阅读说明：本技术 间皮素免疫组化检测试剂盒 (Mesothelin immunologic combined detection reagent kit ) 是由 毛海燕 于占娇 肖童雨 李壮林 房健民 于 2018-05-25 设计创作，主要内容包括：本申请涉及一种间皮素(Mesothelin,MSLN)免疫组化检测试剂盒,其含有MSLN单克隆抗体3-2G6。(This application involves a kind of mesothelin (Mesothelin, MSLN) immunologic combined detection reagent kits, contain MSLN monoclonal antibody 3-2G6.)

1. for detecting the immunohistochemistry antibody compositions of MSLN, containing anti-MSLN monoclonal antibody 3-2G6, the antibody by It is thin with the hybridoma that deposit number CCTCC C201811 is preserved in China typical culture collection center on December 20th, 2017 Born of the same parents generate.

2. immunohistochemistry antibody compositions described in claim 1, the antibody compositions pass through the antibody and antibody is dilute It releases liquid phase to be mixed to get, wherein the antibody diluent includes the phosphate buffer of PH 7.2, it is preferable that the phosphate buffer Concentration is 0.005-0.5M, more preferable 0.01-0.1M.

3. immunohistochemistry antibody compositions as claimed in claim 2, the antibody diluent also includes: the albumen of 0.5%-5% The preservative of matter protective agent, the surfactant of 0.05%-0.5% and 0.01%-0.2%；Preferably, the albumen of 1%-3% The preservative of matter protective agent, the surfactant of 0.05%-0.2% and 0.02%-0.1%；It is highly preferred that 1% protein Protective agent, 0.05% surfactant and 0.05% preservative.

4. immunohistochemistry antibody compositions as claimed in claim 3, wherein the protein protectant includes bovine serum albumin(BSA), The surfactant includes polysorbas20, and the preservative includes 2-methyl-4-isothiazolin-3-one and 5- chloro-2-methyl -4- Isothiazoline -3- ketone.

5. immunohistochemistry antibody compositions as claimed in claim 2, wherein the antibody diluent also includes 0.05-0.5M NaCl, preferably 0.1-0.3M NaCl, more preferable 0.15M NaCl.

6. immunohistochemistry antibody compositions as claimed in claim 2, wherein the antibody diluent includes: 0.05M phosphoric acid buffer Liquid, 0.15M NaCl, 1% bovine serum albumin(BSA), 0.05% polysorbas20 and 0.05% ProClin300.

7. immunohistochemistry antibody compositions of any of claims 1 or 2, wherein the anti-MSLN monoclonal antibody is with antibody-solutions Form exist, wherein antibody concentration be 0.05-20 μ g/ml, more preferable 0.2-10 μ g/ml, most preferably 0.3-5 μ g/ml.

8. immunohistochemistry antibody compositions as claimed in claim 7, wherein the antibody-solutions (initial concentration 1mg/ml) and institute The volume ratio for stating antibody diluent is 1: 200-1: 18000, preferably 1: 1600-1: 6400, more preferable 1: 3200.

9. immunohistochemistry antibody compositions of any of claims 1-8 are preparing mesothelin detection immunohistochemistry reagent Application in box.

10. the immunohistochemical kit for detecting MSLN, it includes immunohistochemistry of any of claims 1-11 Antibody compositions.

11. immunohistochemical kit described in any one of claim 10, also includes: antigen retrieval buffers, immunohistochemistry pen and cell Quality Control piece.

12. immunohistochemical kit described in claim 11, the antigen retrieval buffers include the citric acid or its salt of 0.01M, Its pH is 6.0.

13. immunohistochemical kit described in claim 11, the cytoplasm control wafer includes 4 kinds of cells as control, It is respectively as follows: 293 cells of mesothelin-negative expression, CFPAC-1 cell, the mesothelin positive expression of the expression of mesothelin weakly positive The OVCAR-3-MSLN cell that OVCAR-3 cell, mesothelin strong positive are expressed, wherein the OVCAR-3-MSLN cell is through losing Modification is passed to express the OVCAR-3 cell of external source MSLN.

