anti-CD 23 protein monoclonal antibody, cell line, preparation method and application thereof
阅读说明：本技术 抗cd23蛋白单克隆抗体、细胞系及其制备方法和应用 (anti-CD 23 protein monoclonal antibody, cell line, preparation method and application thereof ) 是由 黄信超 杨清海 高惠然 李恢波 吴茂 王小亚 于 2020-12-09 设计创作，主要内容包括：本发明涉及生物检测领域,提供了一种抗CD23蛋白单克隆抗体,其重链和轻链可变区的氨基酸序列分别是SEQ ID NO.2和SEQ ID NO.3所示的氨基酸序列。用于免疫小鼠的抗原为重组蛋白,所述重组蛋白由SEQ ID No.1所示的核苷酸序列编码,经由大肠杆菌重组表达。发明人还提供了一株分泌抗CD23蛋白的杂交瘤细胞系,所述细胞系为小鼠杂交瘤细胞系9D11B6E6,保藏号为：CGMCC NO.19689。抗CD23蛋白单克隆抗体,具有高特异性、敏感性,可以特异性识别表达CD23蛋白的细胞,适用于免疫学检测,特别是免疫组化检测。(The invention relates to the field of biological detection, and provides an anti-CD 23 protein monoclonal antibody, wherein the amino acid sequences of the heavy chain and light chain variable regions are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3. The antigen for immunizing mice is recombinant protein, the recombinant protein is coded by a nucleotide sequence shown in SEQ ID No.1, and is expressed through escherichia coli recombination. The inventor also provides a hybridoma cell line for secreting anti-CD 23 protein, wherein the cell line is a mouse hybridoma cell line 9D11B6E6 with the preservation number of: CGMCC NO. 19689. The anti-CD 23 protein monoclonal antibody has high specificity and sensitivity, can specifically recognize cells expressing CD23 protein, and is suitable for immunological detection, especially immunohistochemical detection.)
1. The monoclonal antibody for resisting the CD23 protein is characterized in that the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.
2. The monoclonal antibody of claim 1, wherein the heavy and light chain variable region amino acid sequences of the monoclonal antibody are encoded by the nucleotide sequences set forth in SEQ ID No.4 and SEQ ID No.5, respectively.
3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes the CD23 protein.
4. The monoclonal antibody of claim 3, which specifically recognizes the amino acid sequence of the CD23 protein shown in SEQ ID No. 1.
5. The monoclonal antibody according to claim 1, which is produced by a hybridoma cell line having a collection number of CGMCC No. 19689.
6. The monoclonal antibody of claim 1, wherein the anti-CD 23 protein monoclonal antibody is a mouse subtype IgG1 monoclonal antibody.
7. The monoclonal antibody of claim 1, wherein the monoclonal antibody is prepared from an immunogen comprising an antigenic CD23 fragment and a histidine protein tag, and wherein the CD23 fragment is an amino acid fragment from position 48 to position 321 of the human CD23 protein.
8. A hybridoma cell line for secreting anti-CD 23 protein monoclonal antibodies, wherein the cell line is a mouse hybridoma cell line 9D11B6E6, the cell line is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO. 19689.
9. The anti-CD 23 protein monoclonal antibody of any one of claims 1-8, for use in immunodetection of CD23 protein.
10. The immunoassay of claim 9, wherein said immunoassay comprises immunohistochemistry, immunoblotting and enzyme-linked immunoassay.
Technical Field
The invention relates to the field of biological detection, in particular to an anti-CD 23 protein monoclonal antibody, a cell line, a preparation method and application thereof.
