CN102250910B - Human soluble B7-DC quantitative detection kit - Google Patents
Human soluble B7-DC quantitative detection kit Download PDFInfo
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Abstract
The invention discloses a kit capable of quantitatively detecting soluble B7-DC. The kit comprises a horse radish peroxidase marker, tetramethyl benzidine serving as a reaction substrate, bovine serum albumin, an elisa plate, a wash solution and a stop solution, and is characterized by further comprising a coated antibody, standard proteins and a detection antibody, wherein the coated antibody is a mouse anti-human B7-DC monoclonal antibody 8F2; and the detection antibody is a mouse anti-human B7-DC monoclonal antibody 10D6. The kit capable of quantitatively detecting soluble B7-DC has high specificity, can be used for accurately and quantitatively analyzing the concentrations of soluble B7-DC protein factors in liquids such as human cell culturing supernatant, blood serum, blood plasma, hydrothorax and the like, and can be applied to clinical differential diagnosis and treatment effects of asthma.
Description
Technical field
But the present invention relates to the test kit of a kind of detection by quantitative human soluble B7-DC, be specifically related to a kind of enzyme-linked immunologic detecting kit that is used for the quantitative detecting analysis solubility B7-DC factor that utilizes two strain specific murine anti-human B7-DC monoclonal antibody 8F2 and 10D6 and the preparation of B7-DCIg fusion rotein, this detection by quantitative system can be used asthma differential diagnosis and the curative effect judgement field of meeting.
Background technology
Known many receptor/ligand interact and all participate in inducing, setting up and regulate the antigen specific immune reaction.For effectively activated T cell reaction, usually need at least two signals.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 second signal that produces after the costimulatory molecules that also needs T cell and APC to express interacts, i.e. 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 apoptosis.As seen, costimulatory signal is that the amplification of T cell clone, differentiation and performance biological effect institute are requisite.Therefore, first signal has determined the specificity of T cell activation, and second signal determines that then can the cell-mediated immunne response of T 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 molecules 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, such as B7/CD28, LAF1/ICAM-1, ICOS-GL50, PD-1/B7-DC, BTLA/HVEM and CD2/LFA-3 etc.These costimulatory molecules are with the interactional mode conducted signal of receptor/ligand.The receptor/ligand General Expression is on different immunocyte surfaces, and common one is persistent 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 effectively start, the appropriateness of jointly regulating and control organism immune response replied and stopped in good time.
PD-1(CD279) the immunoglobulin superfamily I type transmembrane glycoprotein that is comprised of 288 amino acid is considered to relevant with apoptosis and called after programmed death-1(programmed death-1, PD-1).Research is found, 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 the further activation of Immunosuppression cell.B7-DC is one of two parts of PD-1, and is the same with other member of B7 family, and the B7-DC molecule includes IgV sample district, IgC sample district, cross-film district and a short and conservative cytoplasmic domain afterbody on protein structure.The people B7-DC assignment of genes gene mapping is in 9p24.2,247 amino acid of can encoding, and its cDNA total length is 1.7kb.People's B7-DC mRNA is high expression level in placenta, liver cancer cell, breast cancer cell and neuroblast, express but be low at spleen, lymphoglandula, thymus gland with in becoming fibrous tissue.B7-DC not only expresses the tissue the organa parenchymatosum, also has appropriateness to express on the incomplete antigen presenting cell, as: T cell and the dendritic cell of activation.The distribution of B7-DC is limited to relatively, mainly is expressed in monocytes/macrophages and the dendritic cell (DCs) of activation.But peripheral mononuclear cells is induced the DC high expression level B7-DC of generation through IL-4 and GM-CSF.A large amount of studies show that, the PD-1 significantly biological function of retarding effect T cell that interacts on the T cell of B7-DC and activation, the expression of PD-1 or B7-DC changes and will cause PD-1/PD-L to suppress the approach abnormal, so cause body produce immunologic function hyperfunction/low property disease.