14. immunohistochemical kit described in claim 13, the OVCAR-3-MSLN cell obtains by the following method:

(1) building of destination gene expression carrier

Design primer expands overall length MSLN from the carrier comprising people's MSLN coded sequence, carries out cutting glue using plastic recovery kit Recycling, and digestion is carried out with restriction enzyme, while using endonuclease digestion Lentiviral of the same race；By MSLN gene Segment and expression vector are attached, and connection product is transformed into competent cell, spread plate after amplification, the single bacterium of picking Row bacterium colony PCR is dropped into, the correct bacterium colony of amplified production is chosen and is sequenced；It is the expression built that correct recombinant plasmid, which is sequenced, Plasmid；

(2) packaging of target gene slow virus

293T cell is inoculated in culture bottle, serum free medium is replaced after being incubated overnight, by expression vector, package carrier and packet 293T cell is added after mixing again with Lipofectamine2000 after membrane carrier mixing and (has transfected people's kidney of Adenovirus E1A gene Epithelial cell line) in, culture 48 was as a child collected supernatant and was dispensed after being concentrated；

(3) acquisition of aim cell

Logarithmic growth phase aim cell is added in 24 orifice plates, is incubated overnight；The original culture medium of cell is sucked, virus is added Dilution and the polybrene solution diluted, infection total volume are 250 μ L, are mixed gently；After infection 24 hours, removal contains The culture solution of virus, changes fresh complete medium and continues to cultivate；Virus for carrying puromycin resistance gene is changed The fresh complete medium of purine-containing mycin obtains the cell strain of stable transfection；By the base of the overexpression MSLN mesh of above-mentioned acquisition Because stable cell strain obtains monoclonal using limiting dilution assay, according to MSLN expression intensity in pathological tissue, MSLN expression is selected The consistent cell strain of MSLN high expression intensity in intensity and pathological tissue, so that obtaining OVCAR-3-MSLN is MSLN strong positive table Up to cell.

15. a kind of be directed in vitro paraffin group using immunohistochemistry antibody compositions according to claim 1 to 8 Knit the detection method that MSLN expression in sample carries out non-diagnostic purpose, the step are as follows:

(1) tumor tissues sample is fixed in formalin solution, then carries out graded ethanol dehydration, dewatered tissue is set It is transparent in dimethylbenzene, it is rear to immerse in the low melt point paraffin melted, paraffin embedding is carried out, after wax stone solidification, is cut into thickness 3-5 μm thin slice, picked up using the slide for being coated with poly-D-lysine and dry instrument with drift after tissue and dry；

(2) histotomy after roasting piece 1 hour, carries out dewaxing treatment in 65 DEG C in dimethylbenzene, then carries out graded ethanol aquation；

(3) antigen retrieval buffers are added in pressure cooker, carries out high pressure or hot repair process, naturally cools to room to antigen retrieval buffers Temperature takes out slide, and PBST impregnates；

(4) liquid being sliced around tissue is got rid of, is drawn a circle with immunohistochemistry oil pike in tissue periphery, in delineation region Nei Jiaxin 3% peroxidase of fresh preparation blocks liquid, 37 DEG C of incubations, PBST cleaning slice；Addition according to claim 1 any one of -8 The immunohistochemistry antibody compositions, while negative control, blank control and positive control are set, 37 DEG C are incubated for, and PBST is clear Wash slice；

(5) 1-2 drop detection reagent I covering is added dropwise and is sliced upper tissue, 37 DEG C of incubations are embathed with PBST；It carefully gets rid of and is sliced group The liquid of surrounding is knitted, detection reagent II covering is added dropwise and is sliced upper tissue, 37 DEG C of incubations are embathed with PBST；Above-mentioned detection reagent I, II be respectively reaction amplification agent in polymer iodine secondary antibody system DAB detection kit and Gao Min type enzyme mark anti-mouse/ Rabbit igg polymer；Then the DAB chromogenic reagent of Fresh is used, PBST impregnates color development stopping；Mayer ' s bush uniformly dyeing Core, transparent after being rinsed with water extra haematoxylin, then through being dehydrated, mounting carry out the reading and bat of coloration result with optical microscopy According to.

