200 2205
Lys Pro Thr Thr His Glu Lys Thr Thr Lys Ile Ala Cys Arg Ser
2210 2215 2220
Pro Gln Pro Asp Pro Val Gly Thr Pro Thr Ile Phe Lys Pro Gln
2225 2230 2235
Ser Lys Arg Ser Leu Arg Lys Ala Asp Val Glu Glu Glu Ser Leu
2240 2245 2250
Ala Leu Arg Lys Arg Thr Pro Ser Val Gly Lys Ala Met Asp Thr
2255 2260 2265
Pro Lys Pro Ala Gly Gly Asp Glu Lys Asp Met Lys Ala Phe Met
2270 2275 2280
Gly Thr Pro Val Gln Lys Leu Asp Leu Pro Gly Asn Leu Pro Gly
2285 2290 2295
Ser Lys Arg Trp Pro Gln Thr Pro Lys Glu Lys Ala Gln Ala Leu
2300 2305 2310
Glu Asp Leu Ala Gly Phe Lys Glu Leu Phe Gln Thr Pro Gly Thr
2315 2320 2325
Asp Lys Pro Thr Thr Asp Glu Lys Thr Thr Lys Ile Ala Cys Lys
2330 2335 2340
Ser Pro Gln Pro Asp Pro Val Asp Thr Pro Ala Ser Thr Lys Gln
2345 2350 2355
Arg Pro Lys Arg Asn Leu Arg Lys Ala Asp Val Glu Glu Glu Phe
2360 2365 2370
Leu Ala Leu Arg Lys Arg Thr Pro Ser Ala Gly Lys Ala Met Asp
2375 2380 2385
Thr Pro Lys Pro Ala Val Ser Asp Glu Lys Asn Ile Asn Thr Phe
2390 2395 2400
Val Glu Thr Pro Val Gln Lys Leu Asp Leu Leu Gly Asn Leu Pro
2405 2410 2415
Gly Ser Lys Arg Gln Pro Gln Thr Pro Lys Glu Lys Ala Glu Ala
2420 2425 2430
Leu Glu Asp Leu Val Gly Phe Lys Glu Leu Phe Gln Thr Pro Gly
2435 2440 2445
His Thr Glu Glu Ser Met Thr Asp Asp Lys Ile Thr Glu Val Ser
2450 2455 2460
Cys Lys Ser Pro Gln Pro Glu Ser Phe Lys Thr Ser Arg Ser Ser
2465 2470 2475
Lys Gln Arg Leu Lys Ile Pro Leu Val Lys Val Asp Met Lys Glu
2480 2485 2490
Glu Pro Leu Ala Val Ser Lys Leu Thr Arg Thr Ser Gly Glu Thr
2495 2500 2505
Thr Gln Thr His Thr Glu Pro Thr Gly Asp Ser Lys Ser Ile Lys
2510 2515 2520
Ala Phe Lys Glu Ser Pro Lys Gln Ile Leu Asp Pro Ala Ala Ser
2525 2530 2535
Val Thr Gly Ser Arg Arg Gln Leu Arg Thr Arg Lys Glu Lys Ala
2540 2545 2550
Arg Ala Leu Glu Asp Leu Val Asp Phe Lys Glu Leu Phe Ser Ala
2555 2560 2565
Pro Gly His Thr Glu Glu Ser Met Thr Ile Asp Lys Asn Thr Lys
2570 2575 2580
Ile Pro Cys Lys Ser Pro Pro Pro Glu Leu Thr Asp Thr Ala Thr
2585 2590 2595
Ser Thr Lys Arg Cys Pro Lys Thr Arg Pro Arg Lys Glu Val Lys
2600 2605 2610
Glu Glu Leu Ser Ala Val Glu Arg Leu Thr Gln Thr Ser Gly Gln
2615 2620 2625
Ser Thr His Thr His Lys Glu Pro Ala Ser Gly Asp Glu Gly Ile
2630 2635 2640
Lys Val Leu Lys Gln Arg Ala Lys Lys Lys Pro Asn Pro Val Glu
2645 2650 2655
Glu Glu Pro Ser Arg Arg Arg Pro Arg Ala Pro Lys Glu Lys Ala
2660 2665 2670
Gln Pro Leu Glu Asp Leu Ala Gly Phe Thr Glu Leu Ser Glu Thr
2675 2680 2685
Ser Gly His Thr Gln Glu Ser Leu Thr Ala Gly Lys Ala Thr Lys
2690 2695 2700
Ile Pro Cys Glu Ser Pro Pro Leu Glu Val Val Asp Thr Thr Ala
2705 2710 2715
Ser Thr Lys Arg His Leu Arg Thr Arg Val Gln Lys Val Gln Val
2720 2725 2730
Lys Glu Glu Pro Ser Ala Val Lys Phe Thr Gln Thr Ser Gly Glu
2735 2740 2745
Thr Thr Asp Ala Asp Lys Glu Pro Ala Gly Glu Asp Lys Gly Ile
