CN102250911A - Soluble B7-H1 quantitative detection kit - Google Patents
Soluble B7-H1 quantitative detection kit Download PDFInfo
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Abstract
The invention discloses a kit capable of quantitatively detecting soluble B7-H1, which comprises a horseradish peroxidase label, tetramethylbenzidine serving as a reaction substrate, bovine serum albumin, an elisa plate, washing liquor, stop solution, a coating antibody, a standard protein and a detection antibody, wherein the coating antibody is a mouse anti-human B7-H1 monoclonal antibody and the nucleotide sequence of the heavy chain of the coating antibody is represented by a nucleotide sequence SEQ ID No.3 in the sequence list, and the nucleotide sequence of the light chain of the coating antibody is represented by a nucleotide sequence SEQ ID No.4 in the sequence table. The kit capable of quantitatively detecting soluble B7-H1 has high specificity and can be used for accurately quantitative analysis on soluble B7-H1 protein factor concentration in liquid for human cell culture supernate, serum, plasma, hydrothorax and the like.
Description
Technical field
But the present invention relates to the test kit of a kind of detection by quantitative solubility B7-H1, be specifically related to utilize two strain specific anti-human B7-H1 monoclonal antibody ZH11,10D7 and the development of B7-H1Ig fusion rotein to be used for the method for the enzyme-linked immunologic detecting kit of the quantitative detecting analysis solubility B7-H1 factor, this detection by quantitative system can be applicable to lung cancer differential diagnosis and the field is judged in prognosis.
Background technology
Known have many receptor/ligand interactions all to participate in inducing, setting up and regulate the antigen specific immune reaction.For activated T cell reaction effectively, need two signals usually at least.Wherein the MHC-antigenic compound on T cell antigen receptor (TCR) identification antigen presenting cell (APC) is the antigen-specific signal so that first signal to be provided, the also necessary second signal that obtains the costimulatory molecules interaction back generation of T cell and APC expression.Second signal is costimulatory signal or costimulatory signal.Lack costimulatory signal if the antigen-specific signal is only arranged, the T cell will show as reactionless or immune tolerance state, even cause apoptosis.As seen, costimulatory signal is that the amplification of T cell clone, differentiation and performance biological effect institute are requisite.Therefore, first signal deciding specificity of T cell activation, can second signal then determine the T cell-mediated immune responses effectively carry out.
In recent years, Protocols in Molecular Biology is widely used in immunology research, and costimulatory molecules is constantly found.According to its structure, these costimulatory moleculeses can be divided into two classes: a class is tumour necrosis factor/tumor necrosis factor receptor super family, comprises CD40/CD154, CD27/CD27L, CD30/CD30L, 4-1BB/4-1BBL and Fas/FasL etc.Another kind of is immunoglobulin superfamily, as B7/CD28, LAF1/ICAM-1, ICOS-GL50, PD-1/B7-H1, BTLA/HVEM and CD2/LFA-3 etc.These collaborative stimulation molecules are with the interactional mode conducted signal of receptor/ligand.Receptor/ligand generally is expressed in different immunocyte surfaces, and common one is persistence expression, a feature that is the inducible expression.In the different steps of immunne response, these molecules participate in the signal conduction in mode unique and that be associated separately, and immune response effectively start, the appropriateness of regulating and control body jointly replied and stopped in good time.
PD-1(CD279) the immunoglobulin superfamily I type transmembrane glycoprotein of being made up of 288 amino acid is considered to relevant with apoptosis and called after programmed death-1(programmed death-1, PD-1).Discover, the PD-1 molecule mainly is the inducibility up-regulated expression at T, B, NK cell surface, the specified phase that immature T, B cell are grown in thymus gland and marrow also has weak expression, and the tyrosine residues of PD-1 molecule C-terminal can be with signal transducers effects such as SHP-1, the SHP-2 in downstream and suppressed the further activation of immunocyte.B7-H1(B7-H1 CD274) is one of two parts of PD-1, and is the same with other member of B7 family, and the B7-H1 molecule includes IgV sample district, IgC sample district, strides film district and a weak point and conservative cytoplasmic domain afterbody on protein structure.The B7-H1 extracellular region has 4 conserved cysteine residue, participates in the formation of disulfide linkage in the Ig spline structure, keeps correct space structure.The mRNA of B7-H1 not only is expressed in organa parenchymatosum's tissue, as: heart, lung, liver and placenta etc., also there is appropriateness to express on the incomplete antigen presenting cell, as: activatory B cell and dendritic cell; B7-H1 mRNA is high expression level on the tumor cell line in thymic epithelial cells and multiple epithelium source.On the protein level, B7-H1 goes up the inducible expression at activated T, B, monocyte and polytype tumour cell (as: lung cancer, liver cancer, mammary cancer and squamous cell carcinoma etc.).A large amount of studies show that, the PD-1 significantly biological function of retarding effect T cell that interacts on B7-H1 and the activated T cell, the expression of PD-1 or B7-H1 changes that to cause PD-1/PD-L inhibition approach to take place unusual, so cause body produce immunologic function hyperfunction/low property disease.
Dong H etc. discovers, but the propagation of anti-CD3 monoclonal antibody of the excitated type of low dosage and B7-H1Ig combined utilization effective stimulus T cell, promote the secretion of IL-10, this positivity hormesis is the IL-2 dependency, and the research of mouse B7-H1 has also been obtained similar result.But more studies show that afterwards, B7-H1 can with the propagation and the activation of its negative receptor PD-1 interaction suppressor T cell, especially can significantly reduce the secretion of IL-2, IFN-γ and IL-10.B7-H1 mainly depends on the intensity of TCR and CD28 signal to the influence of T cell function, under suboptimal dose TCR token stimulus, B7-H1 is obvious to the restraining effect of T cell proliferation, and when optimal dose TCR signal, only just promoter action is brought into play in T cell proliferation under the condition that does not have the CD28 costimulatory signal, PD-1/B7-H1 suppresses the effect that approach mediated and can be reversed by CD28 signal or IL-2.Therefore, the significantly activation and proliferation and the production of cytokines of suppressor T cell of PD-1/B7-H1 negativity signal.
Suppress the expression of Bcl-xL after PD-1/PD-L interacts, the expression of the multiple transcription factor of pairing effect T cell produces and suppresses, as: GATA-3, Tbet and Eomes.Studies show that of the B lymphoma cell line of external application transfection PD-1 or Fc γ R II-PD-1 antigen-4 fusion protein gene, PD-1 can suppress the pungency signal of B-cell receptor, thereby reduces the B cell activation.Can suppress Ca after BCR and PD-1 are crosslinked
2+The phosphorylation of the tyrosine residues of interior stream and downstream signal activating molecules such as syk, PI3K, Phospholipid hydrolase-3 and vav.What is interesting is that this retarding effect does not need PD-1 cytoplasmic domain N end to be positioned at the tyrosine residues that the ITIM activation suppresses motif to participate in, but the tyrosine residues and the SHP-2 tyrosine phosphatase bonded result that hold by C.In addition, PD-1 also can suppress the tyrosine residues phosphorylation of the signal activation molecule in downstream-mitogen activated protein kinase (MAPK) more and suppress the propagation of B lymphoma cell line.In addition, be the phosphorylation of the Study of model result crosslinked SHP-2 of causing that also shows TCR and PD-1 with the Jurkat cell strain and raise to the cytoplasmic domain of PD-1.
