A kind of chimeric antibody of anticardiolipin/beta 2 glycoprotein I complexTechnical field
The present invention relates to biotechnology, particularly relate to chimeric antibody of a kind of anticardiolipin/beta 2 glycoprotein I complex and its production and use.
Background technology
The clinical sign of antiphospholipid antibody syndrome (antiphospholipidsyndromeAPS) is not yet fully apparent from, and main clinical manifestation is: thrombosis, habitual abortion, thrombocytopenia and neuropsychic symptom etc..According to existing guide, lupus anticoagulant, anticardiolipin antibody and beta 2 glycoprotein I antibody are the diagnostic methods of main serology Clinical detection anti-phospholipid antibody.If 3 are all positive, then show that this patient or Healthy People antiphospholipid antibody syndrome are high-risk, and if only 1 be the positive, then danger is then relatively low.
Research shows that these 3 inspection items are very sensitive to extrinsicfactor, is difficult to detection method standardization.Lupus anticoagulant is the most relevant to the formation of thrombosis, but, the accuracy of detection lupus anticoagulant is heavily dependent on the process of blood sample and the quality of blood plasma.The anticardiolipin antibody detected is really the antibody being directed to cuorin/beta2 Glycoprotein 1 complex, the ELISA detection of anticardiolipin antibody and anti-beta2 Glycoprotein 1 antibody is insensitive to blood processing procedure, assay method is also not dependent on the function (such as anticoagulant) of antibody, and depends only on the combination of antibody and coated cuorin/beta2 Glycoprotein 1 complex and beta2 Glycoprotein 1.But, ELISA kit testing result widely different that different manufacturers provides, and false positive rate is more.Calibration object that false positive Producing reason may be from adopting in the interference and test kit of non-specific antibody, the preparation of reference substance.
ELISA detection method depends on the calibration object in test kit and reference substance and serum sample to be checked compares analysis and positive differentiation.These calibration objects and reference substance at present, is all by mixing, being configured to certain Concentraton gradient with the positive serum of patient, Healthy Human Serum.This needs exist for stable serum origin, and sera stock is representative.Considering bio-safety problem, serum sample is all performed strict management by various big hospital, causes the production process sera stock short supply of ELISA kit.On the other hand, the clinical sera stock collected has serious individual variation, often results in the fluctuation of the detection data of test kit and the inconsistent of result interpretation in use procedure.This is the problem adopting the ELISA kit that clinical serum produces to be difficult to avoid in application process.
In the detection of anticardiolipin antibody, first with cuorin/beta2 Glycoprotein 1 complex wrapper sheet, it is subsequently adding sample (calibration object), the variable region of the anticardiolipin antibody in sample is combined with coated cuorin/beta2 Glycoprotein 1 complex, then adds enzyme target anti-human IgG Fc antibody.If detecting anticardiolipin IgM type antibody, then the enzyme mark II that can add anti-human IgMFc resists, if to detect anticardiolipin IgA type antibody, then the enzyme mark II that can add anti-human IgAFc resists.
Therefore, anticardiolipin/β 2GP1 complex antibody that preparation Mus/people is fitted together to, it is possible to replace sera stock to prepare the calibration object in ELISA kit and reference substance, thus solving the limited problem causing differences between batches with individual variation of serum origin.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide the chimeric antibody of a kind of anticardiolipin/beta 2 glycoprotein I complex, is used for solving the problems of the prior art.
For achieving the above object and other relevant purposes, the present invention provides the chimeric antibody of a kind of anticardiolipin/beta 2 glycoprotein I complex, the aminoacid sequence of its antibody chain variable region is SEQIDNO:5 or its conservative series of variation, and the aminoacid sequence of its antibody heavy chain variable region is SEQIDNO:1 or its conservative series of variation.
