CN103923881B - Hepatitis A virus (HAV) monoclonal antibody and application thereof - Google Patents
Hepatitis A virus (HAV) monoclonal antibody and application thereof Download PDFInfo
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Abstract
Does is the invention provides a kind of hepatitis A virus (HAV) monoclonal antibody, CGMCC by deposit number? the hybridoma cell strain secretion of No.7859 produces.This monoclonal antibody can efficiently, specifically in and HAV-Ag, the diagnosis, the Prevention and Curation that infect for hepatitis A virus (HAV) are significant.
Description
Technical field
The present invention relates to virological immunology detection field, specifically, relate to hepatitis A virus (HAV) monoclonal antibody and application thereof.
Background technology
Hepatitis A is called for short hepatitis A, is a kind of acute infectious disease caused by hepatitis A virus (hepatitis A virus (HAV), HAV).Hepatitis A virus belongs to pico+ribonucleic acid+virus section, hepatovirus.Virion is spherical in shape, and diameter is about 27nm, without cyst membrane, capsid is made up of 32 capsomeres, and in 20 body cubic symmetry, each capsomere has four polypeptide and is respectively viral protein VP1, VP2, VP3 and VP4, genome is single-stranded positive RNA, about its length is equivalent to 7500 Nucleotide.There is polyadenosine sequence at the 3 ' end of RNA, be connected with virogene histone with covalency form at 5 ' end.According to viral nucleotide sequences analysis, people's hepatitis A virus (HAV) can be divided into four genotype, but its antigenicity is similar, so hepatitis A virus only has a serotype.
Hepatitis A virus is propagated mainly through fecal oral route, and contagium mostly is patient.Virus latency is 15 ~ 45 days, virus be everlasting patient's Cyklokapren raise before the blood being just present in patient for 5 ~ 6 days and ight soil in.Fall ill after 2 ~ 3 weeks, along with the generation of specific antibody in serum, the infectivity of blood and ight soil also fades away.Take carrier for a long time extremely rare.HAV excretes with patient's ight soil, can be caused sporadic popular or be very popular by the propagation of polluted source, food, sea-food (as blood clam etc.), tableware etc.Also by blood transfusion or injection system propagate, but due to HAV time length in blood samples of patients short far beyond hepatitis B virus, so plant circulation way comparatively rare.
After human infection's hepatitis A virus (HAV), first breed in digestive tube, in of short duration viremia, virus can continue again to breed in blood leucocyte, then enters liver, in liver cell, copies breeding.Hepatitis A virus (HAV) only has a serotype and an antigen-antibody system, and therefore its detection antibody can use in countries in the world.The pathogenesis of hepatitis A is unclear, but particularly immunization of cell is relevant to Liver immunity effect.HAV can enter enteron aisle from liver cell through bile and excrete.Therefore, HAV particle can be detected made a definite diagnosis from ight soil, the strongest in the infectivity of latent period or stage of icterus.
Clinical manifestation: hepatitis A is divided into acute icteric, acute anicteric, Subclinical and acute silt courage type, wherein acute silt courage type is the singularity of acute icteric, is because hepatocytes secrete bile function is impaired.Due to no matter from clinical manifestation or from liver function test, all the hepatites virus infections of acute hepatitis A virus infection with other types cannot be distinguished mutually, therefore etiological examination is very important for diagnosing acute hepatitis A virus infection.
The etiological examination of hepatitis A comprises: produce IgM type antibody in early days after (1) anti-HAVIgM:HAV infects, and is the evidence recently infected, and is also the easiest and reliable serologic marker of early diagnosis hepatitis A.After morbidity, a couple of days can be positive, and generally continue 8 ~ 12 weeks, minority can continue 6 months.Adopt enzyme linked immunosorbent assay (ELISA) to detect clinically more.(2) anti-HAVIgG: occur a little later, peaked in 2 ~ 3 months is the mark infected in the past, for many years sustainable or all the life.Other detection methods have: immune electron microscopy and qualification HAV particle, Cell culture invitro isolated viral, detects HAVRNA etc., generally only use in experimental study.
