Process recording slide for immunohistochemical staining
阅读说明：本技术 用于免疫组化染色的过程记录玻片 (Process recording slide for immunohistochemical staining ) 是由 弗雷德里克·克努特·哈舍 岑子祥 于 2018-06-15 设计创作，主要内容包括：一种用于确定在试验过程中,特别是多步免疫组化(IHC)试验中石蜡去除、抗原修复以及使用的一级和二级染色试剂的效果的装置和方法。所述装置包括粘合剂涂覆的显微镜玻片,所述显微镜玻片包含以2D或3D结构作点涂并密封在石蜡涂层下的多种化合物。随后,将组织切片或疏松细胞加到同一玻片上,并且全部经历从组织捕获到盖玻片施加的IHC处理步骤。所述化合物与一级或二级IHC染色试剂反应,以记录共存组织切片或疏松细胞的处理经历。(devices and methods for determining the effect of paraffin removal, antigen retrieval, and the use of grade and secondary staining reagents during the course of an assay, particularly a multi-step Immunohistochemistry (IHC) assay, the devices include adhesive-coated microscope slides containing a variety of compounds spotted in 2D or 3D structures and sealed under a paraffin coating.)
slides comprising a detection zone and a control zone, wherein
The detection area is used for processing a tissue slice or a loose cell through Immunohistochemical (IHC) or Immunochemical (ICC) detection and/or subsequent examination; and
the control zone has control targets that reflect possible errors (if any) present in the intermediate steps of (e.g., 2, 3, 4, 5, 6, 7, 8, 9) of the immunohistochemical or immunochemical detection process and/or provide a reference for qualitatively or quantitatively determining the color density of stained tissue or cells;
preferably, the intermediate step is or more steps selected from the group consisting of paraffin removal, antigen retrieval, grade staining and secondary staining.
2. The glass slide of claim 1, having an adhesive coating to enable the slide to attach molecules such as peptides, proteins, sugars, lipids, inorganic small molecules, more preferably the adhesive coating is covalently bound to glass and present or more end groups selected from the group consisting of-ROH, -R (C ═ O) OH, -RNH3、-R(C＝O)NH2and-RNH2A group of (a); still more preferably, the adhesive coating is slightly hydrophilic.
3. The slide of claim 1 or 2, wherein the control region comprises a -group or groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-group) -grade target array, the -grade target array comprising (e.g., 1-50, 5-45, 10-40, 15-35, or 20-30, specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) -grade target loading spots (which may be any regular or irregular shape, such as circles, ovals, squares, diamonds, etc.), each spot comprising or more (e.g., 1-50, 5-45, 10-40, 15-35, or 20-30, specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) target protein fragments that are immobilized on the 357, 358, 3, 6, 7, 3, 7, 3, 7, 3;
preferably, at least of the -grade targets (or antigenic peptide fragments) in the of the -grade target load points are identifiable by -grade antibodies for the immunohistochemical or immunochemical detection.
4. The slide of any of claims 1-3, wherein the peptide chain of the -grade target is coupled to a carrier protein by a cysteine residue, preferably by way of sulfo-SMCC cross-linking, the bound carrier protein optionally mixed with a pseudoprotein to adjust concentration;
preferably, the carrier protein is selected from Keyhole Limpet Hemocyanin (KLH) and other proteins that are non-reactive with secondary staining reagents, such as Ovalbumin (OVA) from chicken proteins or any equine family, such as horse, donkey or zebra;
still preferably, the -grade target is fixed with a fixative, such as paraformaldehyde or formalin.
5. The slide of claim 4, wherein the -grade target load points form gradient target density pairs, each target pair binding a single reactive antigen, preferably binding peptides are mixed with neutral KLH protein and 4% formalin to make a two to ten-fold (preferably, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold) diluted gradient density series, or
Each target site is composed of two to ten different antigenic peptide fragments, e.g., all at maximum density.
6. The slide of any of of claims 1-5, wherein the control zone further comprises or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) secondary target arrays comprising or more (e.g., 1-50, 5-45, 10-40, 15-35, or 20-30, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) secondary target load points (which may be any regular or irregular shape, such as circles, ovals, squares, diamonds, etc.), each of which is a mixture of host proteins (e.g., IgG) and pseudoproteins (e.g., IgG) immobilized on the slide at a ratio of , wherein the control zone further comprises or more sets of secondary target arrays (e.g., 1, 2, 3, 4, 8, 9, or 10), wherein the secondary target load points are in any regular or irregular shape, such as circles, ovals, squares, diamonds, or the like
The host protein is the same for different points in the same array of different secondary targets, and the pseudoproteins may be the same or different, preferably the same; and
the host proteins are different for different array groups, and the pseudoproteins may be the same or different;
preferably, the secondary target spot array forms a gradient dilution series, preferably a gradient density comprising stepwise dilution increments of 1 to 1000:1, more preferably the gradient density follows a-dBd curve, e.g., from 0 to-80 dBd (100% to 0.01%) the host protein concentration, or any series comprising or more of the same dBd incremental dilutions within 0 to-100 dBd;
still preferably, the secondary target is fixed with a fixative, such as paraformaldehyde or formalin.
7. The slide of claim 6, wherein the host protein is an animal protein that produces -grade antibodies for immunohistochemical or immunochemical detection, preferably the host is selected from the group consisting of mouse, rat, rabbit and goat.
8. The slide glass according to claim 6 or 7, wherein the pseudoprotein is a protein which is sometimes present in a secondary staining kit and does not support non-specific staining; preferably, the pseudoprotein is donkey or horse protein.
9. The slide of of any of claims 1-8, wherein the control zone further comprises (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 3D secondary array target load points, each of the 3D secondary array target load points being formed from a mixture of a backbone-acting polysaccharide and a -defined concentration of a host protein (such as 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or any value in between) and loaded to the slide.
10. The slide of of any of claims 1-9, wherein the control zone further comprises (e.g., 1, 2, 3, 4, or 5) imaging reference load points, preferably the imaging reference load points comprise at least a black target or a white target, more preferably a black target and a white target, e.g., flanking the secondary target.
11. The slide of claim 10, wherein the black reference target is selected from the group consisting of carbon dust; preferably, the size of the carbon compound ranges from 0.1 to 2 microns; and/or
The white reference target is selected from the "metal-lean" oxide or sulfate states in the periodic table, such as titanium oxide, aluminum sulfate, or barium sulfate.
12. The slide of claim 10 or 11, wherein the black target and the white target are both based on an anhydride based epoxy binder that is catalyzed by direct exposure to specified 365nm uv light, preferably but not limited to a uv light initiated anhydride catalyst consisting of methyltetrahydrophthalic anhydride and diphenyliodonium hexafluoroarsenate.
13. A slide as in of claims 1-12, wherein the control zone further comprises or more antigen repair monitor load points for testing under recovery conditions of an antigen repair process, each of the antigen repair monitor load points being a mixture of or more host proteins, preferably a mixture of mouse and rabbit proteins in a ratio of , such as (0-100): 100:0, e.g., about 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 or 90:10, fixed with minimal fixative (e.g., formaldehyde or formalin), more preferably the points being a 50:50 mixture of mouse and rabbit proteins.