Background
CD23, also known as fceri, is a low affinity receptor for IgE in humans and vertebrates, i.e., the type II receptor for IgE; it is a transmembrane glycoprotein widely distributed on cell membrane surfaces of B Cells, Eosinophils (EOS), activated monocytes/macrophages, platelets, follicular dendritic Cells, Langhans Cells, epidermal keratinocytes, thymic epithelial Cells, partial T Cells, natural killer Cells, and the like. The molecular weight is 45 kDa. Belongs to typical II type transmembrane glycoprotein and consists of 321 amino acids, including an intracellular N-terminal consisting of 23 amino acid residues, a transmembrane region consisting of 21 amino acid residues and a C-terminal extracellular region consisting of 277 amino acid residues; the carboxyl terminal is positioned outside the cell, and the amino terminal is positioned inside the cell, belonging to the C-type animal lectin superfamily.
The human CD23 gene is located on chromosome 19 and is a single copy gene, and there are two types, and since the 6 amino acids at the amino terminal of the encoded CD23 molecule are different due to their different promoter genes, two types of CD23 are formed: CD23a and CD23 b. CD23a is expressed primarily in B lymphocytes and eosinophils, whereas CD23B is expressed on activated monocytes/macrophages, B cells, eosinophils, platelets, follicular dendritic cells, langerhans cells, epidermal keratinocytes, thymic epithelial cells, partial T cells, natural killer cells, and certain leukemia cells. Macrophages derived from the marrow cavity matrix highly express CD23 b. Cell membrane surface CD23 can be cleaved by itself or by proteolytic enzymes into soluble fragments of varying sizes, called soluble CD23(sCD23), which are mostly still IgE-binding and exhibit multiple functions unrelated to IgE.
CD23 is a multifunctional receptor molecule/cytokine that is involved in regulating IgE synthesis, promoting B cell activation, proliferation and differentiation, and promoting T cell maturation by mediating B-T cell interactions through binding to membrane CD21, enhancing antigen presentation from B cells to T cells. Soluble CD23(sCD23) can also cooperate with IL-1 to promote proliferation and differentiation of thymocytes, bone marrow precursor cells and germinal center B cells, inhibit monocyte migration, and induce monocytes to release TNF-alpha, IL-1 and IL-6. CD23 is expressed in certain subpopulations of peripheral blood cells, B lymphocytes and EB virus-transfected B lymphoma cell lines, with 0 expression also in chronic lymphocytic leukemia and partial central blast cell lymphoma; it is possible to promote B cell activation, differentiation and maturation, prevent germinal center B cell apoptosis, make B cell appear high reactivity, synthesize and secrete autoantibody, and induce autoimmunity. Soluble CD23(sCD23) is closely related to bronchial asthma and may play an important role in intercellular adhesion, antigen presentation, T and B cell activation, IgE synthesis, transcription of asthma-related genes, and the like.
CD23 has close relation with the proliferation and differentiation of Bc and Tc, and is related to many diseases, such as nasopharyngeal carcinoma and chronic B lymphocyte leukemia, and these cells can express CD23 molecule on their surface. Some anti-human CD23 monoclonal antibodies have been developed internationally, but the antibody types are limited, and the development of the CD23 monoclonal antibody not only can make up for the shortage of the antibody types internationally, but also fills up the domestic blank, and solves the problem of difficult antibody sources.
Disclosure of Invention
The invention provides an anti-CD 23 protein monoclonal antibody, wherein the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.
Furthermore, the heavy chain and light chain variable region amino acid sequences of the monoclonal antibody are respectively encoded by the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.
Further, the monoclonal antibody specifically recognizes the CD23 protein.
Further, the monoclonal antibody specifically recognizes the amino acid sequence shown as SEQ ID NO. 1.
Further, the monoclonal antibody is produced by a hybridoma cell line with the preservation number of CGMCC NO. 19689. The cell strain is a mouse hybridoma cell line 9D11B6E6, and is classified and named as: a mouse hybridoma cell line which has been deposited at 24.04.2020 by the general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at the institute of microbiology, China academy of sciences, No.3, West Lu 1 Hospital, North Cheng, Chaoyang, Beijing.
Further, the anti-CD 23 protein is a mouse IgG1 subtype monoclonal antibody.