Technical field

The present invention relates to a kind of immunohistochemical kits of detection mesothelin (Mesothelin, MSLN).

Background technique

Mesothelin (Mesothelin, MSLN) is a kind of cell surface glycoprotein.Mesothelin gene coding is one The preceding albumen of 71KD, through modification cutting become two sections, be respectively 41KD glycolsyl-phosphatidylinositol cell membrane anchorin and The free segment for being referred to as megakaryocyte-potentiating factor of 30KD.There is research to confirm that the film anchorin of 41KD is comprising one section and another The combined area of one tumor markers CA125 albumen, may be related to cell adherence, thus the anchorin potentially contributes to The transfer of tumour, and cause outcome undesirable.Recent studies have shown that: MSLN in most celiothelioma and cancer of pancreas, It is highly expressed in most oophoromas and lung cancer, in the normal tissue seldom expression, it is general only in pleural mesothelial cell lining, peritonaeum There is a certain amount of expression with pericardium.Although the function of Mesothelin is still inaccurate at present, either cell membrane combined area is also It is that free area is all proved to can be used as the marker and therapy target of the kinds of tumors such as carcinoma mesothelial, cancer of pancreas, oophoroma.

The cardinal principle of ImmunohistochemistryMethods Methods is: applied immunology basic principle --- antigen-antibody reaction, i.e. antigen with The principle that antibody specificity combines makes color developing agent (fluorescein, enzyme, metal ion, the same position of labelled antibody by chemically reacting Element) it develops the color to determine histocyte endoantigen (peptide and protein), it is positioned, qualitative and relative quantification research side Formula.

Immunohistochemistry technique is mainly characterized by: 1. high specificities: immunologic basic principle determines antigen and antibody Between combination have high degree of specificity；2. sensibility is high: antibody, which dilutes thousands of times, up to ten thousand times even more than one hundred million times, can still organize With antigen binding in cell, the antibody antigen of such hypersensitivity reacts, and is conveniently used in ImmunohistochemistryMethods Methods increasingly Routine pathology diagnostic work；3. accurate positioning: the technology, can be in tissue and cell by antigen-antibody reaction and color reaction The accurate positionin of antigen is carried out, thus position observation can be carried out in same tissue or cell to not synantigen simultaneously, thus The research that form and function combines can be carried out, to deeply having great importance for pathological research.In targeted drug During research and clinical use, positioning and relative quantification are carried out to the target antigen in pathological tissue using ImmunohistochemistryMethods Methods Detection, can predict patient whether be possible to from the treatment of the targeted drug be benefited, in targeted drug clinical test process In success of the test rate can be improved, medication precision can be improved during targeted drug clinical use.Currently, having had more Money turns to the adjoint diagnostic kit listing of principle with immune group, such as: VENTANA ALK IHC detection kit is to obtain FDA approval for identification be suitble to targeted drug Trastuzumab treatment patient in-vitro diagnosis IHC detection kit, 2014 The granted listing of China, obtains extensive concern.But still lacks can satisfy the quick to mesothelin protein of market needs at present Express the immunohistochemistry antibody reagent and related detecting method effectively detected.

Summary of the invention

In order to overcome the drawbacks of the prior art, the present invention provides a kind of for the people through the fixed paraffin embedding of formalin The positioning of MSLN and the immunohistochemistry antibody reagent of half-quantitative detection in the tissue such as cancer of pancreas, oophoroma, celiothelioma, can be to swollen Tumor treats mesothelin targeting medication and provides guidance.