Described conservative series of variation is often referred to replaces, by conservation of amino acids, the sequence that change obtains, described conservative is replaced and is typically referred to the aminoacid that another similar aminoacid replacement of character is specified, for example, it is possible to be considered as conservative replace (character is similar) example include but not limited to: a) alanine, serine and threonine；B) glutamic acid and aspartic acid；C) agedoite and glutamine；D) arginine and lysine；E) isoleucine, leucine, methionine and；F) phenylalanine, tyrosine and tryptophan.
Concrete, the chimeric antibody of described anticardiolipin/beta 2 glycoprotein I complex is the chimeric antibody that mouse-anti cardiolipin antibody is formed with people's antibody.More specifically, described chimeric antibody can be mouse-anti cardiolipin antibody variable region fragment respectively with people Ig constant region formed chimeric antibody, described people Ig constant region can be human IgG, IgM, IgA constant region, and the chimeric antibody formed can be IgG, IgM, IgA type chimeric antibody.
In the chimeric antibody of described anticardiolipin/beta 2 glycoprotein I complex, " antibody " uses with broadest sense, is specifically including but not limited to monoclonal antibody (including full length monoclonal antibodies) and its antibody fragment.To those skilled in the art it should be understood that, " antibody fragment " alternative antibody in many cases, the example of antibody fragment includes but not limited to: Fab, Fab', F (ab') 2 and Fv fragment；Bispecific antibody；Linear antibodies；Single-chain antibody molecules；Nano antibody (technology from Ablynx)；Domain antibodies (technology from Domantis)；With the multi-specificity antibody etc. formed by antibody fragment.
Second aspect present invention provides the polynucleotide of a kind of separation, encodes the heavy chain of chimeric antibody of described anticardiolipin/beta 2 glycoprotein I complex and/or the variable region of light chain or full length amino acid.
Third aspect present invention provides one to build body, containing the polynucleotide of described separation.
Concrete, the multiple clone site that described structure body is inserted into expression vector by the polynucleotide of described separation is built-up.
Expression vector in the present invention is often referred to bacterial plasmid well known in the art, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other carriers.
Preferably, the described expression vector one or more combination in pIRES or pcDNA3.1.
Fourth aspect present invention provides the expression system of a kind of chimeric antibody, and described expression system contains the described polynucleotide being integrated with external source in described structure body or genome.
Any cell carrying out expressing suitable in expression vector can serve as host cell.Such as, described host cell can be prokaryotic cell, such as bacterial cell；Or the eukaryotic cell such as low, such as yeast cells；Or higher eucaryotic cells, such as mammalian cell.
Preferably, described host cell is selected from Chinese hamster ovary (ChineseHamsterOvary (CHO)) cell line, multiple COS cell line, HeLa cell line, myeloid cell series are SP2/0 cell line such as, NS0 cell line, YB2/0 cell line etc., and convert B-cell or thin pIRES or the pcDNA3.1 born of the same parents of hybridoma in one or more combination.
The preparation method that fifth aspect present invention provides the chimeric antibody of described anticardiolipin/beta 2 glycoprotein I complex, comprise the steps: when the described antibody of applicable expression, cultivate the expression system of described chimeric antibody, thus giving expression to described chimeric antibody, it is isolated and purified with described chimeric antibody.
Host cell used in the present invention is prior art, commercial sources can be passed through directly obtain, culture medium used in cultivation is also various conventional medium, and those skilled in the art can rule of thumb select the culture medium being suitable for, and cultivates when being suitable to host cell growth.When, after host cell growth to suitable cell density, by the promoter of suitable method (such as temperature transition or chemical induction) induction selection, cell being further cultured for a period of time.Recombinant polypeptide in the above methods can be expressed in cell or on cell membrane or be secreted into extracellular.If it is required, its physics, chemistry separation by various separation methods with other characteristic and the albumen of purification of Recombinant can be utilized.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation processes, processes (salting-out method) with protein precipitant, is centrifuged, permeates the combination of broken bacterium, super process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The present invention screens the variable region gene sequence obtaining purpose antibody from the hybridoma cell strain of Colony Culture, in order to build the carrier for expression of eukaryon of chimeric antibody, the activity of antibody can be rebuild, it is thus achieved that the chimeric antibody of anticardiolipin/beta 2 glycoprotein I complex after expression.