Generation, the development of PD-1/B7-H1 inhibition approach and tumour are closely related.Discover, kinds of tumor cells and tumor tissues composition high expression level B7-H1, as: lung cancer, liver cancer, mammary cancer, squamous cell carcinoma and ovarian cancer.PD-1/B7-H1 suppresses approach to CD8
+The activation and proliferation of T cell has the obvious suppression effect, and tumour cell can be escaped the CTL lethal effect by this approach, weakens antitumor immunity of organism and replys.Can effectively reverse CD4 by the secretion of raising IFN-γ, IL-2, IL-10 with the anti-B7-H1 monoclonal antibody blocking-up of blocking-up type PD-1/B7-H1 signal
+And CD8
+The propagation of T cell is subjected to press down, and the activation degree of T cell and kill capability strengthen simultaneously.This prompting PD-1/B7-H1 approach may be one of reason of tumour cell escape immunity identification and immune clearance.In addition, the tumor microenvironment factor can promote tumor cells expression B7-H1, and the appearance of this molecule and rise help the further growth and the transfer of tumour cell, induces the specific for tumour antigen t cell proliferation of infiltration.Research of Animal Model for Study such as Dong H find, transfection the P815 tumour cell of B7-H1 gene tie up to external cracking of resisting specific CTL, after in its inoculation mouse body, have stronger tumorigenicity and aggressive.Yet these biological characteristicses all can reverse by blocking-up B7-H1.Therefore, can prepare anti-B7-H1 monoclonal antibody of specificity or PD-1 solubility supressor, by blocking-up PD-1/B7-H1 signal the effect of this inhibition approach be sealed, the function of enhanced CT L killing tumor cell effectively suppresses the formation and the growth of tumour.This has opened up new way for immunotherapy of tumors.What is more important, current research show that the expression intensity of B7-H1 can be used as the clinical judgment index of tumour patient prognosis.The PD-1/PD-L approach is being brought into play specific antigen dependency negativity regulating and controlling effect as important cell cycle check point, is one of target molecule of medicine interference body and anti-tumor immunotherapy, has the potential using value.
As the negativity costimulatory signal, no matter range still is the degree of depth to B7-H1 in the immune response process is all playing the part of important role, participates in lymphocyte activation, is bringing into play the effect that can not be substituted aspect immunological tolerance and the immunologic injury.The interaction of PD-1/B7-H1 effectively reduces the expression of IFN-γ and TNF-α, for the treatment of transplant rejection, autoimmune disease and allergy etc. provides some new thinkings.On the one hand, suppress signal to reduce overactivity, the propagation of immune cell by strengthening PD-1, alleviate lymphocyte to the damage of autologous tissue and autoantigen continue reply, also can improve the survival rate of graft by strengthening this negativity signal to alleviate the rejection of host immune system to graft; On the other hand, suppress the responsiveness cellular-restoring biological function that signal makes anergy in the body, promote tumour and virus-specific CD8 by specific inhibition PD-1/B7-H1
+The activation and proliferation of T cell and the secretion of cytokine strengthen the lethality of lymphocyte to the virus of tumour antigen, exotic invasive etc., and enhance immunity power is in time removed tumour cell and virus.PD-1/PD-L is expected to become effective target molecule of immunotherapy of tumors, and also the treatment for HIV and HBV chronic diseases toxinfection provides a new strategy.2006, the U.S. FDA approved one strain Humanized anti-human PD-1 monoclonal antibody, be used for the clinical treatment research of aspects such as tumour and transmissible disease.Along with deepening continuously of research, PD-1/PD-L immunity of organism regulate and the research of all kinds of clinical diseases, prevention, Clinics and Practices in effect will thoroughly be illustrated.
The sickness rate of lung cancer and case fatality rate have all occupied the first place of all kinds of malignant tumours in western countries and China big city.The national for the third time coroner's inquest situation of ministry of Health of China promulgation in 2008 shows: malignant tumour is ranked the first place of the urban population cause of death, and lung cancer ascensional range maximum reaches 465%.Lung cancer local relative hide, cause early diagnosis and prevention difficulty with distant metastasis.Therefore, clinical treatment mainly is that survival rate was only about 10-15% in 5 years for a long time in the face of the medium and advanced lung cancer crowd.Along with oncomolecularbiology and genetic fast development, people recognize that generation, development and the transfer of lung cancer are that a multistage, multistep complex biological rapid, that polygene participates in regulation and control is learned process, and closely related with patient's body's immunological function.Current research is researched and developed effective molecular detection technology except need patients with lung cancer is made the clinical early diagnosis, also needs the signal pathway and the target molecule that play an important role in lung cancer generation, development and immunologic escape are furtherd investigate.In the malignant tumor patient body, the oncocyte that enters lymphatic vessel or blood vessel has only some oncocyte subgroup to be able to dominant growth, obtains metastatic phenotype.How these oncocytes with high metastatic potential escape the immunosurveillance of body, the mechanism of resisting the T cells with antigenic specificity lethal effect is not illustrated as yet fully.In recent years, molecular diagnosis and targeted therapy have become the research focus in lung cancer therapy field, wherein, be used widely in that lung cancer is clinical at the target therapeutic agent epidermal growth factor recipient tyrosine kinase inhibitor (EGFR-TKI) of EGF-R ELISA (EGFR) and vascular endothelial growth factor (VEGF) and VEGF antibody etc., in lung cancer crowd selectively, obtaining obvious existence and benefiting.Along with the understanding to body's immunity regulation mechanism and tumor invasion mechanism is progressively goed deep into, we find that the collaborative stimulation molecule B7-H1 of negativity is at unusual high expression levels of multiple epithelial tumor such as lung cancer, in the developing of tumour, brought into play vital role, be expected to one of important target spot that becomes clinical lung cancer individuation control, and might become the basis, laboratory of lung cancer risk level, curative effect and prognosis evaluation.