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, 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 result of being combined with the SHP-2 tyrosine phosphatase by the tyrosine residues that C holds.In addition, PD-1 also can suppress the tyrosine residues phosphorylation of the signal activation molecule-blocking effect of mitogen activated protein kinases (MAPK) in downstream more and suppress the propagation of B lymphoma cell line.In addition, the result of study take the Jurkat cell strain as model also show TCR and PD-1 the crosslinked SHP-2 of causing phosphorylation and raise to the cytoplasmic domain of PD-1.
The propagation of PD-1/B7-DC energy suppressor T cell under antigenic stimulation and the generation of cytokine, and can mediate the positivity signal that produces by antagonism CD28/B7.The power of TCR and CD28 conducted signal is depended in the combination of B7-DC and its acceptor PD-1 to the restraining effect of T cell, the conduction that strengthens TCR and CD28 can be offset the restraining effect of PD-1/B7-DC.The relative level of positivity signal and negativity signal is determining the threshold value of immunne response.Therefore, if lack the interaction of B7-DC and PD-1, the threshold value of the TCR Inhibitory signal that the T cell activation is required reduces, and easily causes autoimmune disorder.As the negativity costimulatory signal, B7-DC in the immune response process no matter range or the degree of depth all playing the part of important role, participate in lymphocyte activation, bringing into play the effect that can not be substituted aspect immunological tolerance and the immunologic injury.The PD-1/B7-DC approach is as a kind of negative regulate mode, and high expression level plays an important role in the inducing of peripheral tolerance and autoimmune disease on many non-Lymphoid tissues.
Bronchial asthma is in vogue in the world, and particularly the morbidity in developed country rises just year by year, and its morbidity is main relevant with the type Ⅰ allergy due to cell, the molecular immune functional defect.Asthma is to comprise the inflammatory cell of air flue and the chronic airway inflammation disease of Constituent cell (such as eosinophilic granulocyte, mastocyte, lymphocyte, neutrophil leucocyte, human airway epithelial cells, smooth muscle cell etc.) and cellular component participation by various kinds of cell.This chronic inflammatory diseases is relevant with airway hyper-reaction, usually causes extensively changeable reversible airflow limitation, and cause the panting of repeatedly outbreak, shortness of breath, uncomfortable in chest and (or) symptom such as cough, how night and (or) generation in morning.
Asthma is the chronic airway inflammation by the various kinds of cell participation, and wherein the T lymphocyte plays a leading role in initiating and the reaction of adjusting pneumonia, brings into play its effector function thereby the T cell causes inflammatory cell to gather air flue by synthetic and release inflammatory mediator.Under normal circumstances, lung only has a small amount of T cell, and the T cell rolls up during inflammatory reaction.Helper T cell is divided into two crowds of Th1 and Th2 according to the difference of its function.The Th2 type is mainly secreted IL-4(and is promoted the synthetic IgE of B cell), IL-5(promotes the eosinophilic granulocyte Proliferation and differentiation), IL-9(promotes the mastocyte differentiation), IL-13(promotes mucus secretion, induces airway hyperreactivity), promote the generation of allergic inflammation, be considered to cause the main adjusting cell of asthma.The Th1 type has antagonistic action by secretion of gamma-IFN to the Th2 cell.Therefore, the T cell plays an important role in the airway inflammation of asthma, and its activation needs two kinds of signals, and a kind of TCR/CD3 of being is combined the specific antigens stimulus signal of generation with the MHC-antigenic peptide complexes on antigen presenting cell (APCs) surface; Another kind is the heterogenetic antigen stimulus signal, namely our costimulatory signal of often saying.The T cell only has these two kinds of signals of identification could activate, breed, and the performance function then enters not response status if lack costimulatory signal, even apoptosis.
Therefore the propagation of PD-1/B7-DC energy suppressor T cell under antigenic stimulation and the generation of cytokine play an important role in the immunne response of asthma.Matsumoto has found lung dendritic cell and the scavenger cell of PD-L2 high expression level in sensitized mice, and gives anti-PD-L2 antibody in the antigen stimulation stage, has caused the generation of AHR rising and Th2 cytokines to increase.Akbari etc. have set up asthma mouse model, find that by research shortage that PD-L2 expresses has caused mouse AHR to increase and airway inflammation strengthens, so the expression that proposes PD-L2 asthma start and progression in have provide protection.
