CN110531077B - Mesothelin immunohistochemical detection kit - Google Patents
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Abstract
The application relates to an Mesothelin (MSLN) immunohistochemical detection kit, which contains MSLN monoclonal antibody 3-2G6.
Description
Technical Field
The invention relates to an immunohistochemical kit for detecting Mesothelin (MSLN).
Background
Mesothelin (MSLN) is a cell surface glycoprotein. The mesothelin gene encodes a 71KD proprotein which is modified to be cleaved into two segments, 41KD glycosylated phosphatidylinositol cell membrane anchored protein and 30KD free fragment called megakaryocyte stimulating factor, respectively. There are studies demonstrating that the 41KD membrane anchoring protein comprises a binding domain to another tumor marker, CA125 protein, which may be associated with cell adhesion, and thus the anchoring protein may contribute to tumor metastasis and lead to an undesirable prognostic effect. Recent studies have shown that: MSLN is highly expressed in almost all mesothelioma and pancreatic cancers, most ovarian and lung cancers, rarely in normal tissues, and generally only expressed in some amounts in the pleural mesothelial cell lining, peritoneum and pericardium. Although the function of Mesothelin is not exact at present, both the cell membrane binding region and the free region are proved to be capable of being used as markers and therapeutic targets of various tumors such as mesothelial cancer, pancreatic cancer, ovarian cancer and the like.
The main principle of the immunohistochemical method is as follows: the basic principle of immunology, namely the principle of specific binding of antigen and antibody, is used to determine the antigen (polypeptide and protein) in tissue cell by developing the color developing agent (fluorescein, enzyme, metal ion and isotope) of labeled antibody through chemical reaction, and the research modes of locating, qualitative and relative quantification are carried out.
The main characteristics of the immunohistochemical technique are: 1. the specificity is strong: the basic principles of immunology determine that the binding between antigen and antibody is highly specific; 2. the sensitivity is high: the antibody can still be combined with antigen in tissue cells after being diluted by thousands times, tens of thousands times or even hundreds of millions times, so that the high-sensitivity antibody-antigen reaction can lead the immunohistochemical method to be more and more conveniently applied to the conventional pathological diagnosis work; 3. the positioning is accurate: the technology can accurately position the antigen in tissues and cells through antigen-antibody reaction and color reaction, so that different antigens can be positioned and observed in the same tissue or cell at the same time, the research of combining morphology and function can be performed, and the technology has important significance for deep pathological research. In the research and clinical use process of the targeted drug, the immunohistochemical method is adopted to locate and relatively quantitatively detect the target antigen in the pathological tissue, so that whether a patient is likely to benefit from the treatment of the targeted drug can be predicted, the success rate of the test can be improved in the clinical test process of the targeted drug, and the drug administration accuracy can be improved in the clinical use process of the targeted drug. Currently, there are a number of companion diagnostic kits based on the principle of immunohistochemistry, such as: VENTANA ALK IHC the assay kit is an in vitro diagnostic IHC assay kit approved by the FDA for identifying patients suitable for targeted drug herceptin treatment, marketed in the main in 2014 with a broad interest. However, there is no immunohistochemical antibody reagent and related detection method capable of rapidly and effectively detecting the expression of mesothelin protein, which can meet the market demand.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an immunohistochemical antibody reagent for positioning and semi-quantitatively detecting MSLN in tissues such as human pancreatic cancer, ovarian cancer, mesothelioma and the like which are embedded by formalin fixed paraffin, and the reagent can provide guidance for targeting drug administration of mesothelin for tumor treatment.
Because of the change of MSLN antigen structure in formalin-fixed paraffin-embedded tissues, the commercially available anti-MSLN antibodies are difficult to effectively bind and realize detection, and the invention provides a hybridoma cell strain capable of specifically recognizing the monoclonal antibody of the denatured MSLN antigen aiming at the phenomenon. The hybridoma cell strain is preserved in China center for type culture collection (CCTCC for short; address: wuchang Lojia mountain of Wuhan, china) in 12 months and 20 days, and is classified and named as hybridoma cell 3-2G6, and the preservation number is CCTCC C201811.
In one aspect, the present invention provides an immunohistochemical antibody composition for detecting MSLN, comprising anti-MSLN monoclonal antibody 3-2G6, wherein the antibody is produced by hybridoma cells deposited with China Center for Type Culture Collection (CCTCC) C201811, 12 months 20, 2017, with a deposit number.
In some embodiments, the antibody composition of the invention is obtained by mixing an antibody of the invention with an antibody diluent comprising Phosphate (PB) buffer at PH 7.2, preferably at a concentration of 0.005-0.5M, more preferably 0.01-0.1M.
In some embodiments, the antibody diluent further comprises: 0.5% -5% of protein protectant, 0.05% -0.5% of surfactant and 0.01% -0.2% of preservative; preferably, 1% -3% protein protectant, 0.05% -0.2% surfactant, and 0.02% -0.1% preservative; more preferably, 1% protein protectant, 0.05% surfactant, and 0.05% preservative.
In some embodiments, the protein protectant comprises bovine serum albumin, the surfactant comprises tween 20, and the preservative comprises 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one.
In some embodiments, the antibody diluent further comprises 0.05-0.5M NaCl, preferably 0.1-0.3M NaCl, more preferably 0.15M NaCl.
In some embodiments, the antibody diluent comprises: 0.05M phosphate buffer, 0.15M NaCl, 1% bovine serum albumin, 0.05% Tween 20 and 0.05% ProClin300.
In some embodiments, the anti-MSLN monoclonal antibody is present in the form of an antibody solution, wherein the antibody concentration is 0.05-20 μg/ml, more preferably 0.2-10 μg/ml, most preferably 0.3-5 μg/ml.
In some embodiments, the volume ratio of the antibody solution to the antibody diluent is from 1:200 to 1:18000, preferably from 1:1600 to 1:6400, more preferably 1:3200.
In another aspect, the present application relates to the use of an immunohistochemical antibody composition of the invention in the preparation of a kit for the detection of mesothelin.
In another aspect, the present application relates to an immunohistochemical kit for detection of MSLN comprising an immunohistochemical antibody composition as described herein.
In some embodiments, the immunohistochemical kit of the present invention further comprises: antigen retrieval liquid, immunohistochemical pen and cytoplasmatic control sheet.