14. A slide as claimed in , wherein the control zone further comprises another (e.g. 1, 2, 3, 4 or 5) antigen repair monitor load points for the detection of over-recovery conditions of antigen repair processes, each of said antigen repair monitor load points being a mixture of or more host proteins, preferably a mixture of mouse and rabbit proteins in a proportion of , such as (0-100) to (100:0), e.g. about 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 or 90:10, deposited in a 3D scaffold (such as a polysaccharide scaffold) that has been fixed with a fixative (e.g. formaldehyde or formalin; more preferably said points are a 50:50 mixture of mouse and rabbit proteins.
15. The slide of any of claims 1-14, wherein a paraffin coating is applied to one or more biological materials selected from the group consisting of a -grade target, a secondary target, an imaging reference, and an antigen retrieval monitor load point on the slide, or to an inorganic deposit (e.g., a stain), preferably at a coating thickness of 1-5 microns (e.g., 2-3 microns) but not more than 5 microns.
16. The slide of claim 15, the application of the paraffin coating comprising the steps of:
(a) melting the paraffin wax at a temperature in the range of 60 ℃ to 70 ℃ until the paraffin wax is in a liquid state, wherein the melting temperature should not exceed 75 ℃;
(b) adding an aliphatic solvent to the product paraffin obtained in step (a) until a saturated mixture is obtained;
(c) cooling the mixture obtained in step (b) at a temperature in the range of 30-33 ℃ and then by slow addition of an organic solvent until a substantially clear liquid is obtained;
(d) applying the thin film of clarified liquid from step (c) to the biological material or the inorganic precipitate on the slide to form a barrier coating on the slide for barrier coating;
(e) exposing the deposited paraffin wax mixture to infrared heating to melt and evaporate the released solvent, thereby restoring the paraffin wax to its original solid state;
preferably, the paraffin wax is selected from the group consisting of tissuefep and tissuefep 2, of selamer fly el, having a melting temperature of 56 ℃, the parts plast and parts plast plus, of laika, having a melting temperature of 56 ℃, and the parts plast X-tra, of laika, having a melting temperature of 50-54 ℃; and/or
The organic solvent is selected from xylene, aliphatic xylene substitutes (such as mixed xylenes), toluene, paint thinners, turpentine, or a 50:50 mixture of acetone and kerosene.
17. The slide glass according to claim 15 or 16, wherein the paraffin-clarified liquid obtained in step (c) is applied by spraying, ink-jet deposition, transfer printing, screen printing or vapor deposition.
18. The slide of any of claims 1-17, further comprising a label identifying the slide type (preferably on top of the slide label region) and a code identifying an antigen supported by the grade target, and/or
Lot number (preferably just below the label).
19. A method of immunohistochemical staining of a slide according to any one of claims 1 to 18 to comprising the steps of:
b. removing fixative (e.g., formaldehyde) fixation to expose antigenic sites of the tissue section;
c1. applying or more (such as 1, 2, 3, 4, 5) antibodies of grade to bind to any matching antigenic sites found in the tissue section/bulk cells or antibody targets, or more (such as 1, 2, 3, 4, 5) secondary antibodies bound to a moiety capable of producing a color in the presence of a staining agent to bind to antibodies of grade used in tissue section/bulk cells, secondary antigen targets or antigen repair monitors in step c, or
c2. Applying or more (e.g., 1, 2, 3, 4, 5) antibodies that bind to a moiety capable of producing color in the presence of the staining reagent to bind to any matching antigenic sites found in the tissue section/loose cells or -grade antigen target;
e. applying the staining reagent to produce a visible color indication of the presence of the target antigen;
f. optionally, multiple amplification steps to obtain sufficient staining reagent density;
g. quantitatively determining the color density of the stained tissue or cells based on the secondary antigen density gradient, and/or with the aid of the -grade antigen density gradient, if possible;
h. assessing the quality of the detection process based on the staining density of the grade target, the secondary target, and the antigen retrieval monitor;
optionally, if a paraffin coating is selectively applied to the target on the slide, and the tissue section/loose cells are also embedded in paraffin, then the following steps are performed prior to step (b):
a. removing the paraffin from the paraffin-embedded tissue section or loose cells and the paraffin barrier coating overlaid on the target.
20. The method of claim 19, wherein the paraffin removal in step (a) is performed by heating the paraffin at a temperature ranging between 65-75 ℃ for 3-10 minutes to obtain a semi-liquid state, followed by serial liquefaction with organic solvents until rehydration in a buffer solution.
21. The method of claim 19 or 20, wherein the organic solvent is selected from the group consisting of an aliphatic solvent-initiated series solvent, such as xylene or mixed xylenes, absolute ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and salt-based buffer solutions, each solution having an exposure time designated as 3 minutes.
22. The method of any one of claims 19-21, , wherein the removal of formaldehyde fixation can be performed by a heat-induced epitope retrieval (HIER) process, or by a longer multiple-exchange warm distilled water antigen retrieval process.
23. The method of any of claims 19-22, wherein the secondary staining reagent used in step (d) can be selected from the group consisting of an enzyme-labeled secondary antibody, an enzyme-labeled tertiary antibody that reacts with the enzyme-labeled secondary antibody, an APAAP immune complex that reacts with the secondary antibody, an enzyme-labeled (strept) avidin that reacts with the biotinylated secondary antibody, an avidin-/streptavidin-biotin-enzyme complex that reacts with the biotin-labeled secondary antibody, a streptavidin-enzyme complex on the secondary antibody biotinylated in the -class antibody, and a polymer containing the secondary antibody and an enzyme site that binds to the -class antibody.
24. The method of any of , wherein the antigen retrieval buffer used is selected from the range of pH 6-9.
25. The method of any of , wherein the antibody in step (c) is selected from the group consisting of a mouse or rabbit antibody, including common examples: ER, PR, Her2, Ki 67.
26. The method of any of claims 19-25 to , wherein the chromogen is selected from the group consisting of 3,3' -Diaminobenzidine (DAB), amino-9-ethylcarbazole (AEC), DAB + nickel enhancer, fast red, TMB, stay yellow, BCIP/NBT, BCIP/TNBT, naphthol AS-MX phosphate + fast blue BB, naphthol AS-MX phosphate + fast red TR, naphthol AS-MX phosphate + neofuchsin, stay green, and NBT.
27. The method of any of of claims 19-26, wherein the method is cost-effective, repeatable, robust, and facilitates identifying IHC processing steps that lead to a misanalysis.
28. The method of any of , wherein the method is used as a quantitative standard for process control of antigen concentration on the co-existing tissue sections or loose cells.
29. The method of any of claims 19-28, wherein the evaluation criteria for antigen retrieval are as follows:
if the antigen retrieval monitor load point used to detect the target in the restored state is stained, indicating that the AR process has failed;
if the antigen retrieval monitor load point used to detect the over-recovery state is stained, indicating that the AR process is excessive, the slide is not used for diagnostic evaluation;
if 10% to no more than 30% of the secondary target array shows no visible staining, indicating that a normal recovery state has occurred, then the extent of AR damage can be assessed by the amount of low concentration of secondary target that is not stained.