Discover that many collaborative stimulation molecules can exist with cell membranous type and two kinds of forms of solubility respectively, comprise CD40, OX40L, 4-1BBL, CD86, CD30 and CTLA-4 etc.Shla molecule can form by the membranous type molecule on the proteolytic enzyme cleaves cell, for example: B7-H3 and s4-1BBL; Also can produce, for example: sCD86 and sCTLA-4 by single-minded mRNA coding.The expression level of many soluble synergistic stimulation molecules has diagnostic value, and for example, soluble CD 40 L (sCD40L) is activatory CD4
+T cell and the cracking of platelet surface membranous type CD40L molecule, sCD40L level raise in the serum of some autoimmune disease unusually, and the sCD40L level is also higher in primary thrombocytosis and arteriosclerotic's the serum.Solubility CD30 molecule also presents high level expression in some tumour and acquired immune deficiency syndrome (AIDS) (AIDS) patient's serum.Showed already that the molecule on shla molecule and film surface is the same in the body had a corresponding biological function, on the one hand, the molecule on the cytolemma can be by the direct receptor/ligand mediation costimulatory signal that interacts; On the other hand, the soluble proteins factor can participate in blood circulation as cytokine and bring into play important regulatory role in immunne response, they can either influence adjacent cells and can mutually combine with the acceptor of far-end cell surface again, thereby the generation of involved in diseases, development, its breadth and depth that plays a role is considerably beyond the molecule of surface of cell membrane.Up to now, for want of effective detection method at home and abroad there is no solubility B7-H1(sB7-H1) the research report.
Summary of the invention
But goal of the invention of the present invention provides the test kit of a kind of detection by quantitative solubility B7-H1.
To achieve the above object of the invention, the technical solution used in the present invention is: but the test kit of a kind of detection by quantitative solubility B7-H1, comprise: horseradish peroxidase-labeled, the reaction substrate tetramethyl benzidine, bovine serum albumin, enzyme plate, standard substance albumen, washing lotion and stop buffer, described test kit also comprises: coated antibody and detection antibody, wherein, described coated antibody is a mouse-anti people B7-H1 monoclonal antibody, its heavy chain has nucleotide sequence shown in SEQ ID NO:1 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:5 in the sequence table, its light chain has nucleotide sequence shown in SEQ ID NO:2 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:6 in the sequence table; Described detection antibody is mouse-anti people B7-H1 monoclonal antibody, its heavy chain has nucleotide sequence shown in SEQ ID NO:3 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:7 in the sequence table, its light chain has nucleotide sequence shown in SEQ ID NO:4 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:8 in the sequence table.
In the optimized technical scheme, described coated antibody is mouse-anti people B7-H1 monoclonal antibody ZH11, the anti-PD-L1 of hybridoma cell strain ZH11(of the CGMCC No. 1637 that by preserving number is) monoclonal antibody of preparation; Described detection antibody is mouse-anti people B7-H1 monoclonal antibody 10D7, is the secretion mouse anti human PD-L1 molecule monoclonal antibody hybridoma cell strain SIPD-L1(10D7 of CGMCC No. 4802 by preserving number) preparation monoclonal antibody.
In the technique scheme, the anti-PD-L1 of hybridoma cell strain ZH11() preservation information is: preserving number: CGMCC No. 1637, the classification name of suggestion: hybridoma, preservation date: on 03 08th, 2006, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; Hybridoma cell strain SIPD-L1(10D7) preservation information is: preserving number: CGMCC No. 4802, the classification name of suggestion: secretion mouse anti human PD-L1 molecule monoclonal antibody hybridoma cell strain, preservation date: on April 27th, 2011, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
In the technique scheme, described standard substance albumen is standard substance protein B 7-H1Ig albumen, is common B7-H1Ig albumen of the prior art.
In the technique scheme, described washing lotion is selected from: contain the 0.002M imidazole salts solution of polysorbas20 (Tween-20) of volume fraction 0.02% ~ 0.2% or the PBS solution of PH7.4, be preferably the PBS solution of the PH7.4 that contains volume fraction 0.02% polysorbas20 (Tween-20).
In the technique scheme, described stop buffer is selected from: the sulfuric acid of 2M or 1%HCl solution; Be preferably the sulfuric acid of 2M.
It is external that described test kit removes the monoclonal anti that comprises anti-cell of the present invention surface B7-H1 molecule, also can comprise blood sample collection device and related solvents, damping fluid and standard substance etc.
The present invention is claimed a kind of nucleotide sequence simultaneously, and described nucleotide sequence is shown in SEQ ID NO:1.
The present invention is claimed a kind of nucleotide sequence simultaneously, and described nucleotide sequence is shown in SEQ ID NO:2.
The present invention is claimed a kind of nucleotide sequence simultaneously, and described nucleotide sequence is shown in SEQ ID NO:3.
The present invention is claimed a kind of nucleotide sequence simultaneously, and described nucleotide sequence is shown in SEQ ID NO:4.
The present invention is claimed a kind of mouse-anti people B7-H1 monoclonal antibody ZH11 simultaneously, and the heavy chain of described monoclonal antibody is nucleotide sequence shown in SEQ ID NO:1 in the sequence table, or shown in SEQ ID NO:5 aminoacid sequence; The light chain of described monoclonal antibody is nucleotide sequence shown in SEQ ID NO:2 in the sequence table, or shown in SEQ ID NO:6 aminoacid sequence.
The present invention is claimed a kind of mouse-anti people B7-H1 monoclonal antibody 10D7 simultaneously, and the heavy chain of described monoclonal antibody is nucleotide sequence shown in SEQ ID NO:3 in the sequence table, or shown in SEQ ID NO:7 aminoacid sequence; The light chain of described monoclonal antibody is nucleotide sequence shown in SEQ ID NO:4 in the sequence table, or shown in SEQ ID NO:8 aminoacid sequence.
The present invention is claimed a kind of hybridoma cell strain simultaneously, and described hybridoma cell strain is the anti-PD-L1 of hybridoma cell strain ZH11(of secretion mouse anti human B7-H1 monoclonal antibody ZH11), its preserving number is CGMCC No. 1637.
The present invention is claimed a kind of hybridoma cell strain simultaneously, and described hybridoma cell strain is the hybridoma cell strain SIPD-L1(10D7 of secretion mouse anti human B7-H1 monoclonal antibody 10D7), its preserving number is CGMCC No. 4802.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
1. but the test kit of detection by quantitative solubility B7-H1 of the present invention has good specificity, accurately the concentration of the solubility B7-H1 protein factor in the liquid such as quantitative analysis person cells and supernatant, serum, blood plasma and hydrothorax.
2. but the test kit of detection by quantitative solubility B7-H1 of the present invention has good sensitivity, can keep good linear relationship in the sB7-H1 of 0.195 ~ 12.5ng/ml concentration range.