Further, the anti-CD 23 protein monoclonal antibody is prepared by taking a recombinant protein as an immunogen, wherein the recombinant protein comprises an antigenic CD23 fragment and a histidine protein tag, and the CD23 fragment is an amino acid fragment from 48 th to 321 th of a human CD23 protein.
The inventor also provides a hybridoma cell line secreting anti-CD 23 protein, wherein the cell line is a mouse hybridoma cell line 9D11B6E6, the cell line is preserved in the China general microbiological culture Collection center, the preservation date is 2020, 4, 24 days, and the preservation number is: CGMCC NO. 19689.
The inventor also provides the application of the anti-CD 23 protein monoclonal antibody in immunodetection of the CD23 protein.
Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.
In contrast to the prior art, the above-described protocol selects for recombinant expression the region from amino acid 48 to amino acid 321 of CD23 which is suitable for soluble expression and which has good immunogenicity. The histidine tag of 6 His can be used as a purification tag of the fusion protein. Immunizing a mouse, and obtaining a monoclonal cell line 9D11B6E6 which efficiently secretes the anti-CD 23 protein monoclonal antibody and an anti-CD 23 protein monoclonal antibody secreted by the cell line through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the CD23 protein, is suitable for immunological detection, particularly immunohistochemical detection, can reduce misreading and improve the accuracy of diagnosis.
Drawings
FIG. 1: polyacrylamide gel electrophoresis picture after CD23 antigen purification.
FIG. 2: polyacrylamide gel electrophoresis of the purified CD23 monoclonal antibody.
FIG. 3: immunohistochemical staining results for B cell lymphoma (left for (9D11B6E6) CD23, right for commercially available CD23(SP23) rabbit mab).
Detailed Description
EXAMPLE 1 preparation of recombinant CD23 protein fragment
First, Gene cloning
The CD23 protein sequence with the number P06734 was selected as the standard sequence from the Uniprot database (http:// www.uniprot.org). According to the structure of binding with DNA, antigenicity, hydrophilicity and hydrophobicity of amino acid and secondary structure, the region with proper length and special antigenicity is selected as antigenic peptide. The 48 th to 321 th bit sequences of the CD23 amino acid sequence were selected, and the corresponding nucleotide sequence was SEQ ID No. 1. The target protein fragment is connected to a plasmid vector pet beta 2M (pet beta 2M vector is purchased from Wuhan Kerui bioengineering, Co., Ltd., and contains a beta 2M protein sequence and a His label) to synthesize the CD23 recombinant protein plasmid. Transforming into colon bacillus competent cell TOP10, selecting clone on the plate, inoculating, expanding, extracting plasmid DNA, and performing PCR identification. And (4) sequencing and analyzing the clone with positive target gene shown by PCR, and using the clone with completely correct sequence for next experiment.
β 2M is a component of the Major Histocompatibility Complex (MHC) class I, and is involved in presentation of antigenic peptides to the immune system, facilitating antibody production. The histidine tag of 6-His can be used as a purification tag for the fusion protein.
Secondly, recombinant protein expression and purification
And (3) transforming the recombinant plasmid into an escherichia coli competent cell Rosetta, selecting a clone on a plate, inoculating, expanding and culturing, and preserving bacteria. The resulting suspension was inoculated into 4mL of LB liquid medium containing 50. mu.g/mL of kanamycin, and cultured overnight at 37 ℃ under shaking at 270 rpm. Then, the cells were transferred to 200mL of LB liquid medium containing 50. mu.g/mL of kanamycin, and cultured at 37 ℃ and 270rpm for 8H with shaking, and then the cells were collected. The cells expressing the recombinant protein were subjected to ultrasonication. Centrifuging 8000g of the bacterial liquid for 10min, removing a supernatant culture medium, keeping the bacterial liquid, resuspending and washing the bacterial liquid by using a proper amount of PBS buffer solution, and adding a nickel column balance buffer solution into the bacterial liquid for ultrasonic crushing. And (4) carrying out ultrasonic crushing until bacterial liquid is clarified, centrifuging, and respectively collecting precipitate and supernatant. And (4) carrying out SDS-PAGE electrophoresis on the crushed supernatant, the precipitate and the thalli subjected to induced expression, and analyzing the existence position of the recombinant protein.