A kit of comprising a slide as claimed in any one of claims 1 to 18 or or performing a method as claimed in any one of claims 19 to 29 or .
Technical Field
The present invention relates to novel process record slides the present invention specifically relates to process record slides and staining methods for immunohistochemistry the present invention more specifically discloses process record slides that provide control targets for coexisting patient samples to undergo the staining process.
Background
All immunohistochemical methods, as well as other immunochemical methods, are multi-step procedures involving series of reagent exchanges, incubations, and washes.
For both automated and manual methods, there are critical points to consider, care must be taken to avoid sample loss on the slide, thorough washing of the sample during reagent application is highly necessary, particularly to remove unbound antibody, as the residue can be magnified.
In addition, many of the reagents used in immunohistochemical and immunochemical methods, such as enzyme solutions and peroxidase developers, have limited stability at operating temperatures, even at room temperature.
Methods and reagents that improve results and minimize reagent preparation would facilitate both manual and automated immunohistochemical methods. Many improvements can be readily applied to relevant immunochemical methods such as enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays and in situ hybridization.
Reference may be made to "Use of filtered cells as a control for quantitative biochemical analysis of the Estrogen Receptor (ER) in the breast cancer cell, the quick gel method", which discloses that the variation in tissue fixation, handling and staining is the main cause of poor reproducibility of Estrogen Receptor (ER) immunohistochemical tests.cryoagar suspension particles of MCF-7 cells with known ER content are added to each of 55 samples of Invasive Breast Cancer (IBC) as controls image analysis determines the percentage of MCF-7 cells and IBC positive area (total positive nuclei/analyzed) and the percentage of positive staining (sum of optical density of the area of positive nuclei divided by the sum of optical density of all nuclei studied). image analysis by dextran-coated activated carbon, the average of ER in the MCF-7 cells is 150 fmol/mg.55 cells image analysis shows the average area is 70.81, the average of MCF-7 cells is 70.187, the range of the average of ER area is 70.81, and the range of the area of positive nuclei is converted to the control for quantitative control of the amount of ER contained by the molar factors between 0.187 and 1790.
Reference may be made to CN102435728, which discloses a method for preparing positive references for quality control in immunohistochemical process, comprising the steps of pre-adsorbing polypeptides or proteins having different concentrations, which are capable of specifically reacting with antibodies, onto a slide, or pre-placing polypeptides or proteins having different concentrations on a slide, slicing the polypeptides or proteins and pathological tissues while performing a conventional immunohistochemical step, and using the color development result of the polypeptides or proteins as a positive control for the immunohistochemical process.
The above invention reports the problems listed below:
a. since the binding of the peptide fragments to the dextran polymer depends on the viscosity of the mixed solution, the temperature and size of the precipitated polymer particles will vary depending on bath concentration, reaction temperature and NaOH injection reproducibility, so that the target density will not be .
b. Since the peptide concentration available on the polymer particles is unknown, the constructed targets for known reactivity (staining density) are limited the only result is a yes/no grade antibody detector.
c. Although dextran can support protein capture of secondary IgG targets, only yes/no results. Thus, a baseline detection scale supporting digital imaging cannot be established.
d. During antigen retrieval, the target will leak proteins/peptides onto the remaining slides and tissue sections. The target is placed above the tissue section and therefore there is background and tissue contamination from the target during processing.
Reference may be made to Horizon Diagnostics, which makes control slides similar to CN102435728, but the construction of the targets is very different from the preparation of targets from DNA-modified tissue culture cell lines to place naturally the desired antigenic peptides with the cells at , these cell lines can be replicated as required and are called "renewable resources". cells are fixed in formalin and paraffin-grown into tissue blocks in the form of loose cytoplasm.
The above invention reports the problems listed below:
a. controls were limited to only a yes/no result simply because there was too much unknown of the reactive target site density. Simply represent: by changing the cross-sectional section of the cells, the staining intensity results will change. Since the cell mass must pass through the antigen retrieval process, whatever antigen is present will be affected empirically, resulting in unknown variables.
b. Upon cutting the formation of a mixture of known cells (antigen vs. blank) is statistically ineffective because the electrostatic charge on the cells will be different, causing them to separate and aggregate at .
c. The performance of the cell line is not due to the limited replicative life span of cell propagation.
d. Since the tissue block, the cutting block, and the section are manually constructed and used for the slide, it is not cost-effective.
Reference may be made to US2016/0274006a1, which discloses methods and apparatus for use as controls and calibrators for assays of cells and tissues mounted on microscope slides.
This control and calibration solution, while interesting, is impractical in practical use because the stability of the target to the substrate is poor and the target data is difficult to extract because the target material is on sparsely distributed coated beads when a single bead is imaged, the color of the stain changes from the top center of the bead to the edge of the bead, making it difficult to know which points on the bead surface the image data is correct.
Reference may be made to US7271008B2 which discloses apparatus and methods for determining the quality of reagents used in assay procedures, particularly multi-step immunohistochemical assays.
The immunostaining disclosed in the above-mentioned patent document is intended to serve as a quality control slide to evaluate the performance of the secondary staining kit, rather than to support tissue sections that are subjected to immunohistochemical processing. The slide substrate is an aminosilane that does not support covalent bonds that can participate in the antigen retrieval process for any target. In addition, alkaline phosphatase targets are degraded by exposure to antigen retrieval temperatures.
Disclosure of Invention
generally, the aspect of the invention provides procedural record slides for immunohistochemical staining.
In another aspect of the invention, devices and methods are provided for determining the effect of paraffin removal, antigen retrieval, and the use of grade and secondary staining reagents during the course of an assay, particularly a multi-step Immunohistochemistry (IHC) assay.
In another aspect, the invention provides devices comprising an adhesive coated microscope slide containing a plurality of compounds spotted in 2D or 3D structures and sealed under a paraffin coating.
In another aspect, the invention provides process record slides in which tissue sections or loose cells are subsequently added to the same slide and all undergo IHC processing steps applied from tissue capture to coverslips.
In another aspect, the invention provides process record slides in which compounds are reacted with grade or secondary IHC staining reagents to record the processing history of coexisting tissue sections or loose cells.
In another aspect, the invention provides process record slides in which the -grade target consists of bound antigen on a carrier protein.
In another aspect, the invention created process record slides in which the -grade targets consisted of bound antigens on a carrier protein that were mixed with other carrier proteins bound to different antigens to form targets with multiple capture capabilities could use only , but this method expanded the number of -grade targets over the actual number of -grade targets on the slide.
In another aspect, the invention produced process record slides in which the -grade target consists of a bound antigen of a carrier protein that is mixed with other non-reactive proteins to produce a gradient density array of all identical antigens.
In another aspect, the invention provides process record slides wherein two secondary target arrays are used, wherein arrays are mice and another arrays are rabbits.
In another aspect, the invention provides process recording slides wherein a secondary array is applied to the substrate in a 2D gradient density array from 10% to 100% concentration along with a 2D/3D 100% concentration target.
Specifically, there are procedure record slides in accordance with the present invention in which the extrapolation of the secondary array, and if possible, the establishment of a scale or ruler aided by the -level array, objectively measures each antigen concentration on the coexisting tissue sections IHC staining undergoes permanent locking with the coexisting tissue sections or the loose cells to support quality control of the IHC procedure and objective measurement of antigen density.