2750 2755 2760
Lys Ala Leu Lys Glu Ser Ala Lys Gln Thr Pro Ala Pro Ala Ala
2765 2770 2775
Ser Val Thr Gly Ser Arg Arg Arg Pro Arg Ala Pro Arg Glu Ser
2780 2785 2790
Ala Gln Ala Ile Glu Asp Leu Ala Gly Phe Lys Asp Pro Ala Ala
2795 2800 2805
Gly His Thr Glu Glu Ser Met Thr Asp Asp Lys Thr Thr Lys Ile
2810 2815 2820
Pro Cys Lys Ser Ser Pro Glu Leu Glu Asp Thr Ala Thr Ser Ser
2825 2830 2835
Lys Arg Arg Pro Arg Thr Arg Ala Gln Lys Val Glu Val Lys Glu
2840 2845 2850
Glu Leu Leu Ala Val Gly Lys Leu Thr Gln Thr Ser Gly Glu Thr
2855 2860 2865
Thr His Thr Asp Lys Glu Pro Val Gly Glu Gly Lys Gly Thr Lys
2870 2875 2880
Ala Phe Lys Gln Pro Ala Lys Arg Lys Leu Asp Ala Glu Asp Val
2885 2890 2895
Ile Gly Ser Arg Arg Gln Pro Arg Ala Pro Lys Glu Lys Ala Gln
2900 2905 2910
Pro Leu Glu Asp Leu Ala Ser Phe Gln Glu Leu Ser Gln Thr Pro
2915 2920 2925
Gly His Thr Glu Glu Leu Ala Asn Gly Ala Ala Asp Ser Phe Thr
2930 2935 2940
Ser Ala Pro Lys Gln Thr Pro Asp Ser Gly Lys Pro Leu Lys Ile
2945 2950 2955
Ser Arg Arg Val Leu Arg Ala Pro Lys Val Glu Pro Val Gly Asp
2960 2965 2970
Val Val Ser Thr Arg Asp Pro Val Lys Ser Gln Ser Lys Ser Asn
2975 2980 2985
Thr Ser Leu Pro Pro Leu Pro Phe Lys Arg Gly Gly Gly Lys Asp
2990 2995 3000
Gly Ser Val Thr Gly Thr Lys Arg Leu Arg Cys Met ProAla Pro
3005 3010 3015
Glu Glu Ile Val Glu Glu Leu Pro Ala Ser Lys Lys Gln Arg Val
3020 3025 3030
Ala Pro Arg Ala Arg Gly Lys Ser Ser Glu Pro Val Val Ile Met
3035 3040 3045
Lys Arg Ser Leu Arg Thr Ser Ala Lys Arg Ile Glu Pro Ala Glu
3050 3055 3060
Glu Leu Asn Ser Asn Asp Met Lys Thr Asn Lys Glu Glu His Lys
3065 3070 3075
Leu Gln Asp Ser Val Pro Glu Asn Lys Gly Ile Ser Leu Arg Ser
3080 3085 3090
Arg Arg Gln Asn Lys Thr Glu Ala Glu Gln Gln Ile Thr Glu Val
3095 3100 3105
Phe Val Leu Ala Glu Arg Ile Glu Ile Asn Arg Asn Glu Lys Lys
3110 3115 3120
Pro Met Lys Thr Ser Pro Glu Met Asp Ile Gln Asn Pro Asp Asp
3125 3130 3135
Gly Ala Arg Lys Pro Ile Pro Arg Asp Lys Val Thr Glu Asn Lys
3140 3145 3150
Arg Cys Leu Arg Ser Ala Arg Gln Asn Glu Ser Ser Gln Pro Lys
3155 3160 3165
Val Ala Glu Glu Ser Gly Gly Gln Lys Ser Ala Lys Val Leu Met
3170 3175 3180
Gln Asn Gln Lys Gly Lys Gly Glu Ala Gly Asn Ser Asp Ser Met
3185 3190 3195
Cys Leu Arg Ser Arg Lys Thr Lys Ser Gln Pro Ala Ala Ser Thr
3200 3205 3210
Leu Glu Ser Lys Ser Val Gln Arg Val Thr Arg Ser Val Lys Arg
3215 3220 3225
Cys Ala Glu Asn Pro Lys Lys Ala Glu Asp Asn Val Cys Val Lys
3230 3235 3240
Lys Ile Arg Thr Arg Ser His Arg Asp Ser Glu Asp Ile
3245 3250 3255
<210>2
<211>21
<212>PRT
<213> Artificial sequence
<220>
<223> Ki-67 epitope of MIB-1 antibody
<400>2
Thr Pro Lys Glu Lys Ala Gln Ala Leu Glu Asp Leu Ala Gly Phe Lys
1 5 10 15
Glu Leu Phe Gln Thr
20
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