Description of drawings
The anti-PD-L1 of hybridoma cell strain ZH11() preservation information is: preserving number: CGMCC No. 1637, the classification name of suggestion: hybridoma, preservation date: on 03 08th, 2006, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; Hybridoma cell strain SIPD-L1(10D7) preservation information is: preserving number: CGMCC No. 4802, the classification name of suggestion: secretion mouse anti human PD-L1 molecule monoclonal antibody hybridoma cell strain, preservation date: on April 27th, 2011, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
Fig. 1 is that antibody is identified figure among the embodiment one;
Fig. 2,3 is the specificity analyses of antibody among the embodiment one;
Fig. 4 is epitope competitive assay figure as a result among the embodiment one;
Fig. 5 is the figure as a result of the specificity analyses of human soluble B7-H1 ELISA test kit among the embodiment two;
The typical curve of gained concentration and OD value when Fig. 6 A～6B is the analysis chart of human soluble B7-H1 ELISA test kit linear detection range among the embodiment two and test kit quantitative analysis;
Fig. 7 can be used for measuring the figure as a result of the sB7-H1 in the gene transfecting cell culture supernatant for human soluble B7-H1 ELISA test kit among the embodiment two;
Fig. 8 is the typical curve of gained concentration and OD value when using the test kit quantitative analysis among the embodiment three;
Fig. 9 is the figure as a result of the expression level of sB7-H1 in the different tumor cell line culture supernatant of quantitative analysis among the embodiment three;
Figure 10 is the figure as a result of the expression level of quantitative analysis different pathological types and peripheral blood from patients with lung cancer sB7-H1 by stages among the embodiment four;
Figure 11 is the figure as a result of the different tumours sizes of quantitative analysis and nodus lymphoideus transferring rate rank patients with lung cancer periphery sB7-H1 expression level among the embodiment four;
Figure 12 is the figure as a result of the relation of sB7-H1 dynamic change and clinical efficacy in the quantitative analysis peripheral blood from patients with lung cancer among the embodiment four;
The ROC curve of Figure 13 for concerning between quantitative analysis sB7-H1 among the embodiment four and each tumor markers;
Figure 14 analyzes the figure as a result of the expression characterization of this factor in different ages section healthy volunteer peripheral blood for analyzing solubility B7-H1 detection architecture among the embodiment five;
Figure 15 is the figure as a result that Application Example three described test kits are analyzed the expression level of solubility B7-H1 in peripheral blood from patients with lung cancer is clear among the embodiment five;
Figure 16 is the figure as a result that Application Example three described test kits are analyzed the expression level of solubility B7-H1 in the patients with lung cancer hydrothorax among the embodiment five.
Embodiment
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment one:
(1) reagent and material:Calf serum Hyclone company (U.S.); Add calf serum 100ml, L-glutaminate 0.15g, NaHCO in every liter of RPMI1640 or the DMEM substratum (Gibco, the U.S.)
32.0g, Sodium.alpha.-ketopropionate 0.11g, glucose 3.6g, HEPES 4.766g, 2 mercapto ethanol 10.0ml; HAT, HT select HAT, the HT selection substratum (Sigma, the U.S.) of substratum with 50 times of concentration, are working concentration with RPMI1640 or the dilution of DMEM perfect medium; Freund adjuvant (Freund ' adjuvant, Sigma, the U.S.); Cytogamy agent polyoxyethylene glycol (PEG1500); Protein G is affine the layer suction post (Pharmacia, Sweden); Pristane(Sigma, the U.S.).Tissue Culture Flask, plate (Nunc, Denmark); CO2 incubator, whizzer (Jouan, France), inverted microscope (O1ympus, Japan), flow cytometer (Coulter, the U.S.); Changeing people B7-H1 gene cell strain L929/B7-H1(makes up).The all cells strain does not all have mycoplasma contamination after testing; The female Balb/c mouse (Shanghai Experimental Animal Center) in 6 ~ 8 ages in week.
(2) cultivation of cell strain:Employing contains the RPMI1640 substratum of 10% FCS, and resistance screening medicine Zeocin(500 μ g/ml is regularly used in the gene transfecting cell strain) the pressurization cultivation, to keep the stable high expression level of goal gene product.L929/B7-H1 and L929/mock cell are the growth of adherent property, when going down to posterity with 0.25% trysinization; Other cell is the suspension growth, changes liquid at interval in 2 ~ 3 days.
(3) animal immune:Collect well-grown L929/B7-H1 cell, after PBS washing two times, add mitomycin solution (50 μ g/100 μ l/1 * 107), mixing places 37 ℃, 45min, after the PBS washing three times, use the physiological saline of 0.3 ~ 0.4ml to suspend again again, fully emulsified with the IFA of equivalent, respectively at subcutaneous multiple spot of neck and abdominal injection (1 * 10
7/ only); And the 3rd week, the 5th week row immunity again behind initial immunity, change cell quantity into 8 * 10
6/ only, the pre-treatment of cell and the position of injection are the same, and in merging preceding 4 ~ 5 days, abdominal injection 5 * 10 once more
6/ only with booster immunization.
(4) myeloma cell's preparation:Merged preceding 10 ~ 14 days, recovery murine myeloma cell SP2/0 with the DMEM substratum that contains 10% FCS cultivations of going down to posterity, treat that the cell growth is vigorous, form is good, and cell viability can be used for fusion greater than 95%.Merge preceding 24 ~ 36h and cell concn is adjusted into 3 * 10 with fresh culture
5/ ml.
(5) preparation of feeder cell:In merging preceding 1 day, get the Balb/c mouse, the excision eyeball causes death, and the tap water flushing is placed on 10min in 75% alcohol, is fixed in then and dissects on the frame, open the mouse thoracic cavity along breastbone, cut thymus gland, place 200 purpose woven wires, the screen cloth immigration is filled in the plate of 10ml basic medium, grind thymus gland, make it become individual cells and be suspended from the substratum; Draw a little cell suspension counting, 1000rpm, centrifugal 8min, adjusting cell concn with perfect medium is 3 ~ 4 * 10
5/ ml, in 96 well culture plates, every hole adds the 0.1ml cell suspension and (is equivalent to 3 ~ 4 * 10
4/ hole), CO
2Cultivate in the incubator.
(6) preparation of immune mouse spleen cell:The mouse of getting behind the booster immunization is put to death as stated above, handles mouse, opens the abdominal cavity, takes out spleen at left back belly, obtains single spleen cell through grinding.Expect that with tongue blue liquid has nuclear counting as viable cell, centrifugal (1000rpm * 8min) is suspended from sedimentation cell among the 20ml RPMI1640 after washing twice, places incubator standby.
(7) fusion of cell and selectivity are cultivated:RPMI1640 or DMEM basic medium, PEG solution are placed the pre-temperature of 37 ℃ of water-baths.Collect 2 * 10
7Individual well-grown SP2/0 cell is with 1 * 10
8Individual spleen cell is mixed in the 50ml transparent plastics centrifuge tube, with RPMI1640 washing twice, abandons most supernatant behind the centrifugal 8min of 1000rpm, at the bottom of finger attack pipe, makes two kinds of sedimentation cells fully be mixed into pasty state.Plastics tubing is placed 37 ℃ of thermos cups, draw PEG solution 1ml, the tip suction pipe is inserted the cell suspension bottom gently, in 1min, at the uniform velocity add, and the limit edged stirs gently, in water-bath, leave standstill 90s then.The serum-free DMEM that adds 37 ℃ of pre-temperature of 40ml again, the static 5min of room temperature, centrifugal (800rpm * 10min), abandon supernatant.The DMEM that the resuspended gently 40ml of placing of sedimentation cell is contained 2%HAT, 15%FCS cultivates.Drip in containing 96 well culture plates of feeder cell 100 μ l/ holes, 37 ℃, 5%CO behind the mixing
2Cultivate, amount was changed liquid in 3 ~ 4 days half, used the HT substratum after 10 days instead, the DMEM substratum of migrating after 2 weeks and containing 15%FCS.