The research discovery, many costimulatory molecules can exist with Cell model 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 be produced by single-minded mRNA coding, for example: sCD86 and sCTLA-4.The expression level of many soluble synergistic stimulation molecules has diagnostic value, and for example, soluble CD 40 L (sCD40L) is the CD4 of activation
+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.The soluble CD30 molecule also presents high level expression in some tumour and acquired immune deficiency syndrome (AIDS) (AIDS) patient's serum.Already showed, the molecule on shla molecule and film surface is the same in the body has a corresponding biological function, and 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 and bring into play important regulating effect in immunne response as cytokine, they can either affect 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-DC(sB7-DC) 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-DC.
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-DC, comprise: this horseradish peroxidase-labeled, reaction substrate tetramethyl benzidine, bovine serum albumin, enzyme plate, washing lotion and stop buffer, described test kit also comprises: coated antibody, standard substance albumen and detection antibody, wherein, described coated antibody is mouse-anti people B7-DC monoclonal antibody, its heavy chain has nucleotide sequence shown in the SEQ ID NO:1 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:5; The light chain of described monoclonal antibody has nucleotide sequence shown in the SEQ ID NO:2 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:6; Described detection antibody is mouse-anti people B7-DC monoclonal antibody, and its heavy chain has nucleotide sequence shown in the SEQ ID NO:3 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:7; The light chain of described monoclonal antibody has nucleotide sequence shown in the SEQ ID NO:4 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:8.
In the preferred technical scheme, described coated antibody is mouse-anti people B7-DC monoclonal antibody 8F2, the secretion mouse anti human PD-L2 molecule monoclonal antibody hybridoma cell strain SIPD-L2(8F2 of the CGMCC No. 4789 that by preserving number is) monoclonal antibody of preparation; Described detection antibody is mouse-anti people B7-DC monoclonal antibody 10D6, is the secretion mouse anti human PD-L2 molecule monoclonal antibody hybridoma cell strain SIPD-L2(10D6 of CGMCC No. 4790 by preserving number) preparation monoclonal antibody.
In the technique scheme, hybridoma cell strain SIPD-L2(8F2) preservation information is: preserving number: CGMCC No. 4789, Classification And Nomenclature: secretion mouse anti human PD-L2 molecule monoclonal antibody hybridoma cell strain, preservation date: on 04 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; Hybridoma cell strain SIPD-L2(10D6) preservation information is: preserving number: CGMCC No. 4790, Classification And Nomenclature: secretion mouse anti human PD-L2 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-DCIg albumen, and namely common B7-DCIg albumen of the prior art originally.
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.
The mentioned reagent box is external except the monoclonal anti that comprises anti-cell of the present invention surface B7-DC molecule, also can comprise blood sample collection device and related solvents, damping fluid and standard substance etc.
But the test kit of above-mentioned detection by quantitative solubility B7-DC can quantitative analysis human body serum, blood plasma and tissue juice is such as, the expression level of the solubility B7-DC factor in hydrothorax, the ascites; Can be applicable in the differential diagnosis and result for the treatment of of clinical asthma.
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-DC monoclonal antibody 8F2 simultaneously, and the heavy chain of described monoclonal antibody has nucleotide sequence shown in the SEQ ID NO:1 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:5; The light chain of described monoclonal antibody has nucleotide sequence shown in the SEQ ID NO:2 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:6.
The present invention is claimed a kind of mouse-anti people B7-DC monoclonal antibody 10D6 simultaneously, and the heavy chain of described monoclonal antibody has nucleotide sequence shown in the SEQ ID NO:3 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:7; The light chain of described monoclonal antibody has nucleotide sequence shown in the SEQ ID NO:4 in the sequence table, or aminoacid sequence shown in the SEQ ID NO:8.
The present invention is claimed a kind of hybridoma cell strain simultaneously, and described grand antibody hybridoma cell strain is the hybridoma cell strain SIPD-L2(8F2 of secretion mouse anti human B7-DC molecule monoclonal antibody), the CGMCC No. 4789 that its preserving number is.