Sixth aspect present invention provides the chimeric antibody of described anticardiolipin/beta 2 glycoprotein I complex purposes in preparation detection kit.
Concrete, described detection kit can be cuorin/beta 2 glycoprotein I complex and/or anti-cardiolipin antibody detection kit.
Concrete, described detection kit can be anticardiolipin antibody IgG detection kit, and in detection kit, the chimeric antibody of described anticardiolipin/beta 2 glycoprotein I complex is as standard specimen.
Seventh aspect present invention provides a kind of detection kit, including the chimeric antibody of described anticardiolipin/beta 2 glycoprotein I complex.
Inventor obtains the hybridoma cell strain of mouse-anti cuorin/beta2 Glycoprotein 1 complex antibody by hybridoma technology, and clone the variable region sequences of monoclonal antibody, and the chimeric antibody of the anticardiolipin/beta 2 glycoprotein I complex prepared further with technique for gene engineering has good affinity with cuorin/beta 2 glycoprotein I complex, can apply in ELISA detection kit, for instance the ELISA detection kit etc. of cardiolipin antibody.
Accompanying drawing explanation
Fig. 1 is shown as the embodiment of the present invention 2 cuorins/β 2GP1 complex and measures schematic diagram with chimeric antibody relative affinity.
Fig. 2 is shown as the embodiment of the present invention 3 clinical serum (40 parts) detection dependency and compares schematic diagram.
Fig. 3 is shown as the embodiment of the present invention 3 clinical serum (40 parts, 37 DEG C are accelerated 7 days) detection dependency and compares schematic diagram.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art the content disclosed by this specification can understand other advantages and effect of the present invention easily.The present invention can also be carried out by additionally different detailed description of the invention or apply, and the every details in this specification based on different viewpoints and application, can also carry out various modification or change under the spirit without departing from the present invention.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, rather than in order to limit the scope of the invention；In specification and claims of the present invention, unless additionally explicitly pointed out in literary composition, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, between two end points and two end points of each numerical range, any one numerical value all can be selected for.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique are generally understood that.Except the concrete grammar used in embodiment, equipment, material, record according to those skilled in the art's grasp to prior art and the present invention, it is also possible to use similar with the method described in the embodiment of the present invention, equipment, material or that be equal to any method of prior art, equipment and material to realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all adopt the conventional molecular biology of the art, biochemistry, chromatin Structure and analysis, analytical chemistry, cell are cultivated, the routine techniques of recombinant DNA technology and association area.These technology existing improving in existing document illustrates, specifically can referring to the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001；Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, john wiley & sons, NewYork, 1987andperiodicupdates；TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego；Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998；METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999；And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc..
Embodiment 1
The structure of the chimeric antibody IgG type carrier for expression of eukaryon of anticardiolipin/beta 2 glycoprotein I complex and expression (heavy chain and light chain gene synthesize by Han Yu bio tech ltd, Shanghai):
The aminoacid sequence of antibody heavy chain variable region is SEQIDNO:1, and the DNA sequences encoding used in the present embodiment is SEQIDNO:2；The aminoacid sequence of IgG antibody type heavy chain is SEQIDNO:3, and the DNA sequences encoding used in the present embodiment is SEQIDNO:4.