Summary of the invention
The object of this invention is to provide a kind of hepatitis A virus (HAV) monoclonal antibody and application thereof.
In order to realize the object of the invention, the present invention utilizes hepatitis A virus (HAV) (TZ84 strain) whole virus particles to utilize hybridoma technology to prepare monoclonal antibody as antigen, then by SouthernBiotech monoclonal antibody parting kit, hypotype qualification is carried out to obtained monoclonal antibody, by Western blot monoclonal antibody specificity, indirect elisa method is adopted to carry out neutralization test, to obtain in can secreting and hepatitis A virus (HAV) can the clone of specific binding hepatitis A virus (HAV), and the monoclonal antibody produced by this cell line secretes.
In one aspect of the invention, provide a kind of hybridoma cell strain hepatitis A virus (HAV) to high Neutralizing titer, i.e. hepatitis A virus monoclonal antibody hybridoma cell strain HAVmAb-6.This hybridoma cell strain has now been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCCNo.7859, preservation date on July 10th, 2013.
In another aspect of the present invention, provide and secrete by above-mentioned hybridoma cell strain the monoclonal antibody produced.
Further, the present invention also provides the various derivative antibody that (such as heavy chain and variable region of light chain) develops of the antigen binding regions according to described monoclonal antibody, may be used for passive immunotherapy or the infection of prevention hepatitis A virus (HAV), as humanized antibody.
Because monoclonal antibody of the present invention has high-caliber neutralization for hepatitis A virus (HAV), therefore the present invention also provides described monoclonal antibody or its active fragments or conservative variant, or its arbitrary combination is for the preparation of the application detected in the reagent of HAV-Ag, medicament or test kit.
Another aspect of the present invention is provided for detecting HAV-Ag, or the test kit of anti-hepatitis A virus (HAV) IgG or IgM, reagent, medicament, and it comprises monoclonal antibody of the present invention or its active fragments or conservative variant, or its arbitrary combination.Described reagent, medicament or test kit can be used for detecting HAV-Ag.Thus be further used for the infection of diagnosis hepatitis A virus (HAV).
Another aspect of the present invention provides a kind of medicine being used for the treatment of or preventing hepatitis A virus (HAV) to infect, and described pharmaceutical pack is containing described monoclonal antibody or its active fragments or its conservative variant.Preferably, described medicine comprises pharmaceutically acceptable carrier and/or vehicle further.
Another aspect of the present invention provides a kind of method being used for the treatment of and/or preventing hepatitis A virus (HAV) to infect, and is applied to patient comprising by monoclonal antibody of the present invention or its active fragments or conservative variant.
Present invention also offers the method for detecting HAV-Ag, which using monoclonal antibody of the present invention or its active fragments or its conservative mutation body, or the monoclonal antibody of the present invention of mark or its active fragments or its conservative mutation body.Preferably, double-antibody method is adopted to carry out detection HAV-Ag.
For mark of the present invention, it refers to biomarker or chemical labeling.Such as enzyme labelling or fluorescent mark (such as fluorescein-labelled).Preferably, described enzyme labelling is horseradish peroxidase, pyruvate kinase or glucose oxidase enzyme labelling etc.
For of the present invention for preventing or the monoclonal antibody of therapeutic purpose, it is preferably humanized.
The present invention also provides the application of described monoclonal antibody in the humanized antibody infected for the preparation of prevention or treatment hepatitis A virus (HAV).