The present invention is claimed a kind of hybridoma cell strain simultaneously, and described grand antibody hybridoma cell strain is the hybridoma cell strain SIPD-L2(10D6 of secretion mouse anti human B7-DC molecule monoclonal antibody), the CGMCC No. 4790 that its preserving number is.
Because technique scheme is used, the present invention compared with prior art has following advantages:
1. but the test kit of detection by quantitative solubility B7-DC of the present invention has good specificity, accurately the concentration of the solubility B7-DC protein factor in the liquid such as quantitative analysis person cells and supernatant, serum, blood plasma and hydrothorax; Can be applicable in the differential diagnosis and result for the treatment of of clinical asthma.
2. but the test kit of detection by quantitative solubility B7-DC of the present invention has good sensitivity, can keep good linear relationship in the sB7-DC of 3.125 ~ 200ng/ml concentration range.
Description of drawings
Hybridoma cell strain SIPD-L2(8F2) preservation information is: preserving number: CGMCC No. 4789, Classification And Nomenclature: secretion mouse anti human PD-L2 molecule monoclonal antibody hybridoma cell strain, preservation date: on 04 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; Hybridoma cell strain SIPD-L2(10D6) preservation information is: preserving number: CGMCC No. 4790, Classification And Nomenclature: secretion mouse anti human PD-L2 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 Identification of the antibodies figure among the embodiment one;
Fig. 2 is the specificity analyses figure of antibody among the embodiment one;
Fig. 3 is epitope competitive assay figure as a result among the embodiment one;
Fig. 4 is the specificity analyses figure as a result of human soluble B7-DC ELISA test kit among the embodiment three;
Fig. 5 is human soluble B7-DC ELISA test kit linearity test typical curve among the embodiment four;
Fig. 6 is the expression level figure of sB7-DC in asthmatic patient stage of attack and the stationary phase Peripheral Blood among the embodiment four;
Fig. 7 is asthmatic patient treatment front and back sB7-DC dynamic change figure among the embodiment four.
Embodiment
The invention will be further described below in conjunction with drawings and Examples:
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 substratum with HAT, the HT selection substratum (Sigma, the U.S.) of 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.); Turning people B7-DC gene cell strain L929/B7-DC(makes up).The all cells strain after testing, all without mycoplasma contamination; 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 L929/B7-DC and the strain of L929/mock gene transfecting cell) the pressurization cultivation, to keep the stable high expression level of goal gene product.
(3) animal immune:Collect well-grown L929/B7-DC 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 again the physiological saline Eddy diffusion of 0.3 ~ 0.4ml, fully emulsified with the IFA of equivalent, respectively at the subcutaneous multiple spot of neck and abdominal injection (1 * 10
7/ only); And again row immunity of the 3rd week, the 5th week 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 front 4 ~ 5 days, abdominal injection 5 * 10 again
6/ only with booster immunization.
(4) myeloma cell's preparation:Merged front 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 Growth of Cells is vigorous, form is good, and cell viability can be used for fusion greater than 95%.Merge front 24 ~ 36h and with fresh culture cell concn is adjusted into 3 * 10
5/ ml.
(5) preparation of feeder cell:In merging front 1 day, get the Balb/c mouse, the excision eyeball causes death, and the tap water flushing is placed on 10min in 75% alcohol, then is fixed in 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, processes 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 for subsequent use.
(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 abandoning most supernatant behind the centrifugal 8min of RPMI1640 washing twice, 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, tip straw is inserted the cell suspension bottom gently, in 1min, at the uniform velocity add, and the limit edged stirs gently, then in water-bath, leave standstill 90s.The serum-free DMEM that adds again pre-temperature, the static 5min of room temperature centrifugally (abandons supernatant behind the 800rpm * 10min).With sedimentation cell gently the resuspended 40ml of the placing DMEM that contains 2%HAT, 15%FCS cultivate.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, and conversion contains the DMEM substratum of 15%FCS after 2 weeks.