Due to changing through MSLN antigenic structure in the fixed paraffin-embedded tissue of formalin, the antibody of commercially available anti-MSLN Be difficult to effectively combine and realize detection, for this phenomenon, the present invention provides a kind of hybridoma cell strain, the cell can production capacity it is enough The monoclonal antibody of specific recognition denaturation MSLN antigen.And during the hybridoma cell strain is preserved on December 20th, 2017 State's Type Tissue Collection is (referred to as: CCTCC；Address: Wuhan, China city Wuchang Luo Jia Shan), classification naming is that hybridoma is thin Born of the same parents 3-2G6, deposit number are CCTCC C201811.

On the one hand, the present invention provides a kind of immunohistochemistry antibody compositions for detecting MSLN, contains anti-MSLN monoclonal Antibody 3-2G6, the antibody is by being preserved in Chinese Typical Representative culture on December 20th, 2017 with deposit number CCTCC C201811 The hybridoma of object collection generates.

In some embodiments, antibody compositions of the invention are by mutually mixing antibody of the invention with antibody diluent Conjunction obtains, wherein the antibody diluent includes phosphoric acid (PB) buffer of PH 7.2, it is preferable that the phosphate buffer density For 0.005-0.5M, more preferable 0.01-0.1M.

In some embodiments, the antibody diluent also includes: protein protectant, the 0.05%- of 0.5%-5% 0.5% surfactant and the preservative of 0.01%-0.2%；Preferably, the protein protectant of 1%-3%, 0.05%- 0.2% surfactant and the preservative of 0.02%-0.1%；It is highly preferred that 1% protein protectant, 0.05% table Face activating agent and 0.05% preservative.

In some embodiments, the protein protectant includes bovine serum albumin(BSA), and the surfactant includes Polysorbas20, the preservative include 2-methyl-4-isothiazolin-3-one and 5-Chloro-2-methyl-4-isothiazolin-3-one.

In some embodiments, the antibody diluent also includes 0.05-0.5M NaCl, preferably 0.1-0.3M NaCl, more preferable 0.15M NaCl.

In some embodiments, the antibody diluent includes: 0.05M phosphate buffer, 0.15MNaCl, 1% ox blood Pure albumen, 0.05% polysorbas20 and 0.05% ProClin300.

In some embodiments, the anti-MSLN monoclonal antibody exists in the form of antibody-solutions, and wherein antibody is dense Degree is 0.05-20 μ g/ml, more preferable 0.2-10 μ g/ml, most preferably 0.3-5 μ g/ml.

In some embodiments, the volume ratio of the antibody-solutions and the antibody diluent is 1: 200-1: 18000, It is preferred that 1: 1600-1: 6400, more preferable 1: 3200.

On the other hand, this application involves immunohistochemistry antibody compositions of the invention to prepare mesothelin detection immunohistochemistry Application in kit.

On the other hand, this application involves the immunohistochemical kits for detecting MSLN, exempt from it includes described herein Epidemic disease group antibody compositions.

In some embodiments, immunohistochemical kit of the invention also includes: antigen retrieval buffers, immunohistochemistry pen with And cytoplasm control wafer.

In some embodiments, antigen retrieval buffers described in immunohistochemical kit of the invention include the lemon of 0.01M Lemon acid or its salt, pH 6.0.

In some embodiments, immunohistochemical kit of the invention includes cytoplasm control wafer, wherein the cytoplasm Control wafer includes 4 kinds of cells as control, is respectively as follows: 293 cells of mesothelin-negative expression, mesothelin weakly positive is expressed The OVCAR-3-MSLN cell that CFPAC-1 cell, the OVCAR-3 cell of mesothelin positive expression, mesothelin strong positive are expressed, Described in OVCAR-3-MSLN cell be genetically modified to express the OVCAR-3 cell of external source MSLN.

In some embodiments, the protein protectant is preferably BSA (as bovine serum albumin(BSA)).

In some embodiments, the surfactant is preferred Tween-20.

In some embodiments, the preservative is preferred ProClin300.The ProClin300 includes: 2- first Base -4- isothiazoline -3- ketone and 5-Chloro-2-methyl-4-isothiazolin-3-one.