In particular, the invention includes the following embodiments:
a process recording slide for immunohistochemical staining comprising:
optionally, a label on top of the slide label area that identifies the type of slide and a code that identifies antigens supported by the grade target;
optionally, a lot number printed under a label on the slide;
space for processing by IHC and subsequent examination of tissue slices;
an IHC target located below the tissue section;
imaging reference points located on both sides of the protein array; and
optionally, a glass microscope coating.
2. The process recording slide according to
3. The process recording slide according to
4. The process recording slide according to
5. The process recording slide according to
6. The process recording slide according to
3D was fixed with polysaccharide and then 6% formalin.
7. The process recording slide according to
8. The process recording slide according to
9. The process recording slide according to
10. The process recording slide according to
11, A method of immunohistochemical staining of a process recording slide according to
a. removing paraffin from the paraffin-embedded tissue sections and covering the PRS target with a paraffin barrier coating;
b. removing the formaldehyde fixation by an antigen retrieval buffer to expose antigenic sites of the tissue section;
c. applying or two -grade binding antibodies to bind to any matching antigenic sites found in the tissue section or in the -grade antigenic target sites;
d. applying a staining reagent to the exposed antigenic sites obtained in step (b) to produce a visible color indicative of the presence of the target antigen;
e. optionally, multiple amplification steps to obtain a sufficient density of staining reagents;
f. hematoxylin was used to provide a contrasting color (blue) to make the physical form visible.
g. The slide was covered with a coverslip and finally prepared for testing.
12. The method according to embodiment 11, wherein the paraffin removal in step (a) is carried out by heating the paraffin at a temperature ranging between 65-75 ℃ for 3-10 minutes to obtain a semi-liquid state, followed by serial liquefaction with organic solvents until rehydration in a buffer solution.
13. The method of embodiments 11 and 12, wherein the organic solvent is selected from the group consisting of an aliphatic solvent-initiated series solvent, such as xylene or mixed xylenes, absolute ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and salt-based buffer solutions, each solution having an exposure time designated as 3 minutes.
14. The method of embodiment 11, wherein formaldehyde fixation can be removed by an antigen retrieval process, such as: heat-induced epitope removal (HIER) process or antigen retrieval process with multiple water exchanges by longer warm distilled water.
15. The method according to embodiment 11, wherein the secondary staining reagent used in step (d) may be selected from the group consisting of an enzyme-labeled secondary, an enzyme-labeled tertiary antibody reactive with the enzyme-labeled secondary antibody, an APAAP immune complex reactive with the secondary antibody, an enzyme-labeled (strept) avidin reactive with the biotinylated secondary antibody, an avidin-/streptavidin-biotin-enzyme complex reactive with the biotin-labeled secondary antibody, a streptavidin-enzyme complex on the biotinylated secondary antibody in the -grade antibody, and a polymer containing the secondary antibody and an enzyme site binding to the -grade antibody.
16. The method according to embodiment 11, wherein the antigen retrieval buffer used may be selected from the range of pH 6-9.
17. The method according to embodiment 11, wherein the -grade antibody in step (c) is selected from the group consisting of antibodies to mouse or rabbit host proteins, including the common examples ER, PR, Her2, Ki 67.
18. The method of embodiment 11, wherein the chromogen can be selected from the group consisting of 3,3' -Diaminobenzidine (DAB), amino-9-ethylcarbazole (AEC), 3' -diaminobenzidine + nickel enhancer, fast red, 3',5,5' -Tetramethylbenzidine (TMB), Yellow-preserving (Stay Yellow), 5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium (BCIP/NBT), 5-bromo-4-chloro-3-indole-phosphate/tetranitro blue tetrazolium (BCIP/TNBT), naphthol AS-MX phosphate + fast blue BB, naphthol AS-MX phosphate + fast red TR, naphthol AS-MX phosphate + neofuchsin, Green-preserving (Stay Green), and Nitro Blue Tetrazolium (NBT).
19. The method of embodiment 11, wherein the method is cost effective, repeatable, robust, and facilitates identifying IHC processing steps that lead to misanalysis.
20. The method of embodiment 11, wherein said method is used as a quantitative standard for process control of antigen concentration on coexisting tissue sections or loose cells.
Drawings
FIG. 1 shows the types of -grade and secondary targets that may be utilized in accordance with an embodiment of the present invention .
Fig. 2A shows the basic slide fabricated. The slide has the smallest ID by lot number and has a blank area in the colored label area. There are two thick colored long axis stripes that extend beyond the area of the target spot. These bars are recently added to address how the label printer dispenses slides from the bottom of the stack. These strips ensure that the slides stacked on top do not damage the slide coating and more importantly the paraffin mask coating and the target points under the paraffin.
Fig. 2B shows the base slide as it has printed the additional data in the label area: date, 2D barcode and tissue section that has been captured. Note that the tissue sections are more like paraffin. The tissue is substantially transparent and therefore the paraffin color is dominant. The "patient tissue" and "control target" text were not printed on the slide, but rather the reader was instructed here what the paraffin covered area was.
Figure 2C shows the basic slide as it appears after IHC treatment. Both the target and the tissue section are visible and ready for interpretation.
Figure 3 is a close-up view of the effects of AR damage.
Fig. 4 shows the effect on the image when the illumination level is too dark (optimally-5%), best state (+0) and too bright (e.g. +10 or + 15%).
Figure 5 shows how the paraffin barrier coating ensures the sealing of the edge of its deposit.
Detailed Description
As used in the specification including the appended claims, the singular forms "," "," and "the" include the plural forms and reference to a particular numerical value includes at least that particular value unless otherwise expressly indicated, when such a range is expressed in another embodiments, the range may be expressed from "about" or "approximately" another particular values.
The invention is not limited in its application to the details of construction and the arrangement of the components set forth. In or shown in the following description or drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein for the purpose of description should not be regarded as limiting. The use of "including," "comprising," "having," "consisting of …," "involving," and variations thereof, as well as other items.
Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings and the description to refer to the same or like parts.
Definition of
As used herein, the term "slide," also referred to as a "microscope slide," refers to a thin, flat sheet (typically made of glass, and therefore sometimes referred to as a "glass slide") typically 75 x 26mm (3 x 1 inch) thick about 1mm thick for holding an object under examination under a microscope.
The slide in the present invention is also referred to as a "Procedure Record Slide (PRS)", which may be interchangeably referred to as a PRS-IHC slide.
The term "detection zone" as used herein refers to the space in a slide where a sample, such as tissue and loose cells of any biological origin, is placed for subsequent immunohistochemical or immunochemical detection.
The term "control zone" as used herein refers to a space that accommodates known reactive behavior targets for assessing antigen repair status, -grade antibody reagent efficacy, and secondary reagent efficacy, including or more selected from -grade and secondary target arrays, imaging references, and antigen repair monitors.
It should be noted that the "detection zone" and "control zone" have clear label boundaries on the slide; it is preferable to classify them only according to their functions.
As used herein, the term " -grade target" refers to a target to which -grade antibodies used in IHC assays can bind.