Purifying the recombinant protein by adopting a nickel column affinity chromatography. The main purification steps were carried out according to the instructions of Changzhou Tiandi and Biotech Co., Ltd. The purified target protein is dialyzed to remove salt, and is concentrated by an ultrafiltration tube. And finally, determining the protein concentration by an ultraviolet micro-spectrophotometer, and detecting the protein purity by SDS-PAGE electrophoresis. FIG. 1 is a polyacrylamide gel electrophoresis image of purified CD23 antigen.
EXAMPLE 29 establishment of the hybridoma cell line D11B6E6
Immunization
The CD23 protein obtained in example 1 was diluted to 1mg/mL, mixed and emulsified with an equal volume of complete Freund's adjuvant (CFA, Sigma Co.), and 18-20g of Balb/c mice (purchased from Wu's laboratory animal, Fozhou) were immunized by abdominal injection at a dose of 50. mu.g/mouse. Thereafter, the booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (IFA, Sigma Co.) at a dose of 25. mu.g/mouse. The polyclonal titer of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength 450nm) 14 days after the 2 nd boosting immunization, the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by PBS solution with the dosage of 50 mu g/mouse.
Second, cell fusion
Sterile preparation of mouse spleen cell suspension and mouse myeloma cellsp2/0(ATCC) was mixed at a ratio of 5:1, centrifuged at 1000rpm for 10min, the supernatant was discarded, and 1mL of PEG (Sigma) solution preheated to 37 ℃ was added slowly and quickly over 1 min, while gently rotating the tube to bring the cells into contact with PEG. Standing at room temperature for 90s, adding 4mL of serum-free DMEM (Hyclone company) culture medium preheated to 37 ℃ from slow to fast within 2min, then adding 10mL of preheated serum-free DMEM culture medium within 2min, finally adding the rest preheated serum-free DMEM culture medium within 2min, and fixing the volume to 50mL, wherein the centrifugal tube needs to be slowly shaken in the whole adding process to ensure uniform mixing and reduce the damage to cells. Standing at room temperature for 10min, centrifuging (1000rpm, 5min), discarding supernatant, resuspending cells in 10-20mLHAT (Sigma) medium, and diluting with HAT medium to final concentration of 0.5X 106cells/mL, all solutions were transferred to 96-well plates at 200. mu.L/well and labeled. The 96-well plates were carefully transferred to a 37 ℃ 5% CO2 incubator for incubation. The growth state and potential pollution of cells are regularly checked, and the incubator is opened and closed as little as possible to ensure the stability of the culture environment. On day 5 post-fusion, plates were supplemented with HAT medium at 50. mu.L/well.
Third, ELISA screening positive hybridoma cell
When the fused cell diameter is about 1-2mm, 50-200. mu.L of culture supernatant is aspirated for the first cell selection (ELISA, IHC-P and other methods of detection), and HAT medium is added to the culture wells to 200. mu.L. And (3) detecting the culture solution supernatant by ELISA, transferring all cell culture solution in the culture hole with the positive result obtained by detection to a 24-hole culture plate, supplementing HT medium, culturing for 3 days at a concentration of 2 mL/hole.