In some embodiments, the antigen retrieval solution in the immunohistochemical kit of the present invention comprises 0.01M citric acid or salt thereof at a pH of 6.0.
In some embodiments, the immunohistochemical kit of the present invention comprises a cytoplasmatic control sheet, wherein the cytoplasmatic control sheet comprises 4 cells as controls, which are: 293 cells expressing mesothelin negatively, CFPAC-1 cells expressing mesothelin weakly positively, OVCAR-3 cells expressing mesothelin positively, OVCAR-3-MSLN cells expressing mesothelin Pi Sujiang positively, wherein the OVCAR-3-MSLN cells are OVCAR-3 cells genetically modified to express exogenous MSLN.
In some embodiments, the protein protectant is preferably BSA (i.e., is bovine serum albumin).
In some embodiments, the surfactant is preferably Tween-20.
In some embodiments, the preservative is preferably ProClin300. The ProClin300 comprises: 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one.
In another aspect, the invention provides a method for the production of anti-MSLN monoclonal antibodies from hybridoma cells 3-2G6 for the detection of non-diagnostic targets of MSLN in formalin-fixed paraffin-embedded tissue, characterized by:
1. fixing a tumor sample in formalin solution, then dehydrating the tumor sample by gradient ethanol, placing the dehydrated tissue in xylene for transparency, immersing the tissue in melted low-melting-point paraffin, embedding the tissue in paraffin, cutting the tissue into slices with the thickness of 3-5 mu m after the paraffin blocks are solidified, taking out the tissue by using a slide coated with polylysine, and drying the tissue by using a bleaching and drying instrument;
2. roasting the tissue slice at 65 ℃ for 1 hour, dewaxing in xylene, and then hydrating with gradient ethanol;
3. adding antigen retrieval liquid into an autoclave, opening the cover of the autoclave, heating the autoclave on an electromagnetic oven with strong fire until the antigen retrieval liquid boils, stopping the fire, immersing the slices into the retrieval liquid, covering a pot cover, fastening a pressure valve, heating to jet, starting from jet, adjusting to medium fire for 2.5 minutes, stopping the fire, and pulling out a power supply; transferring the pressure cooker onto an experiment table, opening a cooker cover, taking out a slide after the antigen retrieval liquid is naturally cooled to room temperature, and soaking by PBST; the PBST is as follows: phosphate tween buffer;
4. Removing liquid around the tissues on the slices, circling around the tissues by an immunohistochemical oil pen, adding freshly prepared 3% peroxidase blocking solution in the circling area, incubating for 10 minutes at 37 ℃, and washing the slices for 2 times by PBST; adding 3-2G6 antibody solution, setting negative control, blank control and positive control, incubating at 37 ℃ for 60 minutes, and washing the slices by PBST for 2 times;
5. dripping 1-2 drops of detection reagent I to cover the tissue on the slice, incubating for 10 minutes at 37 ℃, immersing and washing for 2 times by using PBST, carefully throwing away liquid around the tissue on the slice, dripping 1-2 drops of detection reagent II to cover the tissue on the slice, incubating for 10 minutes at 37 ℃, and immersing and washing for 2 times by using PBST; the detection reagent I, II is respectively a reaction amplification agent and a high-sensitivity enzyme-labeled anti-mouse/rabbit IgG polymer in a polymer amplification reaction secondary antibody system DAB Detection Kit (Fuzhou maixin, product number: KIT-0016); then using freshly prepared DAB color developing agent to develop color for 1 minute, and terminating the PBST soaking to develop color; mayer's hematoxylin is used for dying the nuclear for 1 minute, excess hematoxylin is washed by water, and then the nuclear is dehydrated, transparent and sealed, and the dying result is read and photographed by an optical microscope.
In some embodiments, the antigen retrieval conditions of the specimen to be tested may be: the sample to be tested can be repaired at high pressure by using a citric acid buffer solution with pH of 6.0, the repair time is 2.5 minutes, and the buffer solution can also be used for thermal repair, wherein the repair condition is 95-97 ℃ for 20 minutes.
In some embodiments, the specimen should be fixed in formalin solution within 30 minutes of ex vivo.
In some embodiments, the antigen retrieval liquid is: citrate buffer at 0.01m pH 6.0, repair conditions were: and repairing under high pressure by adopting the repairing liquid.
In some embodiments, the fire conditions are: 160 w 210 ℃; the medium fire condition is as follows: 800w 130 ℃.
In some embodiments, the 3-2G6 antibody solution described herein is a ready-to-use 3-2G6 antibody solution, preferably at an antibody concentration of 0.05-2. Mu.g/mL, more preferably 0.2-1. Mu.g/mL, most preferably 0.31. Mu.g/mL; the negative control antibody is a mouse monoclonal antibody IgG1 isotype control (CST, catalogNo.5415), clone number G3A1; the positive control sample and the negative control sample can be prepared from 4-in-1 cell quality control sheets, wherein the quality control sheets are prepared by preparing paraffin blocks from 4 cells with different mesothelin expression levels, and re-preparing the paraffin blocks into a new paraffin block according to the requirements of a tissue chip, and slicing the paraffin blocks; wherein the MSLN negative expressed cell line is 293 (0); the MSLN weak positive expression cell is CFPAC-1 (1); MSLN positive expressing cells OVCAR-3 (2); cells expressing strong positive for MSLN OVCAR-3-MSLN (3).
The immunohistochemical antibody reagent for detecting MSLN is used for detecting the expression condition of MSLN in a tissue sample to be detected (tumor tissue subjected to operation or puncture is embedded by formalin fixed paraffin and then cut into slices with the thickness of 3-5 mu m), and the detection result can provide guidance for tumor treatment MSLN targeted medication.
On the other hand, the further detection result is that the expression value of MSLN is obtained by half-quantitatively scoring the tumor cells in the examined tissue sample by a pathologist through immunohistochemical staining conditions and a cytoplasmatic control wafer by an integer of 0-3. Staining intensity was used as a scoring criterion prior to co-clinical with the drug. Wherein 0 is represented by: tumor cells did not appear any form of staining; 1 score: cell membrane staining of tumor cells occurs, but staining intensity < CFPAC-1 cells; the score of 2 indicates: the tumor cells are stained by cell membranes, and the staining intensity is higher than CFPAC-1 and lower than OVCAR-3-MSLN;3 points represent: the cell membrane of the tumor cells is dyed, and the dyeing intensity is more than or equal to OVCAR-3-MSLN. It is clear that the living tumor cells involved in scoring are living and scoring is based on MSLN localized to the cell membrane (i.e., only cells stained with cytoplasm alone are not included in the count range).