The term "antigenic peptide fragment" as used herein refers to a full length or portion of an antigenic protein that has the same or nearly the same antigenic specificity as the antigenic protein, and a halogen.
Typically, a secondary antibody binds to the th grade antibody body in IHC, and thus, secondary targets typically comprise IgG of different origins, such as mouse and rabbit.
The term "host protein" refers to proteins (particularly IgG) of the same origin as the -grade antibody, such as mouse, rat, rabbit, and goat proteins (IgG).
The term "pseudoprotein" refers to a protein that is non-reactive with secondary antibodies and is used in admixture with host proteins to obtain a gradient dilution. Preferably the pseudoprotein is donkey protein (IgG) or horse protein (IgG).
As used herein, the term "loading point" is also interchangeably referred to as "point" and refers to an entity formed by immobilizing a desired peptide or protein on a slide. The "dots" may have any shape such as, but not limited to, circles, ovals, squares, diamonds, and the like.
Glass slide
Typically, Immunohistochemical (IHC) staining is used to assess the presence of specific anti-home sites in patient tissue sections. Subjective interpretation was performed for staining density on tissue sections to determine the level of diagnosis of abnormal or cancerous conditions. In general, it is assumed that IHC treatment always works correctly and that tissue sections will be marked with visible chromogen markers to identify abnormal or cancerous conditions, if any. However, failure of antigen repair or staining reagents does not leave any features that identify artifacts. Thus, there is a great chance that a laboratory technician or pathologist will not be able to make an effective diagnostic decision. In other words, the physical morphology may not be sufficient to reveal an abnormal condition, but if the antigenic sites are not labeled, the slides only provide what is seen on hematoxylin and eosin (H & E) slides.
In an embodiment of the invention, there are disclosed novel adhesive coated slides, which may be interchangeably referred to as "process record slides" (hereinafter adhesive coated slides may be referred to as process record slides with the same scope and meaning.) the above-described "process record slide-immunohistochemistry" (PRS-IHC) slides incorporate targets of known reaction behavior for assessing antigen retrieval status, antibody reagent efficacy and secondary antibody efficacy in another embodiment of the invention, grade antigen target sites are but not limited to deparaffinization, antigen retrieval process, grade antibody performance, secondary amplification of precipitated chromogens and cumulative results of coverslipping, in another embodiment of the invention, secondary target sites are but not limited to the cumulative results of grade antigen target minus grade antibody reagent performance.
In another embodiment of the invention, the stained secondary target set sites described above provide a baseline at which the antigen density of -class sites can be objectively determined.notably, each species of each secondary protein array can also be printed as a 3D scaffold using polysaccharides as 3D scaffolds.furthermore, the ratio of chromogen precipitation between 2D and 3D targets of the same concentration determines a scaling factor that can be applied to -class antigen arrays to enable objective measurement of the antigen concentration on 3D materials.by identifying the 3D antigen density as the degree of chromogen precipitation, a scale or scale can be applied to co-existing tissue sections to objectively quantify the presence of antigen in the tissue sections.
Reference may be made to fig. 1, which shows possible targets employed during an immunohistochemical assay.
Reference may be made to fig. 2, which shows the structure of an adhesive coated slide or process recording slide in which the IHC target is located below the tissue section to reduce the likelihood of protein release from the target material, which may be swept onto the tissue section and processedThe lowest rows of targets are the mouse gradient density array, the middle is the rabbit gradient density array the lowest rows can support twelve targets, which can be a mixture or combination of or grade antigens.the black and white imaging reference point is located to the left of the secondary protein array the right of the colored targets are the 3D mouse and rabbit targets3、-R(C＝O)NH2and-RNH2) End groups and adjustable surface wetting properties during manufacture.
In an embodiment of the invention , the above-described "procedure record slide-immunohistochemistry" (PRS-IHC) slide may incorporate targets of known reaction behavior to evaluate antigen retrieval status, antibody reagent efficacy, and secondary reagent efficacy, the grade antigen target site is the cumulative result of deparaffinization, antigen retrieval process, grade antibody performance, secondary amplification to precipitated chromogen, and cover-slide, the secondary target site is the cumulative result of grade antigen target minus grade antibody reagent performance.
In another embodiment of the invention, the PRS-IHC slide described above incorporates bio-based targets in a gradient density array comprising black and white imaging reference targets in order to conform to the structure of the adhesive coated slide or process recording slide described above, since the targets are of biological origin, a thin film of paraffin can be applied to prevent oxidation and microbial attack.
In the embodiment of the invention, the adhesive coated slide or process recording slide helps to keep the control and patient material co-located so that it is not replaced and lost as with the Laboratory Information System (LIS). The control must therefore go through all of the experience done by capturing a cover slide from the biological material.
In another embodiment of the invention, the adhesive coated slide or process recording slide is repeatable, stable over time, supports or more antigens, each is a gradient density array, is stable to current processing of IHC slides, and is cost effective.
In an embodiment of the invention , the steps of the IHC staining process can be described to understand the immunohistochemical staining performed in an adhesive coated slide or process record slide, embedding the fixed tissue section in paraffin, which must first be heated to a semi-liquid state, then liquefied by xylene (or mixed xylenes), then gradually washed with diluted ethanol and finally removed with buffer to expose the cellular structure of the section, next, formaldehyde must be removed to expose the antigenic sites, most commonly, fixation is removed by a heat-induced epitope repair (HIER) process or longer warm water antigen repair process when the tissue is exposed to buffer (pH 6-10, depending on the tissue type), the HIER process destroys the Schiff base bond between formaldehyde and the tissue by applying heat (optimal 89 ℃ and not more than 95 ℃), at which the antigenic sites are exposed, can use staining reagents to produce a visible color indicating the presence of the target antigen, the running temperature of the water-based antigen repair process is about 10 ℃ higher than the paraffin melting temperature, about 60-65 ℃), and many successive washes dissolve the wax slowly and fix the false stain results in the paraffin process.
Once the antigenic sites are separated from the formaldehyde fixation, either or two -grade conjugated antibody reagents are applied, these will bind to any matching antigenic sites found in tissue sections or PRS -grade antigen target sites, the -grade antibody is conjugated to mouse or rabbit protein, and then acted upon by a secondary staining reagent.
In another embodiment of the invention, to obtain sufficient density of staining reagents for human visual detection, a multi-step amplification process can be performed, from single-step to three-step amplification, with a variety of secondary detection kits, all reaching the same final state of chromogen precipitation, typically, of three commonly used secondary staining reagent sets and several counterstains are used, horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase and nuclear counterstains, or two of the available chromogen colors that can precipitate can be selected from, but are not limited to, those in the following list:
horseradish peroxidase (HRP)
Alkaline Phosphatase (AP)
Nuclear staining
Hematoxylin (most commonly) > > blue
Experiments have shown that the original DAB experiences significant aging in a short period of time, resulting in a significant reduction in colour saturation within 4 hours, the newer version of DAB contains stabilizers, which extend the stability of DAB from hours to days, DAB also has a tendency to be washed out in subsequent buffer wash cycles, and in addition , AEC can remain stable for weeks to months.