And repeatedly screening each cell strain in the 24-pore plate, and removing the culture well cells which are not positive results to obtain the culture well cells with better positive results. Performing subclone screening on the positive well cells obtained from the 24-well culture plate by limiting dilution method, namely adding cell sap obtained by limiting dilution method into 96-well culture plate, and transferring to CO2Culturing in an incubator for 11 days, and repeating cell screening when the diameter of the cloned cell is 1-2 mm. According to the detection result, 4 well-grown monoclonal positive culture wells are selected from each subcloned cell strain and transferred to 24 wellsThe plate was incubated. And after a period of time, screening the positive clone cell strain cloned in the 24-pore plate again, namely the hybridoma cell strain 9D11B6E6 secreting the specific monoclonal antibody. The cell strain is transferred into a T-75 culture flask to be amplified to a logarithmic growth phase for seed preservation or subsequent experiments are carried out.
EXAMPLE 3 preparation of monoclonal antibodies by in vitro culture
First, in vitro culture
After obtaining the stable hybridoma cell line, the monoclonal antibody is obtained mainly by an in vitro culture method.
The hybridoma cells were cultured in DMEM complete medium containing 10% fetal bovine serum, and then centrifuged at low speed to collect culture supernatant, which was stored at 4 ℃ for further use.
Secondly, purification of monoclonal antibody
Antibody purification using rProtein A sepharose Fast Flow (GE) affinity column: filling a proper amount of purchased ProteinA filler into a gravity chromatography column, and washing the gravity chromatography column with an equilibrium buffer solution (0.1M Tris solution, pH7.0) until the balance is achieved; secondly, loading the sample, adding the ascites filtered by the filter membrane of 0.24 mu m into the packed chromatographic column, and controlling the flow rate to be 1 drop/second; thirdly, balancing, and washing the sample solution to be balanced by using a balancing buffer solution after the sample solution is loaded; eluting, adding an elution buffer solution (0.1M citric acid solution, pH3.0) to wash the column and collecting the eluent; fifthly, regenerating, adding an equilibrium buffer solution to wash the column to be balanced after the elution is finished, washing the column with 2 times of column volume of 20 percent ethanol, and storing the column at 4 ℃. And finally, identifying the purity of the antibody (such as a polyacrylamide gel electrophoresis chart of the CD23 monoclonal antibody after purification in figure 2) by adopting an SDS-PAGE method, wherein the purity is over 95 percent, and determining the concentration of the antibody by adopting an ultraviolet micro-spectrophotometer method, wherein the concentration is over 3.0 mg/mL.
EXAMPLE 4 characterization of monoclonal antibodies
Identification of one, two subtypes
The cell supernatants were diluted to 1. mu.g/mL coated ELISA plates, 100. mu.L per well, coated overnight at 4 ℃, the solution was decanted, the plates were washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.L of blocking solution (PBS-T solution containing 2% BSA) was added per well, and incubated for 1h at 37 ℃. The liquid was decanted and washed 3 times with PBS-T. Adding 0.1mL hybridoma cell strain diluted by 5 times into each well for culturingThe culture supernatant was incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Using a confining liquid 1: HRP-labeled goat anti-mouse (kappa, lambda, IgM, IgG1, IgG2a, IgG2b, IgG) diluted 4003IgA) antibody (Southern Biotech) was added in an amount of 0.1mL per well and incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. 100 mu of LTMB (Hiroshi Biotech, Inc., Huzhou) substrate (A, B mixed solution with equal volume) is added into each well for color development, the reaction is carried out for 15min at room temperature, 50 mu L of 1N HCl solution is added into each well to stop the color development reaction, and then the OD value at the wavelength of 450nm is measured by a microplate reader. The results show that the monoclonal antibody of the present invention is a murine monoclonal antibody of the IgG1 type.