(8) screening of hybridoma: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-DC cell with after the PBS washing, minute is 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, add sheep anti-mouse igg two anti-4 ℃ of reaction 30min of PE mark, use facs analysis after the washing, the while with the L929/mock cell as the negative control cell strain.
293T/B7-DC gene transfecting cell with stably express people B7-DC carries out screening and identification, 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-DC molecule, called after 10D6 and 8F2, the flow cytometer detected result shows, this monoclonal antibody can specific recognition people B7-DC, and is not combined (Fig. 1) with the 293T/mock gene transfecting cell.
(9) cloning of hybridoma 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, 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 antibody titer high, be single clonal growth, the good cell of form continues the clone, until the antibody-secreting positive rate is greater than 95%; Enlarged culturing and timely liquid nitrogen cryopreservation.
(10) preparation of monoclonal antibody:Adopt in the ascites body and induce method manufacture order clonal antibody.Get the female Balb/c mouse in 6 ~ 8 ages in week, Intraperitoneal injection Pristane 0.5ml/ only.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 gently mouse web portion, 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 centrifugal removal grumeleuse, saturated (NH
4)
2SO
4Redissolve with PBS behind the solution precipitation antibody protein.(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.Collecting the albumen elution peak uses the Tris-Base of PH9.0 to regulate PH to 7.0, 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 8F2 and 10D6 concentration are respectively 1.55mg/ml and 1.87mg/ml.
(12) antibody titer is measured:Well-grown L929/B7-DC cell is divided in the streaming pipe 5 * 10
5/ pipe.The antibody protein gradient dilution liquid (20 μ l/ pipe) that adds respectively culture supernatant, ascites and the purifying of hybridoma in 4 ℃ of reaction 30min, adds sheep anti-mouse igg-PE after the PBS washing, again in 4 ℃ of reaction 30min.Use flow cytometry analysis after the washing, the maximum dilution multiple when same positive rate and fluorescence intensity occurring is tired as antibody, and result's demonstration: 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, mixing gently after it dissolves naturally, with the qualitative test strip of subclass gently in the tubular stinger, behind 5 ~ 10min, the blue chromoprotein colour developing band corresponding with mAb heavy chain title appears in the one side of test paper, and this i.e. the affiliated mouse Ig subclass of this antibody; And the blue chromoprotein band corresponding with mAb light chain title appears in another side, and this i.e. the light chain type of this mAb; Monoclonal antibody is checked order, records heavy chain nucleotide sequence shown in SEQ ID NO:1 in the sequence table of mouse-anti people B7-DC monoclonal antibody 8F2, or shown in SEQ ID NO:5 aminoacid sequence; The light chain of described monoclonal antibody 8F2 is nucleotide sequence shown in SEQ ID NO:2 in the sequence table, or shown in SEQ ID NO:6 aminoacid sequence; Described detection antibody is heavy chain nucleotide sequence shown in SEQ ID NO:3 in the sequence table of mouse-anti people B7-DC monoclonal antibody 10D6, or shown in SEQ ID NO:7 aminoacid sequence; The light chain of described monoclonal antibody 10D6 is nucleotide sequence shown in SEQ ID NO:4 in the sequence table, or shown in SEQ ID NO:8 aminoacid sequence.
(14) specificity identification of monoclonal antibody:Anti-human B7-DC monoclonal antibody reaction 30min with the gene transfecting cell such as L929/mock, L929/B7-H1, L929/B7-DC and L929/B7-H3 and acquisition, the sheep anti-mouse igg two that adds the PE mark after the PBS washing is anti-, continue reaction 30min, use the fluorescence intensity of facs analysis cell marking after the washing.Make positive control with the anti-human B7-DC monoclonal antibody of commercialization (MIH18).The monoclonal antibody 10D6 that obtains and 8F2 and the gene transfecting cells such as mock, B7-DC, B7-H1 and B7-H3 are carried out immunofluorescence label, the result shows, this monoclonal antibody can specific recognition people B7-DC, and is not combined (Fig. 2) with gene transfecting cells such as mock, B7-H1 and B7-H3.