The above scoring criteria mention that the cytoplasmic control panel is prepared from cells of known MSLN expression levels, including 4 cell clusters: wherein 293 cells are MSLN negative cells, CFPAC-1 cells are MSLN weak positive expression cells, OVCAR-3 cells are MSLN positive expression cells, and all three cells can be purchased through commercial paths; OVCAR-3-MSLN is a strongly positive MSLN expressing cell, and its preparation method is as follows:
1. amplification of the target Gene: designing a primer to obtain the full length of the MSLN of the amplified target gene from the target gene vector;
2. and (3) enzyme cutting of the target gene and the expression plasmid: the amplified target gene MSLN is subjected to gel cutting recovery by using a gel recovery kit, and is subjected to enzyme cutting by using restriction enzymes, and simultaneously, an expression vector pHBLV-CMV-ZsGreen-puro is subjected to enzyme cutting by using the same kind of restriction enzymes;
3. construction of the target gene expression plasmid: connecting the target gene fragment and an expression vector; converting the connection product into competent cells, amplifying and coating the plates; picking single bacterial colony to perform bacterial colony PCR; selecting a colony with correct amplified products for sequencing; sequencing the correct recombinant plasmid to obtain the constructed expression plasmid;
4. packaging target gene lentivirus: inoculation of 1X10 7 The 293T cells were cultured overnight in a T75 flask with replacement of serum-free medium, and the expression plasmid, packaging plasmid and envelope plasmid were mixed and then with Lipofectamine2000 (Lipofectamine) TM 2000 Transfection Reagent product number: 11668027 Mixing, adding into 293T cells, culturing for 48 hours, collecting supernatant, concentrating, and packaging;
5. infection of target cells: taking 5 cm 10 of cells in logarithmic growth phase 4 Adding 0.5 mL/well of each of the above culture medium into a 24-well plate, and culturing overnight; sucking the original culture medium of the cells, adding a virus diluent and a diluted polybrene solution, and lightly mixing the mixture until the total infection volume is 250 mu L; after 24 hours of infection, removing the culture solution containing the virus, and replacing the culture solution with a fresh complete culture medium for continuous culture; for carrying puromycin resistance geneThe virus of (2) is replaced by a fresh complete culture medium containing puromycin to obtain a stably transfected cell strain;
6. obtaining target cells: and (3) obtaining single clone from the obtained steady transgenic cell strain of the over-expressed MSLN target gene by adopting a limiting dilution method, and selecting a cell strain with the MSLN expression intensity consistent with the MSLN high expression intensity in pathological tissues according to the MSLN expression intensity in the pathological tissues, thereby obtaining the OVCAR-MSLN as the MSLN strong positive expression cell.
Finally, the present application also relates to the use of the relevant immunohistochemical antibody reagent or immunohistochemical antibody kit for the preparation of a tumor detection reagent.
The invention has the following technical effects: the instant antibody used in the present application can achieve the following effects by adding components such as a protective protein and a preservative to an antibody buffer: reducing non-specific binding of antibodies to tissue, so that the BSA serum blocking step and the like can be omitted; the antibody is ensured to be stable for a long time under lower concentration, which is beneficial to improving the detection efficiency and sensitivity.
The detection kit or the detection reagent can be used for rapidly and accurately detecting the cell surface mesothelin protein, and is higher than the detection sensitivity and accuracy of the similar kit on the market at present. The application designs and obtains the monoclonal antibody with strong specificity, which can be suitable for immunohistochemical experiments, aiming at the characteristics of the immunohistochemical detection technology, and overcomes the defect that the commercial anti-MSLN antibody is difficult to effectively combine and realize detection because the MSLN antigen structure in the formalin-fixed paraffin-embedded tissue is changed.
The anti-MSLN monoclonal antibody reagent obtained by the method can ensure the reactivity and the validity period of the anti-MSLN monoclonal antibody, and the anti-MSLN monoclonal antibody reagent can be directly used by a user without any operation after obtaining the related product of the method, so that the detection convenience is improved.
Drawings
FIG. 1 shows the results of purity detection profiles of anti-MSLN monoclonal antibodies; through gray scale scanning analysis, the area percentage of the anti-MSLN monoclonal antibody strip is 95%, and the purity meets the requirement.
FIG. 2A shows a comparison of staining effects under different antigen retrieval conditions; wherein (1) and (2) are two different ovarian cancer pathological tissues respectively; 1-3 represent antigen retrieval conditions 1-3, respectively; panel B shows the results of staining OVCAR-3 cell paraffin sections with different dilution ratios of 3-2G6 antibody, wherein 1-6 is 1mg/mL of 3-2G6 antibody: the staining intensity of the OVCAR-3 paraffin sections corresponds to the medium MSLN staining intensity of tumor cells in tissues under the same experimental conditions when the PBST ratio is 1:25, 1:100, 1:400, 1:1600, 1:6400 and 1:25600 respectively; panel C shows the staining results for the primary antibody incubation times of 30 minutes (1) and 60 minutes (2).
FIG. 3A shows a comparison of staining effects in Tris buffer and PB buffer, immunohistochemical staining with 3-2G6 antibody diluted with "pH 7.4.05M TRIS buffer" and "pH 7.4.05M PB buffer", respectively, the images enumerating staining for 4 fields, respectively; panel B shows the results of staining with 5 ion concentration buffers. The total Na ion concentration in the 1-buffer solution is 0.020M, the total Na ion concentration in the 2-buffer solution is 0.095M, the total Na ion concentration in the 3-buffer solution is 0.165M, the total Na ion concentration in the 4-buffer solution is 0.184M, and the total Na ion concentration in the 5-buffer solution is 0.219M; panel C shows the staining results of different degrees of MSLN expressing cells in tumor tissue with the instant 3-2G6 antibody preparation; wherein 1-4 are respectively the staining results of the ready-to-use 3-2G6 antibody preparation prepared by the preferred buffer solution on tumor cells with negative, weak positive, positive and strong positive expression of MSLN in tumor tissues; panel D shows the results of potency detection of 3-2G6 antibody formulations after 7 days, 15 days and 30 days of thermal disruption at 45 ℃; it can be seen that the 4-parameter curve is slightly reduced, but still meets the requirements of immunohistochemical detection stability.
