CN108387731A - A kind of enzyme linked immunosorbent detection and preparation method for people's PD1 albumen - Google Patents

Abstract

The present invention relates to a kind of enzyme linked immunological and its detection methods, are a kind of monoclonal antibody coated elisa plate of anti-human PD1 albumen newly and the preparation method of enzyme linked immunological kit.The monoclonal antibody for the anti-human PD1 albumen of mouse for preparing specificity with gene engineering method is essentially consisted in place of its key, and will be coated on ELISA Plate after this antibody purification, as a part for kit.Kit further includes more anti-, the secondary antibody of horseradish peroxidase-labeled, Sample dilution, antibody diluent, developing solution, the terminate liquids of the recombinant protein standard items of PD1, anti-human PD1 albumen.Kit establishes the assay method of PD1 albumen concentration in fast and simple measurement human serum and blood plasma.Kit is mainly used for using for the research of scientific research and clinical trial.

Description

A kind of enzyme linked immunosorbent detection and preparation method for people's PD1 albumen

Technical field

The invention belongs to biomedicine technical fields, relate in particular to a kind of detection people PD1 albumen（programmed cell death 1）That is the enzyme-linked immunologic detecting kit of apoptosis 1.The invention further relates to the systems of mentioned reagent box Preparation Method.

Background technology

PD1（CD279）The immunoglobulin class I type transmembrane glycoproteins being made of 288 amino acid, are initially logical Cross cut down hybridization technique from mouse be in apoptotic state hybridoma and hematopoietic progenitor cells system clone obtain, be considered withering with cell It dies related and is named as programmed death-1（Programmed death-1, PD-1）.（Chemnitz JM1, et al. J Immunol. 2004 Jul 15;173(2):945-54.）.PD-1 molecule extracellular regions include a lgV spline structures domain, there is 4 Important N connection glycosylation sites, and by high glycosylation.The structure may play an important role in ligand binding.PD- 1 most significant feature is tyrosine residue, the wherein tyrosine residue of N-terminal there are two the areas Bao Jiang of 95 amino acid composition contain ITIM (immunorecepotor tyrosine-based inhibitory are collectively constituted with neighbouring amino acid residue Motifs, immunity receptor Tyrosine Inhibitory Motifs）, the motif can pass through the phosphorylation of tyrosine play antagonism antigen receptor thorn The function of energizing signal,（Ishida Y1. et al. TEMBO J. 1992 Nov;11(11):3887-95.) to Important negative regulation effect is played during immune response.The tyrosine residue of its C-terminal also can be with the SHP-1 in downstream, SHP-2 Equal signal transducers act on and inhibit the further activation of immunocyte.（Latchman Y1, et al. Nat Immunol. 2001 Mar;2(3):261-8.) PD-1 molecules are mainly in T, and B, NK cell surfaces are in inductivity up-regulated expression, In immature T, also there is weak expression in the specific stage that B cell is developed in thymus gland and marrow.（Freeman GJ1, et al. J Exp Med. 2000 Oct 2;192(7):1027-34.）

PD1（B7-H1, CD274）And PD-L2（B7-DC, CD273）It is two ligands of PD-1, the mRNA of PD1 and PD-L2 are not only Expression is organized in organa parenchymatosum, such as：Heart, lung, liver and placenta etc. also have faint expression on incomplete antigen presenting cell, such as：It is living The B cell and Dendritic Cells of change；PD1 mRNA are presented on the tumor cell line of thymic epithelial cells and a variety of epithelial origins Height expression.（Wang Q1, et al. Cytokine. 2006 Oct;36(1-2):23-8. Epub 2006 Dec 12.）. On protein level, PD1 is in the T of activation, B, monocyte and a plurality of types of tumour cells（Such as：Lung cancer, liver cancer, squamous cell Cancer and oophoroma etc.）Upper inducible expression.（Jung HW1, et al. Exp Mol Med. 2004 Feb 29;36(1): 13-22.) 。