The regulatory standards throughout the world seek or persist to suggest that , once this technology is viable and available, can use validated controls to examine reagents, methods and apparatus for treating tissue sections and loose cells for hematology and clinical chemistry such regulatory controls already exist to validate results and ensure quality the results of control tests are plotted in the form of a Levey-Jennings diagram (Westgard et al, 1981), Westgard J, Barry P, Hunt, Groth T (1981) "A Multi-rule Shewhart chart for quality control chemistry". Clin Chem27:493 501.
In another embodiment of the invention, the controls used above must not be significantly affected by the pretreatment steps of paraffin removal and antigen retrieval, the results measure the efficacy of IHC staining reagents and ratios or scales were developed for the determination of antigen concentration on tissue sections.
In another embodiment of the invention, all of the above 2D secondary staining targets are fixed with formaldehyde.
The 2D secondary staining targets incorporate protein gradient density arrays, of which are mice and donkeys and the other are rabbits and donkeys the gradient ratios follow a known profile between 10% and 100% density the donkeys do not support non-specific staining and sometimes are used more often than regular cattle in the second staining kit the ABC secondary staining kit uses goat antibody (mouse or rabbit) as the step reagent and the second staining reagent (containing anti-goat) the goat is too close in species to the cow, supporting capture of the secondary staining reagent of the second step the donkeys or other equine animals to avoid this accidental reaction.
In another embodiment of the invention, different secondary stained and precipitated chromogens vary greatly in color density between type and manufacturer to account for variations, the 2D gradient density array formed the relationship between chromogen density and mouse and rabbit protein concentration, although the secondary gradient array mixture could produce absolute concentration ratios, it did not account for the physical structure of the slide coating.
In another embodiment of the invention, the 2D grade antigen staining target is coupled to a carrier protein by way of cysteine residues and sulfo-SMCC cross-linking using a peptide chain of the desired antigen.
a. Gradient density pairs, where the maximum density exceeds the ability of the antibody to bind, the second is 50% concentration. Each target pair comprises a single reactive antigen.
The b, series of antigen targets, each target containing up to ten different antigens, all antigens having the greatest density, the mix of antigen types in each target is such that only antigens will react during use.
The antigen consists of a peptide chain with cysteine residues and is covalently bound to a carrier protein that has been previously activated by sulfo-SMCC. Keyhole Limpet Hemocyanin (KLH) was used because it is unreactive with any human antibody and is known to support a range of sites for sulfo-SMCC. Other similar proteins with equivalent properties may be used.
The PRS supports each staining reagent kit by using option B described above, better serving the market, since the staining vendors develop their own antibodies in large numbers.
However, in developing new antibodies, it is important that multiple antigen preparations be tested to achieve optimal detection conditions. Option a above may solve this problem better because it is unclear what the optimal antibody concentration and sensitivity are.
In another embodiment of the invention, the 3D target converts the 2D target results to the necessity for measurements that can be applied to tissue sections both between 4 and 10 microns in height.
The aging effect of the DAB reagent causes considerable changes in the staining results in a short period of time. However, the PRS-IHC solution was able to correct the shift reduction in chromogen precipitation only because the 3D antigen concentration ratio was not related to the change in DAB performance. Since the protein and antigen target arrays are composed of known concentrations, the staining results remain the same relative relationship even when compressed. Thus, when the observed intensity (darkness) is diminished, the ratio will continue to provide the same measure of antigen density on the tissue section or the loose cells.
Imaging reference target
In addition to the 2D and 3D secondary protein target arrays, black and white imaging reference targets were printed.
Digital imaging of microscope slides containing stained biological material is being developed to perform pre-screening and possibly comprehensive diagnostic determinations of the stained material. Typically, the imaging system must adjust the illumination light level so that the digital image is not in a compressed state at either the white or black borders. The conventional solution is to place a black and white target at the desired location of the tag. The basic assumption is that white and black targets represent the limits that a slide can assume. However, in doing so, there is a compression in the numerical scale, as black is much darker and white is much whiter than the staining of tissue sections.
In a preferred embodiment of the present invention , microscope slides are disclosed that contain control and reference target standards that coexist with patient tissue sections or loose cell deposits.
In another embodiment of the invention, there are co-existing black and white reference targets between the microscope slide target arrays the black and white reference targets undergo the same reagent exposure and treatment as the other targets and tissue sections.
In another embodiment of the invention, both reference targets, black and white targets, are printed paint deposits that are not reactive with any reagents used to treat the slides.
In another embodiment, both black and white targets are based on anhydride-based epoxy binders that are catalyzed by direct exposure to the designated 365nm UV light, the anhydride catalyst consists of methyltetrahydrophthalic anhydride and diphenyliodonium hexafluoroarsenate.
In another embodiment, the anhydride catalyst eliminates unreacted amine found by the amino silane based catalyst that would otherwise support non-specific staining free amine end groups can and will capture biological material and certain specific staining agents.
In another embodiment, the black pigment uses carbon dust less than 2 microns in diameter, and the white pigment uses aluminum, titanium oxide or barium sulfate beads, preferably barium sulfate for white.
In another embodiment, the preferred epoxy ink/paint formulation avoids surfactants altogether to prevent the ink/paint from reacting to staining and reagents that these slides may experience.
In another embodiment, printing of the target can be accomplished by a stamp or syringe in another embodiment, a syringe is preferred because it supports better control of the feature size of the target deposition.
In another embodiment, the white target is comprised of a metal oxide or sulfate pigment in an anhydride catalyzed epoxy resin, in another embodiment, the black target is comprised of a carbon pigment in an ultraviolet light initiated anhydride catalyzed epoxy resin, in another embodiment, the anhydride catalyst is ultraviolet light initiated by direct ultraviolet exposure.
Shielding coating
Paraffin is typically white or colorless soft solids derived from petroleum, coal or oil shale, consisting of a mixture of hydrocarbon molecules containing twenty to forty carbon atoms, which is solid at room temperature and begins to melt above about 37 ℃ (99 ° F), and which has a boiling point >370 ℃ (698 ° F), common applications for paraffin include lubrication, electrical insulation and candles, dyed paraffin can be made into crayons, unlike kerosene and other petroleum products sometimes referred to as paraffin.
In the pathology laboratory, before slicing thin tissue samples, the tissue is impregnated with paraffin wax, water is removed from the tissue by increasing the alcohol concentration (75% to absolute concentration) and the tissue is removed in an organic solvent, such as xylene, or aliphatic substitutes, such as mixed xylenol.
Embedding tissue sections in paraffin is a routine procedure for long-term preservation of tissue sections. However, paraffin has not been reported to be applied as a thin coating to selected areas of a microscope slide. In the present invention, the target protein deposited on the microscope slide, glass or plastic provides a rich food source for the bacterial or fungal antagonist. In addition, antigenic sites (e.g., epitopes) of the protein are readily oxidized, thereby effectively neutralizing the ability of the detection antibody to bind to the protein. Many of the subsequent reactive binding sites are hydroxyl groups, which can be destroyed by reaction with acids and bases in the air. Typically, slides containing protein deposits are stored at temperatures below those that support microbial growth. However, such constraints limit the effective use of the deposits. In addition, the slide on which the protein is deposited is packaged in a vacuum-sealed container to prevent oxidative damage. The open-air retention period for unprotected protein-deposited slides is between 2 and 5 days, depending on the ambient temperature and the level of contaminants in the air.