FIG. 4 shows immunohistochemical staining patterns for pathological sections of different kinds of tumor cells.
Detailed Description
Example 1: preparation of anti-MSLN monoclonal antibodies
Establishment of hybridoma cell lines
1. Preparation of experimental materials:
a fragment of MSLN-ECD (cat# FCL 1589) protein from G & P Biosciences, which contains 317 amino acids, was used as an immunogen and was located in the region Glu296-Ser592 of the MSLN (UniProt accession #Q13421, isocord 2) sequence; the culture medium used includes but is not limited to HAT, HT selection medium, DEME medium; experimental animals included Balb/c mice; the adjuvant is emulsified Freund complete adjuvant and emulsified Freund incomplete adjuvant.
2. Immunization of animals
Basic immunization: the antigen and Freund's complete adjuvant were fully emulsified in equal volumes and injected subcutaneously in divided doses of 50. Mu.g/mouse, 2 Balb/c mice were immunized.
Boosting: emulsifying the antigen with Freund's incomplete adjuvant; the physiological saline solution containing 50. Mu.g of antigen was directly injected intraperitoneally 3 days before cell fusion was performed.
Tail blood detection: tail blood of immunized mice is taken for detection by an indirect ELISA method.
3. Preparation of hybridoma cells
Myeloma cells are fused with spleen cells of 1 mouse, and 10 96-well cell culture plates are paved on the fused cells; the culture was selected using HAT medium for 1 week, after which the HT medium was replaced for continued culture. Screening positive cell clones by using an indirect ELISA method, performing expansion culture on the selected positive cells, and performing subcloning for 1 time by using a limiting dilution method to obtain 20 monoclonal hybridoma cell strains subjected to subcloning operation for 1 time.
4. Selection and establishment of hybridoma cell lines
The 20 strain 1 subclone cell supernatants were collected, and antibody production was measured and analyzed using denaturing Western-blot, indirect ELISA, immunohistochemistry (antigen retrieval mode is citrate thermal retrieval), and antibody detection performance, respectively. As a result, the antibody secreted by the clone No. 3-2G6 cell line was able to recognize denatured MSLN and to perform differential staining of cells with different amounts of MSLN expression. The selected 3-2G6 cell line was subcloned 2 nd time by limiting dilution to ensure that a monoclonal hybridoma cell line was obtained. Antibodies generated by the 3-2G6 cell strain after 2 subcloning are subjected to denaturation Western-blot, indirect ELISA and immunohistochemical tests, and the detection performance of the antibodies is analyzed and verified. And amplifying and freezing the 2 subclone 3-2G6 cell strain which is qualified by the antibody to obtain a final hybridoma cell strain.
Preparation of (second) anti-MSLN monoclonal antibody
1. Preparation of ascites in mice
Female BALB/c mice of 10 weeks of age were selected, adaptively bred for 2 days, and then each mouse was intraperitoneally injected with 0.5mL of liquid paraffin. After 7 days, mice were inoculated with 1x10 cells 6 The inoculation volume was 300 ul/ml per cell, and the injection was performed intraperitoneally. Observing the growth state and the abdominal size of the mice, taking the ascites of the mice when the abdominal of the mice obviously swells (generally within 7-10 days), using a 12-gauge needle to enter the abdominal cavity of the mice along the position near the groin, so that the ascites naturally flows out, and collecting the ascites by a sterile centrifuge tube.
2. Purification of ascites in mice
The ascites obtained in the above step was centrifuged at a high speed (12000 rpm,20 min) at 4℃and the supernatant was suction-filtered through a microporous membrane of 0.45 μm, followed by purification by a ProteinG SepharoseTM FF affinity column having a volume of 2 mL. The equilibration buffer was 20mM phosphate buffer, pH7.0, the elution buffer was 0.1M glycine, pH3.4, and the stop buffer was 1M Tris-HCl, pH9.0. The buffer in the anti-MSLN monoclonal antibody was replaced with PBS, ph7.2, containing 0.05% proclin300 using ultrafiltration. Ensure that the concentration of IgG is more than or equal to 1mg/ml, and the single band is detected by the modified non-reducing SDS-PAGE. The purity detection profile of the anti-MSLN monoclonal antibody is shown in fig. 1, from which it can be seen that: the area percentage of the anti-MSLN monoclonal antibody strip is 95%, and the purity meets the requirements.
Example 2: anti-MSLN monoclonal antibody 3-2G6 experimental condition optimization
(1) Antigen retrieval conditions were optimized by evaluating different antigen retrieval buffers. Repair condition 1 (Cit 6.0) refers to high pressure repair using acidic ph6.0 citrate buffer for 2.5min. Repair condition 2 (Tris 9.0) refers to high pressure repair using alkaline Tris-EDTA buffer pH9.0 for 2.5min. Repair condition 3 (Tryp) refers to repair with 0.25% pancreatin repair solution at 37℃for 10min. As shown in fig. 2A: in these experimental conditions, the staining effect after Cit 6.0 high pressure repair is superior to that of several other repair modes, and is characterized by clear staining of target cells (tumor cells) and no nonspecific staining of mesenchymal cells. Thus, the method is ultimately selected as an antigen retrieval method for which the present antibodies are suitable.
(2) The staining intensity was evaluated for different concentrations of 3-2G6 to screen for appropriate antibody concentrations. The antibodies were subjected to a series of dilutions ranging from 25-fold to 25600-fold, calculated as an initial concentration of 3-2G6 of 1mg/ml, and immunohistochemical staining was performed on 2+ expressing cells of the target antigen (OVCAR-3) paraffin sections with diluted antibodies. According to fig. 2B: positive staining results were evident for staining results with 1600-fold and 6400-fold antibody dilution concentrations, with relatively little non-specific background reaction, we finally selected 3200-fold antibody dilution for validation experiments.
(3) Different primary antibody incubation times were tested to screen for appropriate antibody incubation conditions. The pathological tissue with weak expression of the target protein is selected for immunohistochemical staining test with primary antibody incubated at 37 ℃ for 30min and at 37 ℃ for 60min, according to the illustration in fig. 2C: the staining results were similar for both incubation times with no significant difference, but to ensure that the incubation time was sufficient to avoid false negative results, experimental conditions were finally selected for incubation for 60min at 37 ℃.