(8) hybridoma screening:When treating that cell clone is covered with 1/3 ~ 1/4 culture hole, can draw supernatant and screen with the indirect immunofluorescence labelling method.Well-grown L929/B7-H1 cell with after the PBS washing, is divided to be added in the streaming pipe (5 * 10
5/ pipe), the culture supernatant (50 μ l/ pipe) that adds hybridoma.In 4 ℃ of reaction 30min, with the PBS washing twice that contains 1% calf serum, the sheep anti-mouse igg two that adds the PE mark is anti-, again in 4 ℃ of reaction 30min, washing back facs analysis, the while with L929/mock, L929/B7.1, L929/PD-L2 cell as the negative control cell strain.
The L929/B7-H1 gene transfecting cell of stably express people B7-H1 is identified, the continuous 3 time cloning cultivations of the clone that repetition measurement is positive, the final hybridoma cell strain that obtains 2 strains energy continuous release specific anti-human B7-H1 molecule, called after ZH11 and 10D7, the flow cytometer detected result shows, this monoclonal antibody energy specific recognition people B7-H1, and do not combine (Fig. 1) with gene transfecting cells such as mock, B7.1 and PD-L2.
(9) hybridoma cloning is cultivated:Adopt limiting dilution assay, behind the positive porocyte accurate counting of antibody response, selecting the substratum gradient dilution with HAT is 50/ml and 10/ml cell, adds 100 μ l/ holes in 96 well culture plates that contain feeder cell.Make every hole on average contain 5 and 1 cell, 37 ℃, 5%CO
2Cultivate.Change liquid in good time and in time carry out multiple sieve by the screening method of the positive colony of having set up according to the growth conditions of cell.Select the antibody titer height, be single clonal growth, the form good cell continues the clone, until the antibody-secreting positive rate greater than 95%; Enlarged culturing and timely liquid nitrogen cryopreservation.
(10) MONOCLONAL ANTIBODIES SPECIFIC FOR:Adopt in the ascites body and induce method manufacture order clonal antibody.Get the female Balb/c mouse in 6 ~ 8 ages in week, the abdominal cavity only injects Pristane 0.5ml/.One all pneumoretroperitoneum injection hybridomas 1 * 10
7/ only, while opposite side abdominal cavity re-injects Pristane and Fu Shi half assistant equal-volume mixture 0.2ml/, massages mouse web portion gently, and hybridoma is scattered in the abdominal cavity.Gather in the crops ascites after 5 ~ 10 days ,-80 ℃ of preservations after the packing of centrifuging and taking supernatant.
(11) antibody purification:Ascites is thawed, and centrifugal removal grumeleuse is got supernatant liquor and is added the saturated (NH of equal-volume by 50% saturation ratio
4)
2SO
4Solution, mixing rearmounted 4 ℃ 4 ~ 6 hours, the centrifugal supernatant of abandoning.Get the PBS redissolution that precipitation adds PH7.4.(NH is removed in dialysis
4)
2SO
4, sample is used PH2.8 glycine-hydrochloric acid wash-out again through the affine layer of Protein G suction post.Collect the Tris-Base adjusting PH to 7.0 of albumen elution peak with PH9.0, measure the content of antibody protein after the PBS dialysis with ultraviolet spectrophotometer, the calculation formula of its concentration is: antibody protein content (mg/ml)=OD280 * 1.55-OD260 * 0.76.Result: monoclonal antibody ZH11 and 10D7 concentration are respectively 2.16mg/ml and 1.93mg/ml.
(12) monoclonal antibody titration:Well-grown L929/B7-H1 cell is divided in the streaming pipe 5 * 10
5/ pipe.Culture supernatant, ascites and the antibody purified albumen gradient diluent (20 μ l/ pipe) that add hybridoma respectively, in 4 ℃ of reaction 30min, PBS washing back adds sheep anti-mouse igg-PE, again in 4 ℃ of reaction 30min.Washing back flow cytometry analysis, the maximum dilution multiple when same positive rate and fluorescence intensity occurring is tiring of antibody.The result shows: tiring of two strain antibodies all reaches more than the 1:10000.
(13) type identification of the grand antibody of monoclonal antibody:Get Hybridoma Cell Culture supernatant 1:50, ascites 1:20000 dilution.Getting the 0.2ml diluent is added in the small test tube that contains the qualitative reagent of subclass, room temperature is placed 1min, treat its dissolve naturally after mixing gently, with the qualitative test strip of subclass gently in the tubular stinger, behind 5 ~ 10min, the one side of test paper occurs and the corresponding blue chromoprotein colour developing band of mAb heavy chain title, and this i.e. the affiliated mouse Ig subclass of this antibody; And another side occurs and the corresponding blue chromoprotein band of mAb light chain title, and this i.e. the light chain type of this mAb.Above-mentioned two monoclonal antibodies are checked order, sequencing result is seen SEQ ID No:1 ~ 8 of Nucleotide/aminoacid sequence table, wherein, the heavy chain of mouse-anti people B7-H1 monoclonal antibody ZH11 has nucleotide sequence shown in SEQ ID NO:1 in the sequence table, or shown in SEQ ID NO:5 aminoacid sequence; Its light chain has nucleotide sequence shown in SEQ ID NO:2 in the sequence table, or shown in SEQ ID NO:6 aminoacid sequence; The heavy chain of mouse-anti people B7-H1 monoclonal antibody 10D has nucleotide sequence shown in SEQ ID NO:3 in the sequence table, or shown in SEQ ID NO:7 aminoacid sequence; Its light chain has nucleotide sequence shown in SEQ ID NO:4 in the sequence table, or shown in SEQ ID NO:8 aminoacid sequence.