Paraffin is inherently referred to as containing antifungal and antibacterial agents that prevent oxidation of the antigenic site and airborne acid/base degradation of the exposed site. The paraffin barrier coating changes the useful life of the biomaterial from 3-5 days to 1-2 years, thereby providing a useful product life to the end user.
The same or similar paraffin formulation was used to protect other deposited material on the same microscope slide, ensuring that no additional slide treatment was required before IHC staining was initiated.
The blend uses laplace X-tra (Paraplast X-tra) or a product comparable to xylene or an aliphatic solvent, such as mixed xylenes, to reduce viscosity and slow the cure rate after deposition.
In another embodiment, the solvent may be selected from, but is not limited to, toluene, paint diluents, turpentine or a 50:50 mixture of acetone and kerosene Paraplast X-tra specifically incorporates the phenolic antioxidants butylated hydroxytoluene to reduce oxidative degradation of proteins, peptides and inorganic targets.
In another embodiment, the paraffin wax is melted to a liquid at a temperature no greater than 75 ℃ above the melting temperature of the wax, then the aliphatic solvent is slowly added until the saturation point is observed (solids are formed.) the mixture is cooled to 45 ℃ and more aliphatic compound is slowly added until it is completely clear.
In another embodiment, the paraffin coating described above is applied to a biological material, which may include but is not limited to a protein, peptide, conjugated protein, protein-coated bead, peptide-coated bead, or conjugated coated bead, and a specific stain-reactive end group that uniquely captures the specific stain material that reacts with the applied antibody and secondary staining reagent, and the specific stain-reactive deposit is first applied to an adhesive that is coated on a microscope slide.
In another embodiment, a paraffin layer is selectively applied to a target on a slide in another embodiment, the paraffin layer can be deposited on a microscope slide including, but not limited to, spray coating, ink jet deposition, transfer printing (such as pad printing), screen printing, and vapor deposition in a preferred embodiment, the paraffin is a thin layer, preferably no greater than 5 microns in another preferred embodiment, the paraffin has a melting temperature of less than 60 ℃, preferably less than 56 ℃, and dissolves when exposed to xylene or mixed xylene (aliphatic substitute) solvents in another preferred embodiment, the paraffin has a similar ambient temperature hardness as the embedded paraffin.
In another embodiment, the tissue mass embedding the paraffin material may include, but is not limited to, tissuePrep and tissuePrep 2(TissuePrep2) from Series fly (Thermo Fisher), melting point 56 deg.C, and Paralast (Paraplast) and Paralast (Paralast plus) from Leica, melting point 56 deg.C, and Paralast X-tra (Paralast X-tra) from Leica, melting point 50-54 deg.C.
In another embodiment, each species is a mixture of purified paraffin wax, synthetic polymer and other materials to determine melt temperature, hardness and viscosity.
In another embodiment, specific staining reagents may include, but are not limited to, Alsinoblue (Alcian Blue), aniline Blue-light-Fast Orange G Solution (Analine Blue-Orange G Solution), azocarmine staining (Azan Stain), Bielschousky silver staining (Bielschowsky silver Stain), Blancei gram-gram staining (Brow & Benn-Grammistain), Tar Violet (Cresyl Viole), Diaminobenzidine (DAB), Totanama Masson (Fontana Masson), Goden-Strigler silver staining (Gordon and Sweet's silver Stain), Gossett silver staining Method (Grocert's dye silver Stain), Holstein Blue (Red-Red Stain), Meloidine Blue (Red-Blue Stain), Meloide Blue (Red-Blue), Meloidine Blue-Red staining (Red-Red), Meloidine Blue-Blue (Red), Meloidine Blue-Red Stain), Meloidine Blue-Red (Red-Red Stain), Meloidine Blue-Red (yellow Stain), Meloidine Blue-Red (Red Blue-Red Stain), Meloidine Blue-Red (Red-Red Stain, Red Blue-Red (yellow-Red Stain), Red-Red Blue-Red (yellow-Red Stain), Red (Red, Red-Red, Red-Red, Red-Red, Red-Red, Red-Red, Red-Red, Red-Red, Red-Red, Red-Red.
In another embodiment, the selection of targets may be, but is not limited to, pigmented deposits, such as black and white, but may include any pigment color.
In another embodiment, the microscope slide on which the paraffin coating described above can be applied can be selected from, but is not limited to, glass, plastic, or any polymeric material in another embodiments, the paraffin can be purified and anhydrous.
In another embodiment, the resulting microscope slide can be post-heated to melt and/or blend the paraffin particles into the bulk surface coating to seal the deposit and the surface of the slide surrounding the deposit.
In another embodiment, the resulting microscope slide is heated after the paraffin is deposited to force the solvent out of the paraffin to ensure it returns to a hardened state, this must be started from the paraffin side of the slide, preferably using infrared light.
Antigen retrieval monitor
In another embodiment of the invention, the antigen retrieval monitor is performed by an antigen retrieval (hereinafter AR) process, which varies greatly between slides and between stains depending on the process used and its embodiment.AR is a open loop process because the direct measurement of AR buffer and buffer temperatures is actually unknown and only estimated.
In another embodiment of the invention, the PRS incorporates two AR targets, ARM3D and ARM2D plus a 2D secondary array.
An insufficient recovery condition is a result of the AR temperature being too low, the exposure time being insufficient, or not running at all. The ARM2D target is, for example, a 50:50 mixed mouse and rabbit protein (or proteins from other species, not limited to mouse and rabbit proteins) at 100% concentration with the lowest formaldehyde fixation rate. If the target stains, the AR treatment fails. Preferably, the protein is IgG.
Conditions > excessive recovery are caused by too high an AR temperature, too much exposure time or an AR buffer pH of more than 9.5 or less than 5.5. ARM3D target was a 50:50 mixed mouse and rabbit protein (or proteins from other species, not limited to mouse and rabbit proteins) at 100% concentration deposited in 3D scaffolds that had been over-immobilized with formaldehyde. If the target is stained, this indicates that the AR process is excessive and the slide is not used for diagnostic evaluation.
Normal recovery occurs when 10% to no more than 30% of the targets on the mouse and rabbit gradient density arrays show no visible staining. The extent of AR damage can then be assessed by the amount of low concentration secondary target that is not stained. This lesion can also be seen in tissue sections.
Antigen imaging ratio extrapolation
It is well known that antibodies of grade consist of processed host serum obtained by inoculating a host animal (e.g., a mouse or rabbit) with the desired antigen fragment.
class targets and secondary targets have well-defined and regular (such as circular) deposition areas onto which a known dispensed volume of target material is applied since protein deposits contain cross-linking coupling agents, they do not sink into the pores in the slide coating beyond the depth of the protein.
Thus, knowing the atomic mass of the protein, the amount of protein per protein type in the deposit, the area of the target, and the active surface protein density of the target can be calculated.
The concentration of antibody deposited can be determined under appropriate conditions, for example, when the concentration is greater than a threshold value of 25% and less than 25% below a saturation value, where the threshold value is defined as a density of target sites insufficient to capture the applied protein concentration, and the saturation value is defined as a concentration at which all applied proteins cannot be captured.