The tumour antigen of tumor cells expression can be identified by host T cell, but cannot effectively be removed in time, tumour On the one hand restraining factors in microenvironment slacken T cell function, on the other hand tumour cell is promoted to mushroom out.Research is found： PD-1/PD1 inhibits the discovery of approach and tumour, development closely related.Various tumor cell strains and tumor tissues are at sense of organization height PD1 is expressed, such as：Lung cancer, liver cancer, breast cancer, squamous cell carcinoma and oophoroma etc..PD-1/PD1 inhibits approach to CD8+ T cells Activation and proliferation have an apparent inhibiting effect, tumour cell can escape CTL lethal effects by this approach, and it is antitumor to weaken body Immune response.（Nishimura H1, Honjo T. Trends Immunol. 2001 May;22(5):265-8.）.It blocks. T cell cannot find tumour cell and send out signal to attack to tumour cell.

Preclinical experiment confirms：Practical antibody blocking PD-1 or PDL1 interactions can restore T cell for swollen Tumor kills, to prevent cancer from invading profit and reduce gross tumor volume.（McDermott DF, et al. Cancer Med 2013; 2:662–73).In future, applies to clinical immunotherapy of tumors and important value is provided.

Invention content

The purpose of the present invention：A kind of new quantitatively detected for people source PD1 in human serum or cells and supernatant is provided ELISA kit.

Technical solution of the present invention is as follows：

1, the preparation of the specific monoclonal antibody coating reaction plate of anti-human PD1, passes through following steps：

（1）The acquisition of 35 KD PD1 protein fragments：

The prokaryotic expression of 35 KD PD1 protein fragments：With BC074740 DNA (be purchased from American type culture collection, ATCC it is) template, with the positive anti-primer of a pair of of specificity, PCR amplification PD1 DNA fragmentations（47nt-913nt）, by gained segment It is cloned in carrier pET28a（Purchased from German Merck companies）, convert BL21（DE3）（Purchased from Merck）Competent E.coli, root According to kalamycin resistance screening positive clone.The Plasmid DNA of extraction positive colony is identified, is obtained and target fragment molecular weight Consistent segment, preliminary judgement are the positive.Plasmid DNA is extracted with PCR positive colonies, is sequenced, the results showed that PD1 CDNA segments are correctly cloned into pET28a, the Complete Open formula reading frame for having 867 bp for one, with people source in GenBank PD1 genes 100% are homologous, calculate that the protein molecular weight of its expression is 35 KD, PI 8.8.

1. the identification of 35 KD PD1 protein fragments：The e. coli bl21 of recombinant plasmid is converted after induction by microorganism collection, Ultrasonic treatment, mycoprotein increase a band newly through SDS-PAGE protein electrophoresis and coomassie dyeing display near 35 KD, Through Western blotting identify, can with the antibody of PD1 occur strong positive reaction, illustrate 35 KD PD1 albumen have compared with Strong immunogenicity.

2. the preparation and purification of the PD1 protein fragments of 35 KD：Through mass propgation, induction, expression, the affine layers of Ni2+ are used Analyse column（Purchased from Pharmacia）PD1 albumen of the purifying with 6 × His labels, obtains a large amount of 35 KD PD1 protein fragments.

（2）Prepare the monoclonal antibody of anti-PD1：

1. the animal immune of PD1 albumen：The PD1 protein fragments of 35KD are mixed with freund adjuvant and after fully emulsified, subcutaneously BALB/c healthy mice of the age of mouse at 8~12 weeks is immunized in injection（Purchased from Hubei Province Animal Experimental Study center）3 to 4, every time Immunization interval is 2 weeks, until serum titer is higher than 40000:1.

2. the preparation of hybridoma：4 days after final immunization, detach mice spleen cell, under PEG existence conditions selection with The Sp2/0 myeloma cell strains of exponential phase（Purchased from China typical culture collection center, CCTCC）With 4:1 ratio is melted It closes, feeder cells come from Peritoneal Cells of Mice.Cell after fusion is selected with the Screening of Media containing HAT after limiting dilution High antibody secretory pit, hole inner cell is cloned, and the ELISA for carrying out antigen-specific is measured, and is selected hypersecretion cell strain and is expanded training It supports or freezes.