(15) biotin labeling monoclonal antibody:Get anti-human B7-DC monoclonal antibody (10D6) 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 lower 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-DC cell (5 * 10
5/ pipe) with adding respectively monoclonal antibody (every pipe 2 μ g) after the PBS washing, hatch 30min for 4 ℃, the monoclonal antibody that adds the biotin mark after the washing, hatch 30min for 4 ℃ again, add streptavidine-PE after the washing, hatch 30min for 4 ℃ and also use flow cytometry analysis after the washing, establish simultaneously the positive and negative control group.
Result's demonstration, the 10D6 of different concns all can not effectively block the combination of 8F2 and the L929/B7-DC of Biotin mark.Therefore, the epitope of 10D6 identification is different from 8F2(Fig. 3).
Preservation is carried out in above-mentioned two strain of hybridoma strains, hybridoma cell strain SIPD-L2(8F2) preservation information is: preserving number: CGMCC No. 4789, Classification And Nomenclature: secretion mouse anti human PD-L2 molecule monoclonal antibody hybridoma cell strain, preservation date: on 04 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; Hybridoma cell strain SIPD-L2(10D6) preservation information is: preserving number: CGMCC No. 4790, Classification And Nomenclature: secretion mouse anti human PD-L2 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-DC comprises following component:
(1) coated antibody: mouse-anti people B7-DC monoclonal antibody (8F2), 30 μ g/ pipe, 1 pipe;
(2) standard substance albumen: B7-DC Ig albumen (R﹠amp; D), 25ng/ pipe, 1 pipe;
(3) detect antibody: mouse-anti people B7-DC monoclonal antibody (Biotin-10D6), 10 μ g/ pipe, 1 pipe;
(4) Streptavidin-HRP (Sigma-Aldrich), 1 μ l//pipe, 1 pipe;
(5) TMB(Sigma-Aldrich)，10ml；
(6) BSA (worker is given birth in Shanghai), 2g/100ml, 30ml;
(7) 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:
Embodiment three:
Analyze the specificity of embodiment two described human soluble B7-DC ELISA test kits:
The continuous doubling dilution of B7.1Ig, OX40Ig, B7-DCIg and B7-H4Ig is become different concentration.Anti-human B7-DC monoclonal antibody (8F2) is adjusted into the coated ELISA check-out console of 3 μ g/ml with carbonate buffer solution (0.01M CBS, pH9.3), and 4 ℃ are spent the night.PBS(contains 0.1% Tween 20) wash 2% BSA room temperature sealing 1h 3 times.The PBS washing adds the good commercialization albumen of above-mentioned dilution 3 times afterwards, room temperature reaction 2h, PBS washing 3 times.Then, add biotin labeled monoclonal antibody biotin-10D6(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 again HRP reaction substrate TMB(100 μ l/ hole), room temperature reaction 10-15min uses 2mol/L H
2SO
4Termination reaction is measured OD with microplate reader
450, each sample arranges 3 multiple holes.
Specificity analyses result as shown in Figure 4.
Embodiment four:
But adopt the test kit quantitative analysis person of embodiment two described detection by quantitative solubility B7-DC
SerumThe concentration of the solubility B7-DC protein factor in the liquid
(1) sample preparation:
Serum sample: 2500 rpm * 10 min, multigelation is avoided in-20 ℃ of preservations.