SEQIDNO:1
VQLQESGAELVKPGASVKLSCKASGYTFTSHWMHWVKLRPGQGFEWIGENNPRNGDTNFNEKFKRKATLTVEKSSNTAYMQLNSLTSEDSAVYYCTIWSFYYGFDYWGQGTSLTVSSAKTT
SEQIDNO:2
GTGCAGCTGCAGGAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCCTCTGGCTACACCTTCACCAGCCACTGGATGCACTGGGTGAAGCTGAGGCCTGGACAAGGCTTTGAGTGGATTGGAGAGAATAATCCTCGCAATGGTGATACTAACTTCAATGAGAAGTTCAAGAGAAAGGCCACACTGACTGTAGAGAAATCCTCCAACACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAATATGGTCCTTCTACTATGGTTTTGACTACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCAGCCAAAACGACA
SEQIDNO:3
MEFGLSWVFLVALFRGVQCEVQLQESGAELVKPGASVKLSCKASGYTFTSHWMHWVKLRPGQGFEWIGENNPRNGDTNFNEKFKRKATLTVEKSSNTAYMQLNSLTSEDSAVYYCTIWSFYYGFDYWGQGTSLTVSSAKTTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO:4
ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTCCGCGGTGTCCAGTGTGAGGTGCAGCTGCAGGAGTCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCCTCTGGCTACACCTTCACCAGCCACTGGATGCACTGGGTGAAGCTGAGGCCTGGACAAGGCTTTGAGTGGATTGGAGAGAATAATCCTCGCAATGGTGATACTAACTTCAATGAGAAGTTCAAGAGAAAGGCCACACTGACTGTAGAGAAATCCTCCAACACAGCCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAATATGGTCCTTCTACTATGGTTTTGACTACTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCAGCCAAAACGACAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCATGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCCCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
The aminoacid sequence of antibody chain variable region is SEQIDNO:5, and the DNA sequences encoding used in the present embodiment is SEQIDNO:6；The aminoacid sequence of IgG antibody type light chain is SEQIDNO:7, and the DNA sequences encoding used in the present embodiment is SEQIDNO:8.
SEQIDNO:5
DIVMTQSHKFMSTSIGDRVSLTCKASQDVSTAVAWYQQKPGHSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYTAPYTFGGGTKLDIKRADA
SEQIDNO:6
GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAATAGGAGACAGGGTCAGCCTCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACATTCTCCTAAACTACTGATTTACTCGACATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTGCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGACATAAAACGGGCTGATGCT
SEQIDNO:7
MDMRVPAQLLGLLLLWLSGARCDIVMTQSHKFMSTSIGDRVSLTCKASQDVSTAVAWYQQKPGHSPKLLIYSTSYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYTAPYTFGGGTKLDIKRADARTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECTAG
SEQIDNO:8
ATGGACATGAGGGTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCTCAGGTGCGCGCTGTGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAATAGGAGACAGGGTCAGCCTCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACATTCTCCTAAACTACTGATTTACTCGACATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTGCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGACATAAAACGGGCTGATGCTCGTACGGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Use PCR reaction that the DNA sequences encoding of above-mentioned light chain and heavy chain is expanded, wherein introduce XhoI and MluI restriction enzyme site respectively at antibody light chain gene 5 ' end and 3 ' ends, forward and reverse primer sequences are respectively as follows: 5 '-GAATCTCGAGATGGACATGAGGGTCCCTG-3 ' (SEQIDNO:9) and 5 '-GAATACGCGTCTAACACTCTCCCCTGTTG-3 ' (SEQIDNO:10), and the coded sequence (SEQID8) of light chain of antibody is template；Introducing XbaI and SalI respectively at antibody heavy chain variable region gene 5 ' end and 3 ' ends, forward and reverse primer sequences are respectively as follows: 5 '-GAACTCTAGAATGGAGTTTGGGCTGAGCTG-3 ' (SEQIDNO:11) and 5 '-GAATGTCGACTCATTTACCCGGAGACAGGGAG-3 ' (SEQIDNO:12) and with the coded sequence (SEQID4) of heavy chain of antibody for template.Adopting PCR reaction, condition is 95 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 75s, 35 circulations, and 72 DEG C extend 10min, and archaeal dna polymerase is TAKARA Products.After pcr amplification, PCR primer is reclaimed purification through agarose gel electrophoresis.Light chain gene adds restricted enzyme XhoI and MluI and carries out enzyme action, and heavy chain gene adds restricted enzyme XbaI and SalI and carries out enzyme action, is purified through DNA purification kit after enzyme action, and with the carrier pIRES of identical digestion with restriction enzyme.Connect product and be transformed into DH5 α escherichia coli, be coated on the 2YT agar culture medium containing 50 μ g/ml Carbenicillins.The positive colony obtained is cultivated in the 2YT fluid medium containing 50 μ g/ml Carbenicillins, after Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's sequence verification, takes out greatly test kit with plasmid and extracts positive colony plasmid.