3. prepared by monoclonal antibody：The hybridoma cell strain for secreting specific monoclonal antibody is subjected to mouse peritoneal inoculation, it is to be generated apparent Ascites is collected after ascites, with the crosslinked affinity column of staphylococcal protein A（Purchased from Pharmacia companies）Purified monoclonal antibody.It is anti- Body is stored among the PBS of 0.01mol/l, pH7.4 containing 55% glycerine, 0.1%BHA and 0.01% thimerosal.

（3）It is coated with reaction plate with gained monoclonal antibody：Monoclonal antibody is diluted to debita spissitudo with the carbonate buffer solution of pH 9.6, packet By 96 orifice plates, per 100 μ l of hole, it is placed in 4 DEG C of wet box 16-24 hour, PBST washings 3 times 30 seconds every time, then pat dry, eliminate hole Interior liquid.

（4）Closing：It is closed with the PBS of the pH7.4 containing 8% calf serum, per 200 μ l of hole, 37 DEG C, after 2 hours are incubated, PBST is washed 3 times, is sealed after dry；The plate is the coated enzyme reaction plate of monoclonal antibody of anti-PD1, can be used for the quantitative inspection of PD1 It surveys.

2, PD1 standard items（PD1 protein solutions）Preparation：

（1）The prokaryotic expression of PD1 albumen：With DNA (being purchased from ATCC) for template, with the positive anti-primer of a pair of of specificity, PCR amplification PD1（47nt-913nt）Target gene converts BL21 by gained gene cloning in carrier pET28a（DE3）Competence large intestine bar Bacterium, according to kalamycin resistance screening positive clone.The Plasmid DNA of extraction positive colony is identified, is obtained and target fragment The consistent segment of molecular weight, preliminary judgement are the positive.Plasmid DNA is extracted with PCR positive colonies, is sequenced, as a result table PD1 The cDNA of protein is correctly cloned into pET28a, for a Complete Open formula reading frame with 867 bp, and in GenBank PD1 genes 100% are homologous, calculate that its molecular weight is 35 KD, PI 8.8.

（2）The identification of PD1 albumen：The e. coli bl21 of recombinant plasmid has been converted after induction by microorganism collection, ultrasound Wave cracks, and mycoprotein increases a band, warp newly through SDS-PAGE protein electrophoresis and coomassie dyeing display near 35 KD Western blotting identifications can occur strong positive reaction with the antibody of PD1, illustrate that PD1 albumen has stronger immunogene Property.

（3）The preparation and purification of PD1 albumen：It is obtained using Ni2+ affinity chromatography column purifications through mass propgation, induction, expression To a large amount of PD1 albumen.

（4）It is prepared by PD1 standard items：PD1 albumen is stored in containing 55% glycerine, 0.1%BHA and 0.01% thimerosal Among the PBS of 0.01mol/l, pH7.4, and make final concentration of 16 ng/mL of PD1 albumen

3, the preparation of PD1 positive control samples：PD1 albumen is dissolved in containing 50% glycerine, 0.5%BSA, 0.1%BHA and 0.01% sulphur Among the PBS of 0.01mol/l, pH7.4 of willow mercury, and make final concentration of 16 ng/ml of PD1 albumen.

4, the how anti-preparation of PD1：

（1）Animal immune：Purified PD1 albumen is taken to be mixed with freund adjuvant and after fully emulsified, it is backbone both sides, symmetrical more Point be subcutaneously injected, be immunized the 3-4 monthly ages, weight 1.7Kg or more healthy Japan large ear rabbit（It is ground purchased from Hubei Province experimental animal Study carefully center）3 to 4, each immunization interval is 2 weeks, and the 8th day ear vein takes blood to survey serum titer after being immunized again after immune January, Until serum titer is higher than 40000:1.