Present invention also offers the detection method of hepatitis A virus (HAV) related antigen, antibody, it comprises: 1, in sample for the detection method of the IgG antibody of hepatitis A virus (HAV): purified virus liquid is adsorbed in solid phase carrier, add the sample to be checked being placed in buffering system again, add again carry can marker detection thing identification detect the second antibody of the IgG antibody of Specimen origin species, then infer the existence of anti-hepatitis A virus (HAV) antibody in sample or how many by the existence of the marker entrained by detecting.Preferred described detectable is horseradish peroxidase or fluorescein-labelled.2, in sample for the detection method of the IgM antibody of hepatitis A virus (HAV): purified virus liquid is adsorbed in solid phase carrier, add the sample to be checked being placed in buffering system again, add again the identification of carrying detectable detect the second antibody of the IgM antibody of Specimen origin species, then infer the existence of anti-hepatitis A virus (HAV) antibody in sample or how many by the existence of the marker entrained by detecting.Preferred described detectable is horseradish peroxidase or fluorescent marker.3, in sample for the detection method of the IgM antibody of hepatitis A virus (HAV): will the antibody (anti-antibody of IgM of anti-species to be checked, the antibody of anti-IgM) be adsorbed on solid phase carrier, add the sample to be checked being placed in buffering system again, add again the identification of carrying detectable detect the enzyme-labelled antigen of sample, then infer the existence of anti-hepatitis A virus (HAV) IgM antibody in sample or how many by the existence of the marker entrained by detecting.Preferred described detectable is horseradish peroxidase or fluorescein-labelled thing.
The invention provides a strain preserving number is CGMCCNo.7859, the hybridoma cell strain producing hepatitis A virus (HAV) monoclonal antibody can be secreted, the monoclonal antibody produced by its secretion can efficiently, specifically in and HAV-Ag, the diagnosis, the Prevention and Curation that infect for hepatitis A virus (HAV) are significant.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The acquisition of embodiment 1 hybridoma cell line
Comprise the steps:
1, immunogenic preparation
Immunogen is HAV-Ag---TZ84 strain, is provided by Beijing Kexing Biotech Products Co., Ltd.By virus inoculation 2BS cell, cultivate, carry out purifying through PEG precipitation with trichloromethane extracting after results, then carry out ultrafiltration and concentration and deactivation to prepare purified virus liquid.
2, animal immune
Adopt hepatitis A virus (HAV) refined solution to carry out BALB/c mouse initial immunity and booster immunization, BALB/c mouse used is female, and the monthly age is 6-8 week, and body weight is 18-22g.Immune programme for children: first immunisation gets hepatitis A virus (HAV) refined solution and Freund's complete adjuvant adjuvant mixing and emulsifying, immune BALB/c mouse, 150 μ g/, subcutaneous multi-point injection; Second time immunity is carried out at interval after 10 days, get hepatitis A virus (HAV) refined solution and Freund's incomplete adjuvant mixing and emulsifying, then the above-mentioned mouse of booster immunization, and dosage is with first time; Take a blood sample after 7 days, adopt indirect elisa method to measure the anti-hepatitis A virus (HAV) activity of antibody.Adjuvant is not added to the mouse detecting antibody titer the highest, carries out abdominal cavity booster immunization with hepatitis A virus (HAV) refined solution.
3, cytogamy
Get immunized mice spleen cell and SP2/0 myelomatosis merges in proportion, spread 96 porocyte culture plates, adopt HAT Selective agar medium to cultivate hybridoma, within the 7th day, adopt indirect elisa method to detect cell conditioned medium to screen positive cell.Indirect method adopts hepatitis A virus (HAV) refined solution bag quilt, screens the cell hole be positive.Indirect elisa method is as follows: by hepatitis A virus (HAV) refined solution coated elisa plate, add positive serum controls, negative serum control, arrange blank control wells simultaneously, and detersive enzyme target after 37 DEG C of reactions, adds goat anti-mouse igg-HRP; Detersive enzyme target after reaction, adds TMB nitrite ion 37 DEG C colour developing 10-20 minute, uses 2MH
2sO
4termination reaction, instrument reads OD
450absorbancy.According to absorbance screening positive cell hole.
4, clone: adopt limiting dilution assay to clone positive cell hole.