(2) operation steps:
1. coated antibody 8F2 is mixed with the coated antibody working fluid of 2.5 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 take 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;
100 μ l 250ng/ml B7-H1Ig sterling albumen are added to first EP pipe, get 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.Serum sample adds elisa plate by 100 μ l/ holes;
It is 0.5 μ g/ml that detection antibody Biotin-10D6 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 and detect antibody Biotin-10D6.Streptavidin-HRP(1:3000), 100 μ l/ holes, room temperature reaction 1h;
6. abandon most reaction solution and contain 0.2% Tween20 with 0.01M PB() wash plate 5-6 time; Add Streptavidin-HRP(1:3000), 100 μ l/ holes, room temperature reaction 1h;
7. abandon most reaction solution and contain 0.2% Tween20 with 0.01M PB() wash plate 8-10 time;
8. add reaction substrate TMB, 100 μ l/ holes, room temperature lucifuge reaction 10-15min; 2M H
2SO
4Stop;
9. using microplate reader selects OD450 nm wavelength to measure the OD reading that elisa plate respectively detects the hole;
(3) 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;
(4) experimental applications is for example:
1. raw data:
2. curvilinear equation:
y = 0.0035x + 0.1276
R
2 = 0.9964
3. make typical curve such as Fig. 5:
(5) Accuracy Analysis:
Method:
1. Accuracy Analysis in criticizing: with in once testing, the sample (100,50,25 ng/ml) of three concentration known is established respectively 20 multiple holes, carry out sB7-DC and detect, analyze the accuracy of this test kit;
2. Accuracy Analysis between criticizing: in this different experiments chamber, with the sample of three concentration known (100,50,25ng/ml) carry out respectively sB7-DC and detect, analyze the accuracy of this test kit;
The result:
Conclusion: variation coefficient CV＜10% confirms that detection method all has good accuracy.
(6) experiment is for example:
1, analyzes the expression level of sB7-DC in asthmatic patient stage of attack and the stationary phase Peripheral Blood, get Fig. 6;
2, analyze asthmatic patient treatment front and back sB7-DC dynamic change, get Fig. 7.
In sum, can use the ELISA test kit of specific assay solubility B7-DC of the present invention asthma is diagnosed and, the effect of asthmatic patient in treatment clinical course to be estimated by stages.Have the expression of sB7-DC in the asthmatic patient serum, and this expression level is along with the improvement rising for the treatment of, until the healthy person level.Described test kit is external except the monoclonal anti that comprises anti-cell of the present invention surface B7-DC molecule, also can comprise blood sample collection device and related solvents, damping fluid and standard substance etc.
Nucleotide and/or aminoacid sequence table
＜110〉University Of Suzhou
＜120〉a kind of human soluble B 7-DC quantitative detection kit
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 420
<212> DNA
＜213〉the unknown
<400> 1
gtgcagctgc agcagtcagg agctgagctg atgaagcctg gggcctcagt gaagatatcc 60
tgcaaggcta ctggctacac attcagtagc tactggatag agtgggtaaa gcagaggcct 120
ggacatggcc ttgagtggat tggagagatt ttacctggaa gtggtagtac taactacaat 180
gagaagttca agggcaaggc cacattcact gcagatacat cctccaacac agcctacatg 240
caactcagca gcctgacatc tgaggactct gccgtctatt actgtgcaag aagcgattac 300
tacggtagtc tgtactactt tgactactgg ggccaaggca ccactctcac agtctcctca 360
gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggaga tacaactggc 420
<210> 2
<211> 344
<212> DNA
＜213〉the unknown
<400> 2
cagattgcga tgacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60
atgacctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagtcaggc 120
acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccagcagtgg agtagtaacc cacccacgtt cggagggggg 300
accaagctgg aaataaaacg ggctgatgct gcaccaactg tatc 344
<210> 3
<211> 420
<212> DNA