（2）Serum obtains：Rabbit middle ear arterial blood extracting, each 40ml are centrifuged, 3000rpm, 5 minutes after standing 30 minutes, on It is serum clearly.

（3）Mostly anti-purifying：With the affinity column of PD1 protein-crosslinkings（Purchased from Pharmacia）Purifying, obtains PD1's It is mostly anti-, it is stored among the PBS of 0.01mol/l, pH7.4 containing 55% glycerine, 0.1%BHA and 0.01% thimerosal, mostly anti-end A concentration of 1mg/ml.

（4）293 cell protein blots of how anti-progress and human tonsil's tissue samples immunohistochemistry, Fig. 3 and Fig. 4 will be obtained, As a result illustrate that how anti-we be made and can be reacted with the PD1 albumen of naive, and with specificity.

5, ELIAS secondary antibody：It is horseradish peroxidase purchased from U.S. R ＆ D systems（horseradish Peroxidase, HRP）The goat anti-rabbit igg of label.

6, this kit includes to the ELISA detecting steps of PD1：

（1）Sample to be tested, standard sample, positive control sequentially add in micropore, are incubated, PD1 by with surface of solid phase carriers Capture antibody --- the antibody being coated in hole in advance is combined.

（2）By washing, by the how anti-adding hole of the anti-PD1 of specificity, in second is incubated, resists more and play the part of detection Antibody, and combined with captured PD1 in first time incubation.

（3）Extra mostly anti-, addition ELIAS secondary antibody is washed away, it is more in ELIAS secondary antibody and previous step in third time is incubated It is anti---- detection antibody is combined, and forms the conjugate of the sandwich shape of 4 components.

（4）Unbonded ELIAS secondary antibody is washed away, 3,3', 5, the 5'- tetramethyl benzidine of substrate of enzyme is added （Tetramethyl benzidine, TMB）, liquid converts au bleu under the catalysis of peroxidase in hole, is added and terminates Liquid converts yellowly.The content of PD1 is proportionate in the depth and sample of color.

（5）With microplate reader absorbance is measured under 450nm wavelength（O.D. it is worth）, calculate PD1 concentration in sample.

This new PD1 ELISA detection kits, it is characterised in that：The kit by above-mentioned specificity PD1 The coated ELISA Plate of monoclonal antibody, PD1 protein standard substances, the positive control sample of PD1, more anti-, horseradish peroxidase-labeleds of PD1 Secondary antibody constitute, concrete component is：（1）ELISA Plate：It is coated with by the monoclonal antibody of PD1；（2）Sample diluting liquid：Containing 8% calf serum The 0.01mol/l PBS of pH7.4；（3）Cleaning solution；（4）PD1 protein standard substances；（5）The positive control sample of PD1；（6）PD1's It is mostly anti-；（7）The secondary antibody of horseradish peroxidase-labeled；（8）Developing solution：TMB-H2O2 systems；（9）Terminate liquid：2mol/l H2SO4 solution.

All indexs of this kit meet the strict demand of U.S.'s R ＆ D systems ELISA kit indices.Mesh Before, there has been no the reports of the ELISA kit of PD1 by external several big ELISA kit manufacturers, and the product of domestic manufacturer is in Initial stage, the positive internal control of PD1, obtained measurement conclusion are not worth discussion substantially.This kit uses 20 feminine genders The average value of sample, as minimum detection line, is 43 pg/ml plus 2 times of standard variances, and the present invention improves detection precision.

The present invention chooses the PD1 segments for containing identification function area, and obtains specificity as mice immunized with antigen High-purity monoclonal antibody.Specific detection, the detection of employment tonsil are carried out to monoclonal antibody（Fig. 5）, verification monoclonal antibody can specificity knowledge Others ensure that the specificity of testing result, utmostly keeps away natural PD1 albumen using this monoclonal antibody as the antibody of capture PD1 False positive is exempted from.The more anti-specificity of verification（Fig. 3 and Fig. 4）, it is then how anti-as detection antibody using specificity, further exclude False positive；ELIAS secondary antibody is added, forms the conjugate of the sandwich shape of 4 components, is eventually adding substrate developing solution, it will be with On obtained correct signal effectively amplify, the standard items and internal reference experiment that comparison synchronous parallel carries out further exclude in experiment Reagent or operating error, finally obtain reliable conclusion.