Example 3: selection of antibody dilutions
A series of evaluations of the individual components of the 3-2G6 antibody dilutions were performed, including matrix, salt ion concentration, pH, protection protein concentration, surfactants, preservatives, etc.
In terms of matrix, immunohistochemical experiments were performed using TRIS buffer at pH 7.4,0.05M and PB (Phosphate Buffer) as buffer to dilute the 3-2G6 antibody, respectively, according to the results shown in fig. 3A: the use of Tris buffer makes non-specific background staining very evident;
in terms of salt ion concentration, it was found that when the nonspecific staining disappeared with increasing ion concentration and the ion concentration increased to 0.15M or more, the specific staining intensity was also decreased by adding 0.015 to 1.2M NaCl to 0.05M TRIS and PB buffer, respectively. Under otherwise identical conditions, too high a salt ion concentration may result in a decrease in tissue staining intensity. Therefore, we need to consider the clarity of target tissue staining and minimize non-specific background staining when selecting the antibody buffer salt ion concentration. The results according to fig. 3B show: the overall staining effect was optimal at 0.15M NaCl concentration. Therefore, we prefer a salt concentration of 0.15M NaCl.
In terms of pH, the effect of 0.05M TRIS (containing 0.15M NaCl) and PB (containing 0.15M NaCl) buffers at pH 6.0, 7.3 and 8.6 on the immunohistochemical staining results was compared, and as a result, the effect of different pH on the staining intensity of the 3-2G6 antibody was not significant.
In the aspect of protein protection, the influence of casein, bovine serum albumin and goat serum on the dyeing performance and stability of the 3-2G6 antibody is compared, and the result shows that the influence of the above components on the immunohistochemical dyeing result is small, and the accelerated stability experiment result shows that the immunohistochemical dyeing performance of the 3-2G6 cannot be changed after the thermal damage at 45 ℃ for 15 days when the addition amount of the protein protective agent is 1% -2%.
In the aspect of the surfactant, two surfactants Tween-20 and Brij-35 (namely, polyoxyethylene lauryl ether) are compared, and the result shows that the two surfactants have no difference on the dyeing performance of 3-2G 6.
In terms of preservative, an accelerated stability test at 45 ℃ for 15 days shows that when the addition amount of ProClin300 (Sigma) is 0.05% -0.1%, the immunohistochemical staining performance of the 3-2G6 product is good.
In summary, we prefer 0.05M PB, pH7.20.15M NaCl, 1% -2% BSA, 0.05% -0.1% Tween-20 and 0.05% -0.1% ProClin300 as an antibody dilution with 3-2G6, the volume ratio of the dilution to 3-2G6 antibody being 3200:1. Mixing the antibody with the diluent to obtain an antibody solution, wherein the diluted antibody solution is the instant 3-2G6 antibody solution.
The staining performance profile of the preferred buffer is shown in fig. 3C, and the experimental results indicate that: the 3-2G6 antibody can specifically bind to the mesothelin protein and can effectively reflect the expression condition of the mesothelin protein; the stability profile is shown in fig. 3D, which shows that: the 3-2G6 antibody preparation has good thermal stability and can meet the requirement of immunohistochemistry on stability.
Example 4: obtaining pathological sections for reading data by using immunohistochemical method
Firstly preparing tumor specimens (such as pancreatic cancer, ovarian cancer and malignant mesothelioma) with MSLN over-expression reported in the prior literature, fixing the specimens in formalin solution within 30 minutes after the specimens are isolated, then dehydrating the specimens by gradient ethanol, placing the dehydrated tissues in xylene to be transparent, immersing the tissues in melted low-melting-point paraffin, embedding the tissues in paraffin, cutting the tissues into slices with the thickness of 3-5 mu m after the paraffin blocks are solidified, taking out the tissues by using a slide coated with polylysine, and drying the tissues by using a bleaching and baking instrument.
Immunohistochemical experiment steps: the tissue slices were baked at 65 ℃ for 1 hour, then subjected to dewaxing treatment in xylene for 10 minutes, then subjected to gradient ethanol hydration, specifically operated such that the dewaxed slices were immersed in absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 5 minutes, and finally immersed in distilled water.
Adding antigen repairing liquid (0.01M citrate buffer solution with pH of 6.0) into an autoclave, opening the cover of the autoclave, heating the autoclave to boil the antigen repairing liquid, stopping fire, immersing the slice into the repairing liquid, covering the cover, fastening a pressure valve, heating the slice to jet air with big fire (160W, 210 ℃) and starting from jet air, adjusting to medium fire (800W, 130 ℃) for 2.5 minutes, stopping fire, and pulling out a power supply; the pressure cooker is carefully and balanced transferred onto a laboratory bench until the automatic lock cylinder falls to the original position, no steam or a small amount of steam is discharged from the exhaust pipe, the pressure valve is opened, the cooker cover handle is rotated anticlockwise, the cooker cover is opened, and the glass slide is taken out after the antigen retrieval liquid is naturally cooled to room temperature (at least 30 min) and soaked in PBST.
Carefully removing the liquid around the tissue on the slice, circling around the tissue with an immunohistochemical oil pen, adding freshly prepared 3% peroxidase blocking solution in the circled area, incubating at 37 ℃ for 10 minutes to eliminate peroxidase activity in the tissue, and washing the slice 2 times with PBST.
Add ready-to-use 3-2G6 antibody solution, incubate at 37℃for 60 min, and PBST wash sections 2 times. Each batch of experiments was run with a negative control, a blank control and a positive control. The ready-to-use 3-2G6 antibody solution was prepared from the antibody dilution described in example 3 at an antibody concentration of 0.31. Mu.g/mL. The negative antibody control was a mouse monoclonal antibody IgG1 isotype control (CST, catalog No. 5415), clone number G3A1. The positive control sample and the negative control sample can be prepared by using a 4-in-1 cell quality control sheet, wherein the quality control sheet is obtained by preparing paraffin blocks from 4 cells with different mesothelin expression levels, and re-preparing the paraffin blocks into a new paraffin block according to the requirements of a tissue chip, and slicing the paraffin blocks. Wherein the MSLN negative expressed cell line is 293 (0); the MSLN weak positive expression cell is CFPAC-1 (1); MSLN positive expressing cells OVCAR-3 (2); cells expressing strong positive for MSLN OVCAR-3-MSLN (3).