＜213〉the unknown
<400> 3
gtcaagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatgtcc 60
tgcaaggctt ctggatacac cttcattgac tactacatga agtgggtgaa gcagagccat 120
gaaaagagcc ttgagtggat tggagatatt aatcctaaca atggtgatac tttctacaac 180
cagaagttca aggacaaggc cacattgact gtagacaaat cctccagcac agcctacatg 240
cagctcaaca gcctgacatc tgaggactct gcagtctatt actgtgcaag ggaatctagg 300
tacgccgcct ggtttgaatg ctggggccaa gggactctgg tcactgtctc tgcagccaaa 360
acaacacccc catcagtcta tccactggcc cctgggtgtg gagatacaac tggttcctcc 420
<210> 4
<211> 381
<212> DNA
＜213〉the unknown
<400> 4
gatgacccag tctccaaatt catgtccact tcactaggag acagagtcag tttcgcctgc 60
aaggccagtc aggatgtggg tcctgctgta gcctggtgtc aagagaaacc aggacaatct 120
cctaaactac tgatttactg ggcatccacc cggcacactg gagtccctga tcgcttcaca 180
ggcagtggat ctgggacaga tttcactctc accattatca atgtgcagtc tgaagacttg 240
gcagattatt tctgtcagca atatagcaac tatccgtaca cgttcggagg ggggaccaaa 300
ctggaaataa aacgggctga tgctgcacca actgtatcaa tctctagagg atccccgggt 360
accgagctcg aattcactgg c 381
<210> 5
<211> 140
<212> PRT
＜213〉the unknown
<400> 5
VQLQQSGAEL MKPGASVKIS CKATGYTFSS YWIEWVKQRP GHGLEWIGEI LPGSGSTNYN 60
EKFKGKATFT ADTSSNTAYM QLSSLTSEDS AVYYCARSDY YGSLYYFDYW GQGTTLTVSS 120
AKTTAPSVYP LAPVCGDTTG 140
<210> 6
<211> 115
<212> PRT
＜213〉the unknown
<400> 6
QIAMTQSPAI MSASPGEKVT MTCSASSSVS YMHWYQQKSG TSPKRWIYDT SKLASGVPAR 60
FSGSGSGTSY SLTISSMEAE DAATYYCQQW SSNPPTFGGG TKLEIKRADA APTVS 115
<210> 7
<211> 140
<212> PRT
＜213〉the unknown
<400> 7
VKLQQSGPEL VKPGASVKMS CKASGYTFID YYMKWVKQSH EKSLEWIGDI NPNNGDTFYN 60
QKFKDKATLT VDKSSSTAYM QLNSLTSEDS AVYYCARESR YAAWFECWGQ GTLVTVSAAK 120
TTPPSVYPLA PGCGDTTGSS 140
<210> 8
<211> 127
<212> PRT
＜213〉the unknown
<400> 8
DDPVSKFMST SLGDRVSFAC KASQDVGPAV AWCQEKPGQS PKLLIYWAST RHTGVPDRFT 60
GSGSGTDFTL TIINVQSEDL ADYFCQQYSN YPYTFGGGTK LEIKRADAAP TVSISRGSPG 120
TELEFTG 127
Claims (5)
1. mouse-anti people B7-DC monoclonal antibody 8F2 is characterized in that, the heavy chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:5; The light chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:6.
2. mouse-anti people B7-DC monoclonal antibody 10D6 is characterized in that, the heavy chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:7; The light chain of described monoclonal antibody is aminoacid sequence shown in the SEQ ID NO:8.
3. hybridoma cell strain is characterized in that, described grand antibody hybridoma cell strain is the hybridoma cell strain SIPD-L2 of secretion mouse anti human B7-DC molecule monoclonal antibody, and its preserving number is CGMCC No. 4789.
4. hybridoma cell strain is characterized in that, described grand antibody hybridoma cell strain is the hybridoma cell strain SIPD-L2 of secretion mouse anti human B7-DC molecule monoclonal antibody, and its preserving number is CGMCC No. 4790.
5. but the test kit of a detection by quantitative solubility B7-DC, comprise: this horseradish peroxidase-labeled, reaction substrate tetramethyl benzidine, bovine serum albumin, enzyme plate, standard substance albumen, washing lotion and stop buffer, it is characterized in that, described test kit also comprises: coated antibody and detection antibody, wherein, described coated antibody is the described mouse-anti people of claim 1 B7-DC monoclonal antibody 8F2; Described detection antibody is the described mouse-anti people of claim 2 B7-DC monoclonal antibody 10D6.
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| WO2018195773A1 (en) * | 2017-04-25 | 2018-11-01 | Sun Yat-Sen University | Use of b7-dc mutant in treatement of asthma |
| JP2022552153A (en) * | 2019-10-04 | 2022-12-15 | デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド | Anti-KIR3DL3 Antibodies and Their Use |
| CN112684185B (en) * | 2020-12-25 | 2024-01-12 | 苏州旭光科星抗体生物科技有限公司 | Soluble B7-H4 quantitative detection kit and application thereof |
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