Compared with the prior art, the present invention has the following advantages and effect：

1, it has wide range of applications：Suitable for human serum, tissue and cell cultivation object；

2, detection speed is fast：Only about 3 hours；

3, easy to use：Complex instrument is not needed；

4, step is simple；

5, acquired results are accurate and reliable：By the antigen of multistep specificity, the compatible reaction of antibody, significantly reduce layer by layer Nonspecific reaction；And in addition to standard items are provided, also provide positive control, it is ensured that the data obtained eliminates numerous reagents, temperature The interference of the factors such as degree.

Description of the drawings：

Fig. 1：Kit body and constituent of the present invention.

Fig. 2：The protein blot electrophoretogram of PD1 recombinant proteins.

Fig. 3：The polyacrylamide gel electrophoresis figure of PD1 albumen is detected with 293 cell.

Fig. 4：The immunohistochemistry figure of PD1 polyclonal antibodies, detection sample are human tonsil's tissue of paraffin embedding, antibody Thinner ratio 1:50.

Fig. 5：The immunohistochemistry figure of PD1 monoclonal antibodies, detection sample are human tonsil's tissues of paraffin embedding, and antibody is dilute It releases than 1:100.

Specific implementation mode

1, reagent：

（1）Coating buffer（The sodium carbonate-bicarbonate buffer solution of 0.02mol/l, pH9.6）：Na2CO3 0.6g, NaHCO3 1.16g, Na2N3 0.2g add distilled water to 1000ml, adjust pH to 9.6.

（2）Sample diluent（0.01mol/l PBS containing 8% calf serum pH7.4）：NaCl 8.0g, NaH2PO4 2H2O 0.3g, Na2HPO4 12H2O 2.9g, KCl 0.2g, thimerosal 0.1g add distilled water to 1000ml, adjust pH to 7.4.

（3）Confining liquid（8% calf serum/PBS solution）：Calf serum 80ml, 0.01mol/l pH7.4 PBS 920ml.

（4）Cleaning solution（pH7.4 PBST）：NaCl 8.0g, NaH2PO4 2H2O 0.3g, Na2HPO4 12H2O 2.9g, Tween 20 0.5g, thimerosal 0.1g add distilled water to 1000ml, adjust pH to 7.4.

（5）ELIAS secondary antibody（The goat-anti rabbit of HRP labels is mostly anti-）：Purchased from U.S. R ＆ D systems, when use, is diluted with PBS To 1:10000 dilutions.

（6）PD1 protein standard substances：The 0.01mol/l PBS solutions of the PD1 albumen of 289 amino acid of overall length, pH7.4 contain 55% glycerine, 0.1%BHA and 0.01% thimerosal, final concentration of 16 ng/ml of PD1, when use, are diluted to sample diluent 8000,4000,2000,1000,500,250,125,0 pg/ml, eight gradients.

（7）The positive control sample of PD1：The PBS solution of 0.01mol/l, pH7.4 of PD1 albumen, containing 50% glycerine, 10% Cow's serum, 0.5%BSA, 0.1%BHA and 0.01% thimerosal, final concentration of 16 ng/ml of PD1, sample diluent when use Make 1:1 dilution.

（8）Anti- PD1's is mostly anti-：Mostly anti-PBS solution, the 0.01mol/ containing 55% glycerine, 0.1%BHA and 0.01% thimerosal L, pH7.4, mostly anti-final concentration of 1mg/ml make 1 when use with sample diluent:10000 dilutions.