Knowing the -grade dilution ratio, the correct -grade target density targets can be selected and the -grade concentration can be verified.
In the embodiment of the invention, each secondary and -level target is a mixture of [ (mouse or rabbit) + (donkey + crosslinker + fungal inhibitor) ] or [ (KLH with antigen A or KLH with antigen B) + (unbound KLH + crosslinker + fungal inhibitor) ].
Mouse IgG 155kDa
Rabbit IgG 150kDa
Donkey IgG 160kDa
KLH subunits conjugated to an antigenic peptide chain, where the subunits are KLH1 and KLH2 ═ 350 and 390 kDa.
In another embodiment of the invention, the 2D secondary target gradient is a stepwise dilution increment of 1 to 1000:1, preferably following a-20 log (dilution) curve where the dilution increment is-3 dBd steps the term-20 log (dilution) ═ dBd refers to describe the dilution in a semi-logarithmic fashion to linearize the data so that the modified terms can be easily applied the term (dilution) refers to dilution X where X is [1..1000], equal to 1:1 to 1000:1, dBd is defined as the dilution or decibel of the dilution intensity the modified terms include antigen repair damage, enzyme gain, -level antibody reagent dilution the individual 2D/3D target is used to measure the dye density increment between the 2D substrate and the 3D particle.
The secondary 100% 2D/3D and 2D targets confirmed that the two deposits were matched in 2D staining density. This demonstrates that the 3D particle component does not consume enough 100% of the protein material to cause movement of the 2D component.
The secondary stain has an enzymatic gain function of between 1 and 20 times that of the stain, which is its structural function. Thus, as the gain is increased, lower concentrations of secondary target will become saturated, and when the gain is decreased to 1, only higher concentrations of secondary target will be visibly stained.
Protein concentration density was established by grade protein due to the considerable size difference between the secondary and grade target proteins.
In the case of an average molecular mass of 150kDa for the -class antibody, the individual antibodies weighed 150kDa (1.6605X 10)12) Equivalent to
The -grade antibody reagent was diluted from its concentrate to an intermediate dilution of 10ug/ml the intermediate dilution was then diluted from 1:1 to 1000:1 and coated onto the slides, which resulted in the deposition of 31.5 to 7.08 antibodies in a dilution ratio of 1:1 to 25.1: 1, respectively, onto the 1 square micron area.
To ensure 100% capture, the grade target should have a safety factor of 100 to 1000 fold.selecting the 1000 fold option, the grade target would need to contain 4 × 106And (c) antigenic sites. Although the KHL subunit is larger than the applied antibody, this increase is not sufficient to increase the number of captured antibodies beyond 1: 1. the average atomic mass of each KLH subunit was 370kDa, corresponding to 614.4X 10-12ng weight.
Can be determined from the molecular weight of the protein and the average specific volume of the protein fractionThe volume of the protein molecule is estimated very simply and reliably. (volume/molecular weight) the average of the soluble globular protein partial specific volumes determined experimentally was 0.73cm3(ii) in terms of/g. This value varies from protein to protein, but the range is narrow. The reaction formula is reduced to (1.212X 10)3×MW)nm3Protein volume of (c). Thus, the individual volume for the KLH subunit is 448.44nm3. If the protein is modeled as a sphere, the diameter of the sphere will become 0.132 x MW1/3In nm. For the KLH subunit, 9.436 nm.
For a target diameter of 1mm, the monolayer of KLH subunits required 11.237 × 1027A protein. For
In an embodiment, the secondary target array is in stepwise dilution increments of 1 to 1000:1 linear slope of dilution dBd ═ 20log (dilution) —, half log range 0dB to-60 dbd for the dilution range listed 1 to 1,000: 1, the-3 dB dilution step is selected and the secondary target dilution becomes: -0, -3, -6, -9, -12, -15, -18, -21 dBd.
The secondary and target arrays are irreversibly immobilized and degrade to a much lesser extent than the tissue or AR target during AR processing protein fragments from cleavable, but not intact, proteins are degraded As AR processing continues on the protein target and tissue section, it can be seen that AR damage is due to a shift in the gradient scale pattern to 100% position, in addition , an increase in the secondary enzyme results in a shift in the gradient array to 10% position, the gains of the enzyme are: 1, 2, 4, 5, 8, 10, 15 and 20.
1.20-fold all target movement-26 dBd
2.15 fold all target movements-23.52
3.10 times all target moves-20
4.5 times all target movements-13.98
5.4 fold all target movement-12.04
6.2 times all target movement-6.02
7.1 times only 2D 100% points near black
Typically, AR damage will cause the secondary array to move three or more spots to 100% of the positions, which is considered excessive, and the slide should be redone using a secondary staining kit with higher enzyme content or higher concentrations of antibody.
The -grade antigen target color density is thus the sum of the antibody concentration times the enzyme gain of the secondary staining kit, whereas the secondary target density is only the enzyme gain times the secondary target protein concentration.
Depending on the digital imaging system, changes in illumination intensity can change the dynamic range of the image to either compression (dimming) or saturation (brightening). These changes will change the color scale of the antigen, while the numerical scale of the antigen density will not change. Thus, the digital scale is independent, while the color scale depends on the illumination intensity.
In an embodiment of the invention, the above-described secondary protein target array forms two lines, mouse iggs and another rabbit iggs mixed with virtual IgG serum proteins to form a five or more component gradient density series from a maximum density to a minimum density in a-20 log (dilution) linear slope, where the dilution can vary from 1:1 to 1000:1 after an initial 1000:1 dilution.
In another embodiment, in the final processing step, those antigenic sites identified are discolored by chromogen precipitation, thus, the mouse and rabbit target arrays reflect a-20 log (dilution) linear slope of the chromogen precipitation of the secondary staining kit.
In another embodiment, a preferred solution for a method of forming -scale antigen density ratios is to successfully compose target mixtures based on depositing them onto an adhesive-coated slide with covalent bonds between the adhesive and the target material.
In another embodiment, it is concluded that the target array was successfully applied and that and secondary staining reagents perform reasonably, so a fitted curve between data sets can be readily obtained by computer algorithms in another embodiment, grade staining can be selected from antibodies to any IHC-approved mouse or rabbit host protein used, which also do not bind to fluorescent markers or to enzyme sites (such as HRP or AP), in another embodiment, secondary staining agents can be selected from, but are not limited to, secondary staining agents with 1-fold to 25-fold enzyme gain, which are unique between mouse and rabbit, respectively, each using a different color chromogen.
In another embodiment of the invention, it is noted that the performance results based absolutely on slides may be different from those of another slides at another times due to the different performance of the secondary staining kit from that of the grade bound grade antibody, however, any procedure records the performance of the slides, the antigen ratio is valid and is quite equivalent to that of another slides done with different staining reagents.
In another embodiment, a -scale antigen concentration ratio is applied to coexisting tissue sections to approximate tissue sections of a detected cellular defect, such as cancer.
It will be apparent to those skilled in the art that various modifications may be made and other embodiments may be used without departing from the broader scope of the invention presented herein.
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