Utilize the surge system of BioRad company, by linearizing plasmid DNA transfection to Chinese hamster ovary celI.Chinese hamster ovary celI after transfection is diluted colonized culture, cultivates in CDCHOAGT culture medium (Invitrogen Products) at the G418 containing 800ug/mL and screens, thus obtaining monoclonal cell system.
Being tied up to by the monoclonal cell of acquisition in CDCHOAGT culture medium (Gibco company of the U.S.) and carry out shake-flask culture, expression time is generally 7-14 days, the harvesting culture fluid supernatant when viable cell density is lower than 30%.The goat-anti people's constant region of light chain utilizing goat anti-human igg Fd (Meridian Products) and horseradish peroxidase-labeled carries out the detection of double sandwich-ELISA method and expresses the content of antibody in supernatant, using untransfected supernatant as negative control, the IgG sterling of people is as standard substance.Experimental result shows, expressing the destination protein obtained by two antibody recognition, can have IgG antibody feature, and the chimeric antibody expression built is at 200～500 μ g/ml, has higher expression.ProteinA affinity column is adopted to separate purification purpose antibody from cells and supernatant.
Embodiment 2
Anticardiolipin/β 2GP1 complex chimeric antibody Function Identification
Affinity of antibody is identified:
By cuorin/β 2GP1 complex (being mixed according to 1:2 equimolar ratio with beta 2 glycoprotein I by cuorin), being coated in ELISA Plate (working volume 30ul) by the cuorin concentration of 2 μ g/ml, 4 DEG C stand overnight.3 times are washed with the PBS (PBST) of the Tween20 containing 0.05%.BSA room temperature with 0.1% is closed 1 hour.Wash 3 times with PBST, the antibody that embodiment 1 prepares is configured to 4 μ g/ml concentration, and (diluent is: PBST+1%BSA+0.05%ProClin), totally 8 gradients, and every hole adds 30 μ l, and room temperature stands 1 hour to carry out 3 times of gradient dilutions.Washing 3 times with PBST, add the goat anti-human igg Fc of the horseradish peroxidase-labeled of 30 μ l1:4000 dilutions, room temperature stands 1 hour.Wash 4 times with PBST, add TMB colour developing, and with the H of 2M2SO4Terminate reaction.With microplate reader reading at 450 nm.Can be seen that from result, anticardiolipin/β 2GP1 complex is had obvious affinity by the antibody that screening obtains, and concrete outcome is as shown in Figure 1.
Embodiment 3
Anticardiolipin/β 2GP1 complex chimeric antibody application in ELISA detection kit
Prepared by the material in ELISA application:
Commercial ELISA kits calibration after antibody antibody dilution buffer (PBST+1%BSA+0.05%ProClin) dilution that embodiment 1 is prepared, it is thus achieved that the relative RU value of counterpart's serum cardiolipin antibody.
Quality controlled serum in ELISA application, prepares for cardiolipin antibody IgG positive clinical serum, 100RU/ml.
Precision serum in ELISA application, for cardiolipin antibody IgG clinical serum sample (60RU/ml and 150RU/ml).
The clinical serum sample (20 examples are positive, 20 examples are negative) evaluated in ELISA application derives from clinical collection serum sample.