（9）Substrate developing solution（TMB-H2O2 systems）：

1. substrate solution A（3', 3', 5,5'- tetramethyl benzidine tetramethyl benzidine, TMB）：200 milligrams of TMB, Absolute ethyl alcohol 100ml, adds distilled water to 990ml；

2. substrate solution B（H2O2 urea）：Na2HPO4 12H2O 36.8g, citric acid 9.3g, 1% hydrogen peroxide urea 4.8ml add Distilled water adjusts pH to 5.2 to 1000ml.

3. being added before enzyme mark hole 10 minutes by both substrate solution A and substrate solution B 1:1 mixing, as substrate developing solution.

（10）Terminate liquid（2mol/l H2SO4 solution）：600ml distilled waters are previously added in beaker, by concentrated sulfuric acid 100ml It is slowly added dropwise, is stirred continuously, 900ml is settled to after being cooled to room temperature.

2, key instrument：

（1）Microplate reader：BIO-RAD Model 680.

（2）ELISA reaction plates：96 Well EIA/RIA plate of U.S. Corning Incorporated costar R.

（3）Water-bath：Purchased from Napco companies of the U.S., Model 203.

3, method：

（1）The selection of the most suitable titre of ELIAS secondary antibody

1. 100 ng/ml rabbit iggs are coated with reaction plate, washing；

2. ELIAS secondary antibody sample diluent is made a series of dilutions, in adding hole, 37 DEG C, it is incubated after forty minutes, PBST is washed 3 times；

3. substrate colour developing is added, terminate liquid is added after twenty minutes；

4. read light absorption value, take light absorption value be 1.0 when ELIAS secondary antibody dilution --- 1:10000 as the best of ELIAS secondary antibody Dilution.

（2）Chessboard method selects the best coating titre of monoclonal antibody

1. after monoclonal antibody is made a series of dilutions with coating buffer, 4 DEG C are coated with reaction plate 16-24 hours, and PBST is washed 3 times, eliminates hole Interior liquid；

2. standard sample and PD1 positive controls, negative reference solution is added, it is incubated, washing；

3. how anti-solution is added, it is incubated, washing；

4. ELIAS secondary antibody is added, it is incubated, washing；

5. substrate colour developing is added, terminate liquid is added after twenty minutes；

6. reading light absorption value, it is 0.8 to 1.0 to choose PD1 positive control sample light absorption values, and the light absorption value of negative control is less than 0.1 Monoclonal antibody is coated with dilution --- and 1:10000 are used as optimum dilution degree.

（3）Monoclonal antibody is coated with the preparation method of reaction plate：

1. diluting monoclonal antibody with carbonate buffer solution, 96 orifice plates are coated with, per 100 μ l of hole, are placed in 4 DEG C of wet box 16-24 hours, PBST is washed It washs 3 times, 30 seconds every time, then pats dry, eliminate liquid in hole；

2. being closed with the PBS of the pH7.4 containing 8% calf serum, per 200 μ l of hole, 37 DEG C, 2 hours, PBST was washed 3 times, after dry Sealing；The plate is the coated ELISA Plate of monoclonal antibody of anti-PD1, can be used for the specific detection of PD1.

（4）The composition of kit：

The kit by specific monoclonal antibody coating reaction plate made from ELISA Plate, sample diluent, cleaning solution, ELIAS secondary antibody, PD1 standard items, PD1 positive control samples, more anti-, substrate, terminate liquid compositions, concrete component are：

1. ELISA Plate：The coated ELISA Plate of monoclonal antibody of PD1；

2. sample diluent：0.01mol/l PBS containing 8% calf serum and pH7.4；

3. cleaning solution：pH7.4 PBST；

4. ELIAS secondary antibody：The goat anti-rabbit igg of HRP labels；

5. PD1 standard items：The PBS solution of PD1 albumen, final concentration of 16 ng/ml of PD1；

6. PD1 positive control samples：The PBS solution of PD1 albumen, final concentration of 16 ng/ml of PD1；

7. mostly anti-：Mostly anti-PBS solution, the final concentration of 1mg/ml of antibody；

8. substrate developing solution：TMB-H2O2 systems；

9. terminate liquid：2mol/l H2SO4 solution.