On the other hand, the present invention provides produce anti-MSLN monoclonal antibody for detecting warp by hybridoma 3-2G6 The detection method on the non-diagnostic mesh ground of MSLN in the fixed paraffin-embedded tissue of formalin, it is characterised in that:

1. tumor sample is fixed in formalin solution, graded ethanol dehydration is then carried out, dewatered tissue is set It is transparent in dimethylbenzene, it immerses in the low melt point paraffin of thawing, carries out paraffin embedding later, after wax stone solidification, be cut into thickness 3-5 μm of thin slice uses drift to dry instrument drying after picking up tissue using the slide for being coated with poly-D-lysine；

2. histotomy after roasting piece 1 hour, carries out dewaxing treatment in 65 DEG C in dimethylbenzene, graded ethanol water is then carried out Change；

3. antigen retrieval buffers are added in pressure cooker, the pressure cooker high fire on electromagnetic oven of uncapping is heated to antigen retrieval buffers boiling It rises, ceases fire, slice is submerged into and repairs pot cover on liquid middle cover, pressure valve is buckled, is heated to jet, since jet, be adjusted to Fiery timing 2.5 minutes ceases fire, pulls up power supply；Pressure cooker is transferred on experimental bench, pot cover is opened, to antigen retrieval buffers nature It is cooled to room temperature taking-up slide, PBST impregnates；The PBST are as follows: phosphate Tween buffer；

4. getting rid of the liquid being sliced around tissue, drawn a circle around tissue with immunohistochemistry oil pike, in delineation region 3% peroxidase of Fresh is added to block liquid, 37 DEG C are incubated for 10 minutes, and PBST cleaning slice 2 times；Add 3-2G6 antibody molten Liquid, while negative control, blank control and positive control are set, 37 DEG C are incubated for 60 minutes, and PBST cleaning slice 2 times；

5. 1-2 drop detection reagent I covering, which is added dropwise, is sliced upper tissue, 37 DEG C incubation 10 minutes, embathe 2 times with PBST, carefully The liquid being sliced around tissue is got rid of, 1-2 drop detection reagent II covering is added dropwise and is sliced upper tissue, 37 DEG C are incubated for 10 minutes, use PBST embathes 2 times；Above-mentioned detection reagent I, II is respectively polymer iodine secondary antibody system DAB Detection Kit (good fortune State steps new, article No.: KIT-0016) in reaction amplification agent and Gao Min type enzyme mark anti-mouse/rabbit igg polymer；Then using new The DAB chromogenic reagent of fresh preparation 1 minute, PBST impregnate color development stopping；Mayer ' s bush uniformly dyeing core 1 minute, is rinsed with water more Transparent after remaining haematoxylin, then through being dehydrated, mounting carries out the reading of coloration result with optical microscopy and takes pictures.

In some embodiments, the antigen retrieval condition of sample to be detected may is that pH6.0 can be used in sample to be measured Citric acid solution carry out Pressure method, repair time is 2.5 minutes, and the buffer also can be used, and to carry out hot repair multiple, Repairing condition is 95-97 DEG C, 20 minutes.

In some embodiments, the sample should be fixed in formalin solution in vitro 30 minutes.

In some embodiments, the antigen retrieval buffers are as follows: the citrate buffer of 0.01M pH 6.0 repairs item Part is: being repaired under high pressure using above-mentioned reparation liquid.

In some embodiments, the high fire condition are as follows: 210 DEG C of 1600W；The moderate heat condition are as follows: 800W 130 ℃。

In some embodiments, herein described 3-2G6 antibody-solutions are instant 3-2G6 antibody-solutions, and antibody is dense Degree is preferably 0.05-2 μ g/mL, more preferable 0.2-1 μ g/mL, most preferably 0.31 μ g/mL；Negative control antibody is mouse monoclonal 1 Isotype control of IgG antibody (CST, CatalogNo.5415), clone G3A1；Positive control and negative control sample can 1 cytoplasm control wafers are closed with 4, and Quality Control piece is that the cell of 4 different mesothelin expressions is prepared to paraffin mass, and according to tissue core The requirement of piece is prepared in again in a new paraffin mass, is sliced；Wherein the cell line of MSLN feminine gender expression is 293 (0)；MSLN weakly positive expression cell is CFPAC-1 (1)；MSLN positive expression cell OVCAR-3 (2)；The expression of MSLN strong positive Cell OVCAR-3-MSLN cell (3).