Above quality controlled serum, precision serum, clinical serum sample standard deviation meet bio-safety detection regulation, and cardiolipin antibody IgG value is measured (Ou Meng, EA1621-9601G) by business ELISA reference reagent box.
nullThe application in ELISA kit is detected: cuorin/β 2GP1 complex (mol ratio 1:2) is diluted to CBS buffer the cuorin concentration of 2ug/ml at cardiolipin antibody IgG，4 DEG C are coated 12-17 hour，PBST washs 3 times，The PBST confining liquid of the new-born calf serum containing 1% 37 DEG C is closed 1.5 hours，PBST washs 3 times，Solution after the antibody calibration that embodiment 1 is prepared，It is diluted to 200RU/ml、20RU/ml、2RU/ml (the RU value relative to human serum cardiolipin antibody)，As the calibration object in ELISA method，Serum sample serum sample dilution buffer (PBST+1%BSA+0.05%ProClin) dilutes 201 times，Precision serum serum sample dilution buffer (ibid) dilutes 201 times，Quality controlled serum directly uses；By above calibration object, precision serum, clinical serum sample 100ul/ hole, duplicate hole, room temperature 30 minutes, PBST washes 3 times, adds goat anti-human igg's Fc ELIAS secondary antibody of 5000 times of dilutions, room temperature 30 minutes, washes 4 times with PBST, adds TMB colour developing, and with the H of 2M2SO4Terminate reaction.With microplate reader reading at 450 nm.Calibration object fit standard curve after the antibody calibration prepared by embodiment 1, calculates the cardiolipin antibody IgG content of 2 parts of precision serum, quality controlled serum and 40 parts of clinical serum samples.
Reproducibility: detection method is ibid, 2 parts of precision serum of calibration object duplicate detection after the antibody calibration prepared by embodiment 1, batch between duplicate detection 20 time, batch duplicate detection based on 6 days, every day repeat 3 times detect, testing result shows such as table 1, table 2,37 DEG C accelerate within 7 days, refer specifically to by detection after sample be placed at 37 DEG C 7 days again with microplate reader reading at 450 nm), the CV repeated in crowd, lower than 10%, repeats CV lower than 15% between batch.
Table 1
Reproducibility withinrun precision (n=20)
Table 2
Reproducibility betweenrun precision (n=18)
Coincidence rate is evaluated: detection method is ibid, 40 parts of clinical serum samples of calibration object duplicate detection (20 parts of positives, 20 parts of feminine genders) after the antibody calibration prepared by embodiment 1, testing result shows as shown in table 3, same commercial ELISA Kit (the Ou Meng of yin and yang attribute result of determination of 40 parts of clinical serum pattern detection results, EA1621-9601G) result of determination coincidence rate is 100%, with the dependency r value of reference reagent box testing result more than 0.98 (such as table 3, Fig. 2 and Fig. 3).
Table 3
Coincidence rate and relativity evaluation (40 parts of clinical serum samples, 20 parts of feminine genders, 20 parts of positives)
Estimation of stability: after the calibration object after antibody calibration embodiment 1 prepared adds antibody dilution buffer, carry out 37 DEG C of accelerated stability detections, detect after accelerating the 7th day, detection method is ibid, detection content is with repeated withinrun precision and coincidence rate evaluation, testing result shows (such as table 1, table 3, Fig. 3), calibration object after the calibration of antibody that embodiment 1 prepares adds antibody dilution buffer 37 DEG C and detects precision serum and clinical serum sample as calibration object after 7 days, and repeatability and coincidence rate evaluation are basically unchanged.
In sum, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
Above-described embodiment is illustrative principles of the invention and effect thereof only, not for the restriction present invention.Above-described embodiment all under the spirit and category of the present invention, can be modified or change by any those skilled in the art.Therefore, art has usually intellectual such as modifying without departing from all equivalences completed under disclosed spirit and technological thought or change, must be contained by the claim of the present invention.