（5）The method for detecting PD1 concentration in tissue or cell culture, serum using mentioned reagent box：

1. sample to be tested, standard items, positive control sample are handled：Blood serum sample is carried out 1 with sample diluent:1, positive Property control 1:1 dilution；Standard items are diluted to 8000,4000,2000,1000,500,250,125,0 pg/ with sample diluent Eight gradients of ml；

2. sample to be tested, standard sample, positive control are sequentially added in enzyme mark hole, per 100 μ l of hole, 37 DEG C are incubated 1 hour, PBST Washing 3 times, every time washing pat dry liquid in hole；

3. by the how anti-addition enzyme mark hole of anti-PD1, per 100 μ l of hole, 37 DEG C are incubated 1 hour, and PBST is washed 3 times；

4. ELIAS secondary antibody is added, per 100 μ l of hole, 37 DEG C are incubated 40 minutes, and PBST is washed 3 times, and washing every time pats dry liquid in hole；

5. the substrate of enzyme is added, per 100 μ l of hole, terminate liquid is added in incubation at room temperature after twenty minutes, per 50 μ l of hole；

6. measuring absorbance under 450nm wavelength with microplate reader, the light absorption value for making various concentration standard items is Y-axis, dense with its The logarithm of degree is that standard curve is made in X-axis.

7. result of calculation：If the measured value and sign value of positive control sample（16 ng/ml）It compares, variation lines Number is less than 10%, illustrates that continuous mode is reliable, can establishing criteria curve calculate PD1 concentration in institute sample.

Claims (2)

1. the preparation method of the monoclonal antibody coated elisa plate of people PD1, is prepared through the following steps：

(1) using BC074740 DNA as template, PCR amplification PD1 DNA fragmentations（47nt-913nt）Genetic fragment, with carrier PET28a connections convert BL21, express a large amount of 35 KD PD1 protein fragments；

(2) by 35 KD PD1 protein immunization small white mouses, its splenocyte is taken to be merged with rat bone marrow tumour cell, selects positive hybridoma Cell is cultivated in mouse ascites fluid, and purifying protein obtains the monoclonal antibody of anti-PD1；

(3) 96 orifice plates, 100 holes μ l/, 4 DEG C of 16-24 will be coated with after the carbonate buffer solution of the pH9.6 dilution of gained monoclonal antibody Hour, PBST is washed 3 times, 30 seconds every time, then eliminates liquid in hole；

(4) with the PBS closings of the pH7.4 containing 8% calf serum, per 200 μ l of hole, 37 DEG C, 2 hours, PBST was washed 3 times, dry After seal；The plate is the coated ELISA Plate of monoclonal antibody of anti-PD1, can be used for the specific detection of PD1.

2. the ELISA detection kit of the monoclonal antibody coated elisa plate of application people PD1 described in claim 1, it is characterized in that：By weighing Profit require the coated ELISA Plate of monoclonal antibody of the anti-PD1 described in 1, PD1 standard items, the positive control sample of PD1, anti-PD1 it is more it is anti-, Sample diluting liquid, cleaning solution, developing solution, terminate liquid are constituted, and concrete component is：（1）ELISA Plate：It is coated with by the monoclonal antibody of PD1；（2） Sample diluting liquid：The 0.01mol/l PBS of pH7.4 containing 8% calf serum；（3）Cleaning solution：pH7.4 PBST；（4）Enzyme mark two It is anti-：The goat anti-rabbit igg of horseradish peroxidase-labeled purchased from U.S. R ＆ D systems；（5）PD1 standard items：The PBS of PD1 Solution, final concentration of 16 ng/ml of PD1；（6）PD1 positive control samples：The PBS solution of PD1, final concentration of 16 ng/ml of PD1； （7）Anti- PD1's is mostly anti-：Mostly anti-PBS solution, mostly anti-final concentration of 1mg/ml；（8）Developing solution：TMB-H2O2 systems；（9） Terminate liquid：2mol/L H2SO4 solution.