1-2 drops of detection reagent I were added dropwise to cover the tissue on the sections and incubated at 37℃for 10 minutes. The cells were rinsed 2 times with PBST. The tissue on the section was carefully removed by shaking, 1-2 drops of detection reagent II were added dropwise to cover the tissue on the section, incubated at 37℃for 10 minutes, and rinsed 2 times with PBST. The detection reagents I, II are a reaction amplifier of a polymer amplified reaction secondary antibody system DAB Detection Kit (available from Fuzhou Miss, cat# KIT-0016) and a highly sensitive enzyme-labeled anti-mouse/rabbit IgG polymer, respectively. The color was then developed using freshly prepared DAB color developer for 1 minute and PBST was stopped. Mayer's hematoxylin is used for dying the nuclear for 1 minute, excess hematoxylin is washed by water, and then the nuclear is dehydrated, transparent and sealed, and the dying result is read and photographed by an optical microscope. The staining patterns of different pathological sections are shown in fig. 4, and the results show that: the immunohistochemical reagent provided by the application can clearly characterize the expression condition of mesothelin protein aiming at pancreatic cancer, ovarian cancer and mesothelioma cells.
EXAMPLE 5 interpretation of MSLN expression values
The pathologist combined with the cytoplasmatic control slide scored a semi-quantitative whole number from 0 to 3 for immunohistochemical staining of tumor cells in the examined tissue specimens. Staining intensity was used as a scoring criterion prior to clinical study with MSLN targeted drug. Wherein 0 is represented by: tumor cells did not appear any form of staining; 1 score: cell membrane staining of tumor cells occurs, but the staining intensity is less than or equal to that of CFPAC-1 cells; the score of 2 indicates: the tumor cells are stained by cell membranes, and the staining intensity is higher than CFPAC-1 and lower than OVCAR-3-MSLN;3 points represent: the tumor cells are stained by cell membranes, and the staining intensity is more than or equal to OVCAR-3-MSLN. It is clear that the living tumor cells involved in scoring are living and that scoring is based on MSLNs localized to the cell membrane (i.e., only cells stained with cytoplasm alone are not included in the count range) the expression values of MSLNs in fig. 4 are interpreted according to the above criteria, as follows:
| organization name | Scoring results |
| |
2 |
| |
2 |
| |
3 |
EXAMPLE 6 preparation of cell Mass control sheet
6.1 Preparation of OVCAR-3-MSLN cells
Construction of the Gene expression vector of interest
Primers were designed to amplify full length MSLN from a vector containing the human MSLN coding sequence (pCMV 3, purchased from Equipped China), cut-gel recovery was performed using a gel recovery kit, and restriction enzyme was used to cut, while lentiviral expression vectors (pHBLV-U6-MCS-CMV-ZsGreen-PGKL-PURO, purchased from Hantao) were cut using the same restriction enzyme. The MSLN gene fragment and the expression vector are ligated, the ligation product is transformed into competent cells, and the plates are spread after amplification. And picking a single colony for colony PCR, and selecting a colony with correct amplified product for sequencing. And sequencing the correct recombinant plasmid to obtain the constructed expression vector.
Packaging of target gene lentiviruses
The 293T cells are inoculated in a T75 culture flask, the serum-free culture medium is replaced after overnight culture, the expression vector, the packaging vector and the envelope vector (pHBLV-U6-MCS-CMV-ZsGreen-PGKL-PURO, paPAX2, pMD.2G, purchased from Henry) are mixed and then mixed with Lipofectamine2000, 293T cells (human kidney epithelial cell line transfected with adenovirus E1A gene) are added, and the supernatant is collected after 48 hours of culture and concentrated and split charging.
Obtaining of cells of interest
Taking cells in logarithmic growth phase, adding the cells into a 24-well plate, and culturing overnight; sucking the original culture medium of the cells, adding a virus diluent and a diluted polybrene solution, and lightly mixing the mixture until the total infection volume is 250 mu L; after 24 hours of infection, the virus-containing medium was removed, replaced with fresh complete medium (Gibco RPMI1640-A10491-01 medium+20% FBS+0.01mg/ml insulin) and the culture continued; the virus carrying puromycin resistance gene is replaced by fresh complete culture medium containing puromycin, and a stably transfected cell strain is obtained.
And (3) obtaining a monoclonal by adopting a limited dilution method to the obtained steady transgenic cell strain of the over-expressed MSLN target gene, and selecting a cell strain with the MSLN expression intensity consistent with the MSLN high expression intensity in pathological tissues according to the MSLN expression intensity in the pathological tissues, thereby obtaining the OVCAR-3-MSLN as the MSLN strong positive expression cell.
6.2 Paraffin embedding
Experimental materials:
the MSLN four-in-one cytoplasmatic control sheet comprises four cell lines: 293 (
Number：CRL-1573 TM )、CFPAC-1(/>
Number：CRL-1918 TM )、OVCAR-3(/>
Number：HTB-161 TM ) OVCAR-3-MSLN (prepared according to the above method). Materials used in cell culture and embedding processes are commercially available.
The experimental process comprises the following steps:
(1) Cell culture
Cell name: 293 (dyeing intensity is 0);
culture medium: gibco RPMI1640 medium+10% FBS;
cell name: CFPAC-1 (staining intensity 1);
culture medium: gibco IMDM medium+10% FBS;
cell name: OVCAR-3 (staining intensity 2);
culture medium: gibco RPMI1640-A10491-01 medium+20% FBS+0.01mg/ml insulin;
cell name: OVCAR-3-MSLN (staining intensity 3);
culture medium: gibco RPMI1640-A10491-01 medium+20% FBS+0.01mg/ml insulin;
wherein the FBS is fetal bovine serum, and the Gibco RPMI1640 (ATCC modified) medium is the RPMI1640 medium with the product number of A1049101 of Gibco company in the United states (namely Gibco RPMI 1640-A10491-01); gibco IMDM medium is Gibco company, U.S. IMDM medium under the accession number 12440053; the Gibco RPMI1640 common medium is a Gibco company's medium with the product number of 11875135;
Culture conditions: performing stationary culture at 37 ℃ with 5% CO 2; the cells proliferated to 80-90% of the bottom of the dish for one passage 2-3 days.