(14) specificity of monoclonal antibody is identified:With cell pyrolysis liquid or human IgG, B7-H1Ig, PD-L2Ig, B7.1Ig and CD28Ig albumen carbonate buffer solution (0.01M such as L929/mock, L929/B7-H1, L929/PD-L2, L929/B7.1 and L929/B7.2, PH9.3) be adjusted into 0.2 μ g/ml bag by the ELISA check-out console, 4 ℃ are spent the night.Seal 1h for 37 ℃ with 2% BSA after washing 2 times, wash the mouse-anti people B7-H1 monoclonal antibody that adds the development acquisition after 2 times, 37 ℃ of reaction 1h, the washing back adds HRP-sheep anti-mouse igg two and resists (1:1000,100 μ l/ holes), 37 ℃ are continued reaction 1h, and PBS washing 6 times adds freshly prepared substrate TMB(100 μ l again, Sigma, the U.S.), 37 ℃ of reaction 15min use 2mol/L H
2SO
4(50 μ l/ hole) stops the reaction of enzyme-to-substrate, measures OD with microplate reader
450
With cell pyrolysis liquid bags such as L929/mock, L929/B7-H1, L929/PD-L2, L929/B7.1 and L929/B7.2 by elisa plate, detected result shows, but B7-H1 albumen (Fig. 2) in ZH11 that development obtains and the 10D7 monoclonal antibody specific recognition cell pyrolysis liquid; Equally, but also specific recognition commercialization B7-H1Ig recombinant protein (Fig. 3) of this two strains monoclonal antibody.
(15) biotin labeling monoclonal antibody:Get anti-people B7-H1 monoclonal antibody (10D7) 200 μ g to be marked, add and contain in the 0.01M PH=9.3 carbonic acid buffer dialysis tubing of 1ml, after 4 ℃ of following dialysis equilibriums spend the night, adding concentration is the BNHS-DMSO solution 40 μ l of 1mg/ml, room temperature lucifuge reaction 4 hours, again in PH7.2 PBS, dialysing 72 hours-20 ℃ of preservations after the packing under 4 ℃.
(16) antibody competition experiment:With L929/B7-H1 cell (5 * 10
5/ pipe) with the monoclonal antibody (every pipe 0.016 ~ 10 μ g) that adds different concns after the PBS washing respectively, hatch 30min for 4 ℃, the washing back adds the monoclonal antibody (every pipe 1 μ g) of Biotin mark, hatch 30min for 4 ℃ again, the washing back adds streptavidine-PE, hatch 30min for 4 ℃ and also use flow cytometry analysis in the washing back, establish the positive and negative control group simultaneously.
With L929/B7-H1 is that the competition target cell is analyzed the antigen site that monoclonal antibody ZH11 and the 10D7 that obtains discerned, competition through immunofluorescence label suppresses experiment, the result shows that the 10D7 of different concns all can not effectively block the ZH11 of Biotin mark and combining of L929/B7-H1.Therefore, the 10D7 identified epitope is different from ZH11(Fig. 4).
Above-mentioned two hybridoma cell strains are carried out preservation, the anti-PD-L1 of hybridoma cell strain ZH11() preservation information is: preserving number: CGMCC No. 1637, the classification name of suggestion: hybridoma, preservation date: on 03 08th, 2006, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; Hybridoma cell strain SIPD-L1(10D7) preservation information is: preserving number: CGMCC No. 4802, the classification name of suggestion: secretion mouse anti human PD-L1 molecule monoclonal antibody hybridoma cell strain, preservation date: on April 27th, 2011, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
Embodiment two:
But the test kit of a kind of detection by quantitative solubility B7-H1 comprises following component:
(1) coated antibody: embodiment one described mouse-anti people B7-H1 monoclonal antibody (ZH11), 30 μ g/ pipe, 1 pipe;
(2) standard substance albumen: B7-H1Ig albumen (R﹠amp; D), 25ng/ pipe, 1 pipe;
(3) detect antibody: embodiment one described mouse-anti people B7-H1 monoclonal antibody (Biotin-10D7), 10 μ g/ pipe, 1 pipe;
(4) this horseradish peroxidase-labeled Streptavidin-HRP (Sigma-Aldrich), 1 μ l//pipe, 1 pipe;
(5) reaction substrate: tetramethyl benzidine TMB (Sigma-Aldrich), 10ml;
(6) bovine serum albumin Bovine Serum Albumin BSA (worker is given birth in Shanghai), 2g/100ml, 30ml;
(7). enzyme plate elisa plate (Costar), 1;
(8). washing lotion: 10 * PBS, 100 ml; Tween-20,0.5 ml;
(9). stop buffer: 2M H
2SO
4, 5 ml;
Storage requirement:
The specificity analyses of mentioned reagent box:
1. coated antibody ZH11 is mixed with the coated antibody working fluid of 3 μ g/ml concentration with 0.01M CBS (pH9.6) damping fluid, adds elisa plate by 100 μ l/ holes, in 4 ℃ of standing over night;
2. abandon coating buffer, wash plate 3 times with 0.01M PB; Add 2%BSA 200 μ l/ holes, room temperature sealing 1h; Contain 0.5%BSA with PBS() be sample diluting liquid, prepare the proteic solution of sB7-H1, sB7.1, sPD-L2, sB7-H3 of a series of concentration known, prepare working sample simultaneously;
3. discard confining liquid, 0.01M PB washes plate 3 times, adds the proteic solution of sB7-H1, sB7.1, sPD-L2, the sB7-H3 100 μ l/ holes of a series of concentration known, room temperature reaction 2h; Cells and supernatant, blood serum sample add elisa plate by 100 μ l/ holes;
4. discard reaction solution, 0.01M PB washes plate 2 times, and it is 1 μ g/ml that detection antibody Biotin-10D7 is adjusted to final concentration with antibody diluent, adds elisa plate, room temperature reaction 2h by 100 μ l/ holes;
5. wash plate 3 times with 0.01M PB after abandoning most sample; Add Streptavidin-HRP(1:5000), 100 μ l/ holes, room temperature reaction 1h;
6. abandon most reaction solution and contain 0.2% Tween20 with 0.01M PB() wash plate 8-10 time;
7. add reaction substrate TMB, 100 μ l/ holes, room temperature lucifuge reaction 10-15min; 2M H
2SO
4Stop;
8. using microplate reader selects OD450 nm wavelength to measure the OD reading that elisa plate respectively detects the hole;
Obtain the graph of relation of OD reading and each protein concentration, as shown in Figure 5.
Then, analyze the linear detection range of above-mentioned test kit, dispose the proteic solution of sB7-H1 of a series of concentration known, detect then, obtain the graph of relation of OD reading and sB7-H1 protein concentration, shown in Fig. 6 A ~ 6B.
Use the sB7-H1 in the mentioned reagent box mensuration gene transfecting cell culture supernatant, specifically may further comprise the steps: L929/mock, L929/PD-L2 and L929/B7-H1 cell (5 * 10
4/ hole) cultured continuously in 24 well culture plates, every interval 12h collects culture supernatant.(0.01M CBS pH9.3) is adjusted into 3 μ g/ml bag by the ELISA check-out console, and 4 ℃ are spent the night with carbonate buffer solution with anti-B7-H1 monoclonal antibody (ZH11).PBS(contains 0.1% Tween 20) wash 2% BSA room temperature sealing 1h 3 times.After washing 3 times, PBS adds commercialization standard substance B7-H1 albumen, room temperature reaction 2h, PBS washing 3 times.Then, add biotin labeled monoclonal antibody biotin-10D7(1 μ g/ml, 100 μ l/ holes), room temperature continues reaction 1h, after the PBS washing 3 times, add Streptavidin-HRP(1:3000,100 μ l/ holes), room temperature reaction 1h, PBS washing 6-8 time, add HRP reaction substrate TMB(100 μ l/ hole again), room temperature reaction 10-15min uses 2mol/L H
2SO
4Termination reaction is measured OD with microplate reader
450, each sample is provided with 3 multiple holes.Finally obtain Fig. 7.