Tissue specimen to be detected is detected (for the tumor group performed the operation or punctured using the immunohistochemistry antibody reagent of detection MSLN Knit and be cut to the thin slice with a thickness of 3-5 μm after the fixed paraffin embedding of formalin) in MSLN expression, testing result can be right Oncotherapy MSLN targets medication and provides guidance.

On the other hand, further testing result is that the expression value of MSLN is, virologist passes through immunohistochemical staining feelings Condition combination cell Quality Control piece carries out being obtained by the integer marking of 0-3 for sxemiquantitative to the tumour cell in examined tissue specimen.In Before the common clinic of drug, first using staining power as standards of grading.Wherein 0 point of expression: tumour cell does not occur any shape The dyeing of formula；1 point of expression: there is cell membrane dyeing in tumour cell, but the staining power of staining power < CFPAC-1 cell；2 Dividing indicates: there is cell membrane dyeing in tumour cell, and staining power is greater than CFPAC-1, is less than OVCAR-3-MSLN；3 points of expressions: swollen Oncocyte cell membrane dyes, staining power >=OVCAR-3-MSLN.It should be clear that participating in scoring is tumour living Cell, and give a mark be carried out based on the MSLN that is located on cell membrane (that is: the cell for only having cytoplasm individually to dye is not included in Count range).

It mentions cytoplasm control wafer in above-mentioned standards of grading to be prepared by the cell of known MSLN expression, including 4 Cell mass: wherein 293 cells are MSLN negative cells, and CFPAC-1 cell is the cell of MSLN weakly positive expression, and OVCAR-3 is MSLN positive expression cell, three of the above cell can be bought by commercialization approach；OVCAR-3-MSLN is MSLN strong positive Expression cell, preparation method are as follows:

1. the amplification of target gene: design primer obtains amplifying target genes MSLN overall length from target gene carrier；

2. target gene and expression plasmid digestion: the target gene MSLN after amplification carries out cutting glue using plastic recovery kit Recycling, and digestion is carried out with restriction enzyme, while using endonuclease digestion expression vector pHBLV-CMV- of the same race ZsGreen-puro；

3. the building of destination gene expression plasmid: target gene fragment and expression vector are attached；By connection product It is transformed into competent cell, spread plate after amplification；Picking single bacterium colony carries out bacterium colony PCR；It is correct to choose amplified production Bacterium colony is sequenced；It is the expression plasmid built that correct recombinant plasmid, which is sequenced,；

4. the packaging of target gene slow virus: inoculation 1x10⁷A 293T cell is in T75 culture bottle, after being incubated overnight more Change serum free medium, by expression plasmid, packaging plasmid and envelope plasmid mix after again with Lipofectamine2000 (Lipofectamine^TM2000 Transfection Reagent article No.s: 11668027) being added in 293T cell after mixing, Culture 48 was as a child collected supernatant and was dispensed after being concentrated；

5. aim cell infects: logarithmic growth phase aim cell 5*10⁴The hole a/mL, 0.5mL/ is added in 24 orifice plates, It is incubated overnight；The original culture medium of cell is sucked, the polybrene solution that viral dilution is added and has diluted, infection is totally Product is 250 μ L, is mixed gently；After infection 24 hours, the culture solution containing virus is removed, fresh complete medium is changed and continues to train It supports；Virus for carrying puromycin resistance gene changes the fresh complete medium of purine-containing mycin, obtains stable transfection Cell strain；

6. the acquisition of aim cell: the overexpression MSLN target gene stable cell strain of above-mentioned acquisition is used limiting dilution Method obtains single clone, according to MSLN expression intensity in pathological tissue, selects MSLN high in MSLN expression intensity and pathological tissue The consistent cell strain of expression intensity, so that obtaining OVCAR-MSLN is MSLN strong positive expression cell.