(2) Cell collection and immobilization
When the cells are cultured until 80-90% of the bottom of the cell culture dish is fully paved, the cells are collected. Pouring out the culture medium, washing with 1-2ml PBS once, adding 1-2ml 0.25% pancreatin to digest for 3-5min, observing cell rounding under microscope, adding 1ml cell culture medium to stop digestion,the cells were collected and counted by pipetting, washing with 3-5ml PBS, centrifuging at 1000rpm for 5min, discarding the supernatant, resuspending the cells with 4% paraformaldehyde or 10% neutral formalin fixative, the addition of fixative being 2X 10 7 cells/1ml fixative, 16h-24h at room temperature.
(3) Agarose supporting cells
Agarose gel preparation: weighing agarose and PBS to prepare a solution with the concentration of 3-4%, covering tin foil paper, heating in a microwave oven until the agarose is completely dissolved, taking out and placing in a water bath with the temperature of 60 ℃ for heat preservation for later use.
Mixing cells with agarose: 1-2×10 7 cells were subjected to a fixation procedure, naturally settled to a volume of about 100. Mu.l, and the supernatant was carefully removed with a pipette. Preheating a centrifuge tube containing cells in a water bath at 60 ℃, transferring agarose gel with the same volume as the cells by using a preheated pipettor, adding the agarose gel into the centrifuge tube, uniformly mixing the agarose gel with the cells (the uniform mixing action needs to be gentle, the whole process is preferably controlled within 3min, and the influence of water evaporation is reduced), transferring the uniformly mixed agarose and cell mixture into a 1ml syringe needle tube with a needle removed, and cooling and forming at room temperature.
(4) Dehydration of cell mass
The cell mass mixed with agarose was removed, cut into small pieces of 0.5cm thickness, and placed in a labeled dewatering basket. Sequentially immersing the tissue blocks into ethanol with different concentration gradients to dehydrate the tissues: 70% ethanol overnight- & gt 80% ethanol 1.5 h- & gt 90% ethanol 1.5 h- & gt 95% ethanol 1.5 h- & gt 100% ethanol I50 min- & gt 100% ethanol II 50min.
(5) Cell mass is transparent
The cell pellet subjected to gradient ethanol dehydration is immersed into xylene I and xylene II in turn for transparency, and the transparency time is 20min each time.
(6) Cell mass wax dipping
Placing the paraffin blocks in a 65 ℃ oven in a paraffin dipping cup until paraffin is completely melted, immersing the tissue blocks with transparent tissues in melted paraffin for tissue paraffin dipping, replacing the paraffin after 1 time of paraffin dipping, and carrying out paraffin dipping for 1 time each time for 1 hour.
(7) Cell mass entrapment
The manual embedding process was performed in an oven at 60 ℃. Pouring melted paraffin into a tissue embedding bottom die, taking out the cell mass after the paraffin dipping from the corresponding dehydration basket, rapidly putting the cell mass into the paraffin of the tissue embedding bottom die, avoiding air bubbles as much as possible during putting, adjusting the position of the cells, covering an embedding frame with an up-down mark on the embedding box, transferring the embedding frame to a cooling table, and cooling for 30min to obtain the tissue embedding paraffin block. Before using the paraffin block, a small block of paraffin was cut vertically off from the outer edge of the 293 cell site and used as a slice orientation marker.
6.3 method for producing slices
(1) Fixing: the slicer is used according to the instructions provided by the manufacturer, and should ensure that the blade is sharp before use, and then the wax block is fixed to the holder, taking care to ensure that the chamfer is located at the upper right corner of the wax block.
(2) Repairing: in the trimming mode, the thickness of the trimming slice can be adjusted to 15-20 μm, and the trimming wax block can be cut until all the four embedded cell clusters can be cut. The number of repairs should be avoided as much as possible and the continuity of the organization should be ensured.
(3) Slicing: the thickness of the slice is adjusted to be about 3-5 mu m, and then the slice is cut, so that the slice is required to be complete and uniform, has proper thickness, and has no knife mark, no fold and no crack.
(4) Spreading: the slice is gently picked up by a writing brush, moved into the water surface in the bleach-dryer by tweezers and spread (the temperature is 37-40 ℃ and the slice can rotate but can not turn over in the process of transferring the slice to the water surface of the bleach-dryer).
(5) Fishing: and sticking the unfolded slice on a glass slide, and holding a glass slide sand grinding area by a right hand during the slice dragging process, so that the paraffin notch is kept at the upper left part, and bubbles are prevented from being generated between the slice and the glass slide as much as possible. The slide was tilted to 45. To the point where excess water on the slices runs down.
(6) Baking slices: the slides were placed in an oven and baked at 60 ℃ for 30min to make tissue paraffin sections, which were stored for later experiments.
Claims (25)
1. An immunohistochemical antibody composition for detecting MSLN, comprising anti-MSLN monoclonal antibody 3-2G6 produced by hybridoma cells deposited with the chinese collection center at accession number cctccc C201811 at 12.20.2017, wherein the tissue detected by the immunohistochemical antibody composition is formalin-fixed paraffin-embedded human pancreatic, ovarian, malignant mesothelioma tissue.
2. The immunohistochemical antibody composition of claim 1, obtained by mixing the antibody with an antibody dilution liquid, wherein the antibody dilution liquid comprises phosphate buffer of PH 7.2.
3. The immunohistochemical antibody composition of claim 2 wherein the phosphate buffer concentration is 0.005-0.5M.
4. The immunohistochemical antibody composition of claim 3 wherein the phosphate buffer concentration is 0.01-0.1M.
5. The immunohistochemical antibody composition of claim 2, the antibody diluent further comprising: 0.5% -5% of protein protectant, 0.05% -0.5% of surfactant and 0.01% -0.2% of preservative.
6. The immunohistochemical antibody composition of claim 2, the antibody diluent further comprising: 1% -3% of protein protecting agent, 0.05% -0.2% of surfactant and 0.02% -0.1% of preservative.
7. The immunohistochemical antibody composition of claim 2, the antibody diluent further comprising: 1% protein protectant, 0.05% surfactant, and 0.05% preservative.
8. The immunohistochemical antibody composition of claims 5-7 wherein the protein protecting agent comprises bovine serum albumin, the surfactant comprises tween 20, the preservative comprises 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one.
9. The immunohistochemical antibody composition of claim 2 wherein the antibody diluent further comprises 0.05-0.5M NaCl.
10. The immunohistochemical antibody composition of claim 2 wherein the antibody diluent further comprises 0.1-0.3M NaCl.