5, detect: the cell conditioned medium in clone's plate is detected, adopt hav antigen refined solution coated elisa plate to detect.
6, secondary cloning, with the operation of step 4.
7, the detection after secondary cloning, with the detection method of step 5, adopts indirect elisa method that the cell conditioned medium of second time clone is carried out the positive detection for HAV-Ag, selects unicellular positive hole to carry out enlarged culturing, obtain a large amount of hybridoma.
The screening of embodiment 2 monoclonal antibody and preparation
1, monoclonal antibody ascites preparation
Hybridoma enlarged culturing adopts intraperitoneal injection of mice to prepare ascites.Wherein ascites preparation flow is as follows: cultivate hybridoma, and in the BALB/c mouse of pneumoretroperitoneum injection paraffin sensitization, within about 7 ~ 14 days, collect ascites, 1200 revs/min centrifugal, and removing precipitation, collects upper strata monoclonal antibody ascites.
2, the preliminary screening of monoclonal antibody and preparation
Adopt the rear coated elisa plate respectively of hepatitis A virus (HAV) refined solution 1:100 dilution, 100 μ l/ holes, wrap and are spent the night; With 0.01MPBST-20(containing 0.5v/v ‰ tween 20) washing lotion wash plate 3 times, add 10% calf serum, 0.01MPBST-20 solution (containing 0.5v/v ‰ tween 20), 200 μ l/ holes, close, discard solution in plate for 37 DEG C.Add the upper strata monoclonal antibody ascites of the above-mentioned acquisition of serial dilution, 100 μ l/ holes, its small mouse negative serum is negative control; Hatch 1 hour for 37 DEG C, wash plate by above-mentioned washing lotion, add 1:10000 and be diluted in 10% calf serum, 0.01MPBST-20(containing 0.5v/v ‰ tween 20) goat anti-mouse igg-HRP of solution, 100 μ l/ holes, hatch 1 hour, wash plate for 37 DEG C, add the TMB nitrite ion of 100 μ l, adopt 2MH
2sO
4termination reaction, 450nm place reading, is total to the monoclonal antibody hybridoma cell that screening place 6 strain is reacted for HAV-Ag, is numbered 1#, 2#, 3#, 4#, 5#, 6# respectively.
The hybridoma cell strain of above-mentioned screening is built and is, Liquid nitrogen storage.By 6 strain of hybridoma strain injection mouse peritoneals of primary dcreening operation, obtain monoclonal antibody.
Adopt the indirect elisa method prescreening method of hepatitis A virus (HAV) bag quilt in the present embodiment, save workload and time.
Embodiment 3 monoclonal antibody hypotype is identified
According to the operation of antibody subtype identification kit (purchased from SouthernBiotech company) specification sheets, hypotype qualification is carried out to the monoclonal antibody filtered out in embodiment 2.Experimental result is as shown in table 1.
Table 16 strain hepatitis A antibody mab hypotype qualification result
From table 1,1# monoclonal antibody is IgM type, this subclass antibodies poor specificity; 2# monoclonal antibody is IgG3 type, and under limited experiment condition, this subclass antibodies is not easy to purifying; Therefore 1#, 2# monoclonal antibody is not suitable as the starting material of ELISA enzyme labelled antibody.In all the other 4 strain monoclonal antibodies, 3#, 5# monoclonal antibody is IgG2a type, and 4#, 6# monoclonal antibody is IgG2b type, can be used as the candidate target of ELISA enzyme labelled antibody material.