Finally, the application further relate to related immune group antibody reagent or immunohistochemistry antibody kit be used to prepare it is swollen Purposes in tumor detection reagent.

The present invention achieves following technical effect: instant antibody used in this application, i.e., by antibodies buffer The ingredients such as protected protein and preservative are added, can be realized following effect: reducing the non-specific binding of antibody and tissue, thus It can be omitted BSA serum closing step and its similar step；Antibody is set to guarantee steadily in the long term, to be conducive to improve under low concentration The efficiency and sensitivity of detection.

It can be realized quick and precisely using the detection kit or detection reagent of the application for cell surface mesothelin egg White detection, higher than the detection sensitivity and accuracy of similar kit on the market at present.The application is detected for immunohistochemistry The characteristics of technology design and obtain can be suitable for immunohistochemical experiment high specificity monoclonal antibody, overcome due to It changes through MSLN antigenic structure in the fixed paraffin-embedded tissue of formalin, the antibody of commercially available anti-MSLN is difficult to effectively combine And realize the defect of detection.

The application adaptive immune group antibody reagent can guarantee reactivity and the validity period of anti-MSLN monoclonal antibody, and User just can be used directly after obtaining the application Related product without carrying out any operation, improve the convenience of detection.

Detailed description of the invention

Fig. 1 shows anti-MSLN monoclonal antibody purity detecting map result；It is analyzed through gray scale scanning, anti-MSLN monoclonal Antibody band area percentage is 95%, and purity meets the requirements.

Dyeing effect compares under the conditions of Fig. 2 .A figure shows different antigen retrievals；Wherein (1) and (2) be respectively two not Same oophoroma pathological tissue；1-3 respectively represents antigen retrieval condition 1-3；B figure shows different dilution ratio 3-2G6 antibody To the coloration result of OVCAR-3 cell paraffin section, wherein 1-6 is the 3-2G6 antibody of 1mg/mL: PBST ratio is respectively 1: 25,1: 100,1: 400,1: 1600,1: 6400 and 1: 25600 when to the coloration result of OVCAR-3 cell paraffin section, identical The staining power of OVCAR-3 paraffin section is corresponding with the medium MSLN staining power of tumour cell in tissue under experiment condition；C figure Show the coloration result of 30 minutes (1) and 60 minutes (2) of primary antibody incubation time.

Fig. 3 .A figure shows the comparison of dyeing effect under Tris buffer and PB buffer, and " pH7.4 has been respectively adopted 0.05M TRIS buffer " and " pH7.4 0.05M PB buffer " diluted 3-2G6 antibody incubation carry out immunohistochemical staining, Picture lists the staining conditions in 4 visuals field respectively；B figure shows the coloration result of 5 ionic concentration buffer liquid.Condition 1- is slow Total Na ion concentration is 0.020M in fliud flushing, and total Na ion concentration is 0.095M in condition 2- buffer, in condition 3- buffer Total Na ion concentration is 0.165M, and total Na ion concentration is 0.184M in condition 4- buffer, in condition 5- buffer total Na from Sub- concentration is 0.219M；C figure shows instant 3-2G6 antibody preparation to MSLN expression cell different degrees of in tumor tissues Coloration result；Wherein, 1-4 is respectively the instant 3-2G6 antibody preparation of preferred buffer preparation to MSLN table in tumor tissues Up to the coloration result for the tumour cell expressed for feminine gender, weakly positive, the positive and strong positive；D figure shows that 3-2G6 antibody preparation exists 45 DEG C of potency testing results after heat damage 7 days, 15 days and 30 days；It can be seen that 4- parameter curve is declined slightly, but still conforms to and exempt from The requirement of epidemic disease groupization detection stability.

Fig. 4 shows the immunohistochemical staining figure to variety classes tumour cell pathological section.

Specific embodiment