11. The immunohistochemical antibody composition of claim 2 wherein the antibody diluent further comprises 0.15M NaCl.
12. The immunohistochemical antibody composition of claim 2 wherein the antibody diluent comprises: 0.05M phosphate buffer, 0.15M NaCl, 1% bovine serum albumin, 0.05% Tween 20 and 0.05% ProClin300.
13. The immunohistochemical antibody composition according to claim 1 or 2 wherein the anti-MSLN monoclonal antibody is present in the form of an antibody solution wherein the antibody concentration is 0.05-20 μg/ml.
14. The immunohistochemical antibody composition of claim 13 wherein the antibody concentration is 0.2-10 μg/ml.
15. The immunohistochemical antibody composition of claim 13 wherein the antibody concentration is 0.3-5 μg/ml.
16. The immunohistochemical antibody composition of claim 13 wherein the initial concentration of the antibody solution is 1mg/ml, the volume ratio of the antibody solution to the antibody diluent is 1:200-1:18000.
17. the immunohistochemical antibody composition of claim 16, wherein the volume ratio of the antibody solution to the antibody diluent is 1:1600-1:6400.
18. The immunohistochemical antibody composition of claim 17, wherein the volume ratio of the antibody solution to the antibody diluent is 1:3200.
19. Use of an immunohistochemical antibody composition according to any one of claims 1-18 in the preparation of a mesothelin detection immunohistochemical kit.
20. An immunohistochemical kit for detecting MSLN comprising the immunohistochemical antibody composition of any one of claims 1-19.
21. The immunohistochemical kit of claim 10, further comprising: antigen retrieval liquid, immunohistochemical pen and cytoplasmatic control sheet.
22. The immunohistochemical kit of claim 21, wherein the antigen retrieval solution comprises 0.01M citric acid or a salt thereof at a pH of 6.0.
23. The immunohistochemical kit of claim 21, wherein the cytoplasmatic control patch comprises 4 cells as controls, which are: 293 cells expressing mesothelin negatively, CFPAC-1 cells expressing mesothelin weakly positively, OVCAR-3 cells expressing mesothelin positively, OVCAR-3-MSLN cells expressing mesothelin Pi Sujiang positively, wherein the OVCAR-3-MSLN cells are OVCAR-3 cells genetically modified to express exogenous MSLN.
24. The immunohistochemical kit of claim 23, wherein the OVCAR-3-MSLN cells are obtained by the method of:
(1) Construction of the Gene expression vector of interest
Designing a primer to amplify full-length MSLN from a vector containing a human MSLN coding sequence, performing gel cutting recovery by using a gel recovery kit, performing enzyme cutting by using a restriction enzyme, and simultaneously performing enzyme cutting by using the same incision enzyme to obtain a lentivirus expression vector; connecting the MSLN gene fragment and the expression vector, converting the connection product into competent cells, coating a flat plate after amplification, picking a single colony for colony PCR, and selecting a colony with the correct amplified product for sequencing; sequencing the correct recombinant plasmid to obtain the constructed expression plasmid;
(2) Packaging of target gene lentiviruses
Inoculating 293T cells into a culture bottle, culturing overnight, replacing a serum-free culture medium, mixing an expression vector, a packaging vector and an envelope vector, then mixing with Lipofectamine2000, adding the mixture into the 293T cells, culturing for 48 hours, collecting supernatant, concentrating and packaging, wherein the 293T cells are human kidney epithelial cell lines transfected with adenovirus E1A genes;
(3) Obtaining of cells of interest
Taking cells in logarithmic growth phase, adding the cells into a 24-well plate, and culturing overnight; sucking the original culture medium of the cells, adding a virus diluent and a diluted polybrene solution, and lightly mixing the mixture until the total infection volume is 250 mu L; after 24 hours of infection, removing the culture solution containing the virus, and replacing the culture solution with a fresh complete culture medium for continuous culture; replacing the virus carrying puromycin resistance gene with fresh complete culture medium containing puromycin to obtain a stably transfected cell strain; and (3) obtaining a monoclonal by adopting a limited dilution method to the obtained steady transgenic cell strain of the over-expressed MSLN target gene, and selecting a cell strain with the MSLN expression intensity consistent with the MSLN high expression intensity in pathological tissues according to the MSLN expression intensity in the pathological tissues, thereby obtaining the OVCAR-3-MSLN as the MSLN strong positive expression cell.
25. A method of detection of non-diagnostic interest for MSLN expression in an isolated paraffin tissue sample using an immunohistochemical antibody composition according to any one of claims 1 to 18, the steps of:
(1) Fixing a tumor tissue sample in formalin solution, then dehydrating the tumor tissue sample by gradient ethanol, placing the dehydrated tissue in xylene for transparency, immersing the tissue in melted low-melting-point paraffin for paraffin embedding, cutting the tissue into slices with the thickness of 3-5 mu m after the paraffin blocks are solidified, taking out the tissue by using a glass slide coated with polylysine, and drying the tissue by using a bleaching and drying instrument;
(2) Roasting the tissue slice at 65 ℃ for 1 hour, dewaxing in xylene, and then hydrating with gradient ethanol;
(3) Adding an antigen retrieval liquid into an autoclave, performing high-pressure or thermal retrieval treatment, taking out a slide after the antigen retrieval liquid is naturally cooled to room temperature, and soaking by PBST;
(4) Removing liquid around the tissues on the slices, circling the tissues by an immunohistochemical oil pen, adding freshly prepared 3% peroxidase blocking solution in the circling area, incubating at 37 ℃, and cleaning the slices by PBST; adding the immunohistochemical antibody composition of any one of claims 1-8, simultaneously providing a negative control, a blank control and a positive control, incubating at 37 ℃, and PBST washing the sections;
(5) Dripping 1-2 drops of detection reagent I to cover tissues on the slice, incubating at 37 ℃, and soaking and washing with PBST; carefully throwing away liquid around the tissue on the slice, dripping a detection reagent II to cover the tissue on the slice, incubating at 37 ℃, and immersing and washing with PBST; the detection reagent I, II is respectively a reaction amplifier and a high-sensitivity type enzyme-labeled anti-mouse/rabbit IgG polymer in a DAB detection kit of a polymer amplification reaction secondary antibody system; then using freshly prepared DAB color developing agent to develop color, and terminating the PBST soaking; mayer's hematoxylin is used for dying the nucleus, excess hematoxylin is washed by water, dehydrated, transparent and sealed, and the dying result is read and photographed by an optical microscope.
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