Second, determination of affinity constant
The CD23 protein was coated at a concentration of 100. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 1h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was diluted to the following concentrations (unit: ng/mL): 2000. 500, 125, 62.5, 31.25, 15.625, 3.125, 0.625, incubation at 37 ℃ for 1h, PBS-T wash 3 times. HRP-labeled goat anti-mouse secondary antibody 1: diluted at 5000, 100. mu.l/well, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. Mu.l of TMB (Hiroshi Biotech, Inc., England, Huzhou) color developing solution was added to each well, and the reaction was stopped by adding 100. mu.l of 1.0N salt solution for color development for 13 min. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. The OD values were plotted against the antibody dilution factor to find 1/2 the antibody concentration a corresponding to the "plateau OD value". The affinity constant was calculated to be 6.15X 10 using the following formula9L×mol-1。
Example 6 tissue chip staining and characterization
First, tissue wax block preparation process
HE section staining was performed on the sample tissue to determine the tumor lesion site. And (5) circling the lesion site and preparing to punch. When the receptor wax block is manufactured, a plastic frame is placed on a mold, melted paraffin (the melting point is 56-58 ℃) is poured into the mold, the tissue block is placed into wax liquid in the mold, then a proper amount of wax liquid is added to enable the tissue block to be completely embedded in the wax liquid, the mold is placed into a refrigerator with the temperature of-20 ℃ for 6min after being cooled to the room temperature, the wax block is taken out of the mold, and the section is sliced or placed into the refrigerator with the temperature of 4 ℃ for storage. After trimming, continuous slicing is carried out, the thickness is determined to be 4 mu m, the continuous slicing is rinsed in 40% alcohol, the slices are naturally unfolded, then the separated slices are transferred to warm water at 45 ℃ for 30 seconds, a glass slide treated by 2% APES acetone solution is used for mounting the slices, the prepared tissue chip is placed into an oven at 60 ℃ for baking for 2 hours, the slices are taken out for cooling at room temperature, and the tissue chip is placed into a refrigerator at-4 ℃ for storage.
Second, IHC staining and analysis
Conventional xylene dewaxing was performed 3 times for 6 minutes each time with 100%, 95%, 85% gradient ethanol hydration for 3 minutes each time and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3min with PBS. Dropwise adding 3% H2O2Incubate for 10min and wash with PBS for 3 × 3 min. The PBS was spun off and the peroxidase blocker was added dropwise and incubated for 10 minutes at room temperature. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 20 seconds, PBS turns blue. Dehydration was carried out in a gradient of 85% (3 min), 95% (3 min), 100% (3 min) and 100% (3 min) in order, and finally two times xylene was cleared for 10min, followed by sealing with neutral gum.
The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:
1. the sample is weakly positive; marked "+";
2. the sample is moderately positive; marked "+";
3. the sample is highly positive; marked "+ + + +";
4. the sample was negative and marked "-".
Thirdly, sample detection results:
the results of simultaneous detection using the anti-CD 23 protein monoclonal antibody (9D11B6E6) and the control antibody, commercially available anti-CD 23(SP23) rabbit monoclonal antibody, on 49 cases of B cell lymphoma are shown in the following table.
The result shows that the staining of the anti-CD 23 protein monoclonal antibody (9D11B6E6) is accurate in location, clear in staining, free of non-specific staining and clean in background, and the anti-CD 23 protein monoclonal antibody (9D11B6E6) is strong in specificity. In 49 cases of B cell lymphoma, the number of positive cells and the positive intensity using the monoclonal antibody against CD23 protein (9D11B6E6) were higher than those using the control reagent, indicating that the monoclonal antibody against CD23 protein (9D11B6E6) had higher sensitivity and affinity than the commercial antibody.
And the monoclonal antibody (9D11B6E6) of the anti-CD 23 protein and a control antibody, namely a commercial rabbit polyclonal antibody are used for synchronous detection on a normal tissue chip, and the positive and negative results of a sample are consistent, which indicates that the specificity of the antibody in normal tissues is equivalent to that of the commercial antibody.
FIG. 3 is a graph of the immunohistochemical staining results for B cell lymphoma (left (9D11B6E6) for CD23 and right for commercially available CD23(SP23) rabbit mab). Among them, the number of positive cells and positive intensity of the staining of 9D11B6E6 with CD23 were significantly higher than that of the staining of commercial CD23(SP23) rabbit monoclonal antibody, indicating that the sensitivity was higher.
Sequence listing
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