In sum, the specificity of this test kit is good, and has good linear relationship between 195-12500pg.
Embodiment three: embodiment two described test kits can be applicable to the concentration of the solubility B7-H1 protein factor in the liquid such as quantitative analysis person cells and supernatant, serum, blood plasma and hydrothorax.
The method of sample preparation is respectively:
1. cells and supernatant: 2500 rpm * 10 min, multigelation is avoided in-20 ℃ of preservations.
2. serum sample: 2500 rpm * 10 min, multigelation is avoided in-20 ℃ of preservations.
3. plasma sample: 2500 rpm * 10 min, multigelation is avoided in-20 ℃ of preservations.
The detection by quantitative operation steps:
1. coated antibody ZH11 is mixed with the coated antibody working fluid of 3 μ g/ml concentration with 0.01M CBS (pH9.6) damping fluid, adds elisa plate by 100 μ l/ holes, in 4 ℃ of standing over night;
2. abandon coating buffer, wash plate 3 times with 0.01M PB; Add 1%BSA 200 μ l/ holes, room temperature sealing 1h;
3. discard confining liquid, 0.01M PB washes plate 3 times; Contain 0.5%BSA with PBS() be sample diluting liquid, prepare working sample.Standard protein:
Get 7 EP pipes, every pipe adds 100 μ l diluents and places on the EP pipe support;
The pure product albumen of 100 μ l 50ng/ml B7-H1Ig is added to first EP pipe, gets l to the second EP pipe of 100 μ behind the mixing again and carry out doubling dilution, the rest may be inferred to the 7th pipe.Cells and supernatant, blood serum sample add elisa plate by 100 μ l/ holes;
It is 1 μ g/ml that detection antibody Biotin-10D7 is adjusted to final concentration with antibody diluent;
4. add standard substance and testing sample 100 μ l/ holes, room temperature reaction 2h;
5. wash plate 3 times with 0.01M PB after abandoning most sample; Add Streptavidin-HRP(1:5000), 100 μ l/ holes, room temperature reaction 1h;
6. abandon most reaction solution and contain 0.2% Tween20 with 0.01M PB() wash plate 8-10 time;
7. add reaction substrate TMB, 100 μ l/ holes, room temperature lucifuge reaction 10-15min; 2M H
2SO
4Stop;
8. using microplate reader selects OD450 nm wavelength to measure the OD reading that elisa plate respectively detects the hole.
Interpretation of result:
1. standard substance OD value adopts the matched curve of GraphPad Software software utilization the best to list only functional equation;
2. the OD value substitution equation of sample/control wells, and multiply by corresponding extension rate, calculate the respective concentration of sample and control wells;
3. sample well is got the mean value in multiple hole.
Experimental applications is given an example:
1. raw data:
2. curvilinear equation:
y= 0.0447x + 0.0938，
R2=0.9922; Wherein, y represents the OD value, and x represents sB7-H1 concentration, and unit is ng/ml;
3. the typical curve of Hui Zhiing as shown in Figure 8.
Analysis of the accuracy:
Method:
1. analysis of the accuracy in the plate: with in the experiment once, the sample (12.5,6.25,3.125 ng/ml) of three concentration known is established 20 multiple holes respectively, carry out sB7-H1 and detect, analyze the accuracy of this test kit;
2. analysis of the accuracy between plate: in these different experiments chambers 24, the sample (12.5,6.25,3.125 ng/ml) of three concentration known is carried out B7-H3 respectively detect, analyze the accuracy of this test kit;
The result:
Conclusion: variation coefficient CV＜10% confirms that detection method all has good accuracy.
Practical application is given an example:
1, the expression level of sB7-H1 in the different tumor cell line culture supernatant of quantitative analysis gets Fig. 9;
2, the expression level of quantitative analysis different pathological types and peripheral blood from patients with lung cancer sB7-H1 by stages gets Figure 10;
3, different tumour sizes of quantitative analysis and nodus lymphoideus transferring rate rank patients with lung cancer periphery sB7-H1 expression level get Figure 11;
4, the relation of sB7-H1 dynamic change and clinical efficacy in the quantitative analysis peripheral blood from patients with lung cancer gets Figure 12;
5, the ROC curve that concerns between quantitative analysis sB7-H1 and each tumor markers gets Figure 13;
In sum, can use the ELISA test kit of specific assay solubility B7-H1 of the present invention that lung cancer is carried out differential diagnosis, the progress of patients with lung cancer in treatment clinical course estimated.Have the sB7-H1 high expression level in the patients with lung cancer serum, and the tumor load of this high expression level and lung cancer is relevant with extent of disease, and the sB7-H1 level is higher than serum content in the focal as pernicious hydrothorax of tumour.Clinical Follow-up discovers that the expression level of this soluble factor and clinical efficacy are negative correlation in the patients with lung cancer serum.It is external that described test kit removes the monoclonal anti that comprises anti-cell of the present invention surface B7-H1 molecule, also can comprise blood sample collection device and related solvents, damping fluid and standard substance etc.
Embodiment five:
Application Example two described test kits, analyze solubility B7-H1 detection architecture and analyze the expression characterization of this factor in different ages section healthy volunteer peripheral blood, get Figure 14, Figure 14 shows, in healthy human serum, have the expression of sB7-H1, and the expression level of sB7-H1 in human serum increases along with the increase at age.Among the crowd, 3-10 year children's expression level is minimum, and the elderly in 51-70 year then has than high expression level.Two intermediate ages sections adult's expression level is then placed in the middle.
Application Example two described test kits are analyzed the expression level of solubility B7-H1 in peripheral blood from patients with lung cancer is clear, get Figure 15, the ELISA of Figure 15 display application specific detection people sB7-H1 carries out quantitatively the expression level of this factor in the periphery serum of patients with lung cancer, the result shows, compare with the healthy volunteer, the content of sB7-H1 significantly raises in the patients with lung cancer serum, and the average content of this factor is more than the twice of healthy people's contrast.
Application Example two described test kits are analyzed the expression level of solubility B7-H1 in the patients with lung cancer hydrothorax, get Figure 16, the ELISA of the specific detection people sB7-H1 that Figure 16 display application is set up analyzes 10 routine patients with lung cancer hydrothorax and periphery serum, the result shows that the expression amount of sB7-H1 is significantly higher than the level in the peripheral blood in the patients with lung cancer hydrothorax.
Nucleotide and/or aminoacid sequence table
＜110〉University Of Suzhou
＜120〉a kind of solubility B7-H1 detection by quantitative test kit
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 420
<212> DNA
＜213〉the unknown
<400> 1
gtcaagctgc agcagtcagg agctgagctg gtgaagcctg gggcttcagt gaagctgtcc 60
tgcaagactt ctggcttcac cttcaacagt agctatataa gttggttgag gcaaaagcct 120
ggacagagtc ttgagtgtat tgcatggatt tatgctggaa ctggcggtac taactataat 180
cagaagttca cagacaaggc ccaactgact gtagatacat cctccagcac agcctacatg 240
caattcagca gcctgacaac tgaggactct gccatctatt actgtgcaag acacccccgg 300
agttttttct atggtatgga ctactggggt caaggaacct cagtcaccgt ctcctcagcc 360
aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 420
<210> 2
<211> 380
<212> DNA
＜213〉the unknown
<400> 2
cacattgtga tgacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60
atgacctgca gtgccagctc aagtgtaact tccatgcact ggtaccagca gaagtcaggc 120
acctccccca aaggatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccagcagtgg agtaataacc cactcacgtt cggtgctggg 300
accaagctgg agctgaaacg ggctgatgct gcaccaactg tatcaatcgt cgacctgcag 360
gcatgcaagc ttggcgtaat 380
<210> 3
<211> 396
<212> DNA
＜213〉the unknown
<400> 3
gagcagtcag ggggagactt agtgaagcct ggagggtccc tgaaactctc ctgtgcagcc 60
tctggattca ctttcagtta ttatgccatg tcttgggttc gccagactcc agagaagagg 120
ctggagtggg tcgcatccat tagtagtcgt ggtgacacct actatccaga cattgtgaag 180
ggccgattca ccatctccag agataatgcc aggaacatcc tgtaccttca aatgagaagt 240
ctgcggtctg aggacacggc cttgtattac tgtgcaagag gggggggaaa ctacgaggga 300
ttggattatt ggggtcaagg aacctcattc accatctcct cagccaaaac gacaccccca 360
tctgtctatc cactggcccc tggatctgct gcccaa 396
<210> 4
<211> 353
<212> DNA
＜213〉the unknown
<400> 4
tcgacgattc agattgggat gacccagtct ccagcaatca tgtctgcatc tccaggggag 60
aaggtcacca tgacctgcag tgtcagctca agtattactt tcatgtactg gttccagcag 120
aagccaggat cctcccccag actcctgatt tatgacacat ccaagctggc ttctggagtc 180
cctgttcgct tcagtggcag tgggtctggg acctcttact ctctcacaat cagccgaatg 240
gaggctgaag atgctgccac ttattactgc cagcagtgga gtggttaccc gtacacgttc 300
ggagggggga ccaagctgga aataaaacgg gctgatgctg caccaactgt atc 353
<210> 5
<211> 140
<212> PRT
＜213〉the unknown
<400> 5
VKLQQSGAEL VKPGASVKLS CKTSGFTFNS SYISWLRQKP GQSLECIAWI YAGTGGTNYN 60
QKFTDKAQLT VDTSSSTAYM QFSSLTTEDS AIYYCARHPR SFFYGMDYWG QGTSVTVSSA 120
KTTPPSVYPL APGSAAQTNS 140
<210> 6
<211> 127
<212> PRT
＜213〉the unknown
<400> 6
HIVMTQSPAI MSASPGEKVT MTCSASSSVT SMHWYQQKSG TSPKGWIYDT SKLASGVPAR 60
FSGSGSGTSY SLTISSMEAE DAATYYCQQW SNNPLTFGAG TKLELKRADA APTVSIVDLQ 120
ACKLGVX 127
<210> 7
<211> 132
<212> PRT
＜213〉the unknown
<400> 7
EQSGGDLVKP GGSLKLSCAA SGFTFSYYAM SWVRQTPEKR LEWVASISSR GDTYYPDIVK 60
GRFTISRDNA RNILYLQMRS LRSEDTALYY CARGGGNYEG LDYWGQGTSF TISSAKTTPP 120
SVYPLAPGSA AQ 132
<210> 8
<211> 118
<212> PRT
＜213〉the unknown
<400> 8
STIQIGMTQS PAIMSASPGE KVTMTCSVSS SITFMYWFQQ KPGSSPRLLI YDTSKLASGV 60
PVRFSGSGSG TSYSLTISRM EAEDAATYYC QQWSGYPYTF GGGTKLEIKR ADAAPTVS 118
Claims (9)
1. a nucleotide sequence is characterized in that, described nucleotide sequence is shown in SEQ ID NO:1.
2. a nucleotide sequence is characterized in that, described nucleotide sequence is shown in SEQ ID NO:2.
3. a nucleotide sequence is characterized in that, described nucleotide sequence is shown in SEQ ID NO:3.
4. a nucleotide sequence is characterized in that, described nucleotide sequence is shown in SEQ ID NO:4.
5. a mouse-anti people B7-H1 monoclonal antibody ZH11 is characterized in that, the heavy chain of described monoclonal antibody is shown in SEQ ID NO:1 in the sequence table shown in the nucleotide sequence, or shown in SEQ ID NO:5 shown in the aminoacid sequence; The light chain of described monoclonal antibody is shown in SEQ ID NO:2 in the sequence table shown in the nucleotide sequence, or shown in SEQ ID NO:6 shown in the aminoacid sequence.
6. a mouse-anti people B7-H1 monoclonal antibody 10D7 is characterized in that, the heavy chain of described monoclonal antibody is shown in SEQ ID NO:3 in the sequence table shown in the nucleotide sequence, or shown in SEQ ID NO:7 shown in the aminoacid sequence; The light chain of described monoclonal antibody is shown in SEQ ID NO:4 in the sequence table shown in the nucleotide sequence, or shown in SEQ ID NO:8 shown in the aminoacid sequence.
7. a hybridoma cell strain is characterized in that, described hybridoma cell strain is the anti-PD-L1 of hybridoma cell strain ZH11(of secretion mouse anti human B7-H1 monoclonal antibody ZH11), its preserving number is CGMCC No. 1637.
8. a hybridoma cell strain is characterized in that, described hybridoma cell strain is the hybridoma cell strain SIPD-L1(10D7 of secretion mouse anti human B7-H1 monoclonal antibody 10D7), its preserving number is CGMCC No. 4802.
9. but the test kit of a detection by quantitative solubility B7-H1, comprise: horseradish peroxidase-labeled, reaction substrate tetramethyl benzidine, bovine serum albumin, enzyme plate, standard substance albumen, washing lotion and stop buffer, described test kit also comprises: coated antibody and detection antibody, it is characterized in that described coated antibody is the described mouse-anti people of claim 5 a B7-H1 monoclonal antibody; Described detection antibody is the described mouse-anti people of claim 6 B7-H1 monoclonal antibody.
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