Embodiment 4 Western blot monoclonal antibody specificity
The 4 strain monoclonal antibodies filtered out are examined and determine to hypotype and carries out specific detection.Reduced form SDS-polyacrylamide gel electrophoresis (12% separation gel, 5% concentrated glue) is carried out to HAV-Ag refined solution.By gel transferring film 1-2 hour under 80-100V constant voltage after electrophoresis, protein isolate on glue is transferred on pvdf membrane.Containing the 0.01mol/LPBS4 DEG C of closed pvdf membrane that spends the night of 3%BSA.The odd contradictive hydroperitoneum incubated at room of being diluted with 1:100-1:200 respectively by film is after 2 hours, PBS washs three times, add the sheep anti-mouse igg-HRP incubated at room 1-2 hour of 1:1000 dilution, PBST20 washs three times, and HRP-DAB substrate colouring reagents box (purchased from sky root TIANGEN) develops the color.
Hepatitis A viral antigen refined solution SDS-PAGE immunoblotting detects hepatitis A virus refined solution result.Result display 3# monoclonal antibody has non-specific binding to the impurity protein in viral purification liquid, therefore this strain monoclonal antibody can not be selected as enzyme labelled antibody starting material.All the other 3 strains (4#, 5#, 6#) all can specific binding on the VP2 albumen of hepatitis A virus (HAV), this 3 strain monoclonal antibody can be used for the screening of follow-up enzyme labelled antibody material.
The identification of embodiment 5 monoclonal antibody antigen epi-position
According to hypotype qualification and the experimental result of immunoblotting, weed out 3 strain monoclonal antibodies (1#, 2#, 3#) altogether, other 3 strain candidate monoclonal antibody caprylic acid-ammonium is carried out purifying, get partial purification liquid and carry out HRP mark, monoclonal antibody refined solution is blocked by the enzyme plate of HAV-Ag by adding to wrap after 1:100 dilution, then each monoclonal antibody adding HRP mark carries out pairing and between two cross match certainly, analyzes the HAV-Ag of each monoclonal antibody in conjunction with epi-position according to itself block and Cross-blocking results.This tests replication twice, statistical experiment result.
Namely blocking-up rate >=50% is considered as blocking successfully, and the blocking-up rate of < 50% will not show.Concrete detected result is in table 2.
Table 2 hepatitis A MAbs blocking result statistics (blocking-up rate %)
From table 2,4#, 5#, 6#3 strain monoclonal antibody can intersect and mutually blocks, and shows to identify identical epitope.
The Neutralizing titer of embodiment 6 monoclonal antibody detects
By the 3 strain monoclonal antibody refined solutions Sterile Filtration after 8 times of dilutions filtered out, then 2 times of serial dilutions, are diluted to 1000CCID with equivalent
50the hepatitis A virus (HAV) mixing of/ml, with 1-2 hour in 37 DEG C, is inoculated in the 25cm growing up to individual layer 2BS cell
2in Tissue Culture Flask, 1ml/ bottle, each extent of dilution inoculates 2 bottles, 37 DEG C of absorption 2-3 hour, add cell maintenance medium, cultivate abandoning supernatant after 21 days for 33-35 DEG C, clean cell surface by PBS solution, add 1-2mlVersen liquid, after multigelation 5 times, gather in the crops virus.Adopt ELISA double antibody sandwich method qualitative detection hav antigen, with can completely in and 1000CCID
50the most high dilution of/ml hepatitis A virus (HAV) is the Neutralizing titer of this strain monoclonal antibody.Experimental result is as shown in table 3.
Table 3 monoclonal antibody Neutralizing titer detected result
| Monoclonal antibody is numbered | 4# | 5# | 6# |
| Neutralizing titer | 1:4096 | 1:1024 | 1:4096 |
From table 3,4# and 6# monoclonal antibody Neutralizing titer is higher, can reach 1:4096, and 5# monoclonal antibody Neutralizing titer is lower, is 1:1024.The hybridoma cell strain HAVmAb-6 finally choosing secretion 6# monoclonal antibody carries out preservation, and its deposit number is CGMCCNo.7859.
Sequencing result shows, the nucleotide sequence of coding 6# monoclonal antibody IgG light chain and variable region of heavy chain is respectively as shown in SeqIDNo.1 and 2.
According to above description, tool of the present invention has the following advantages:
(1) 6# monoclonal antibody of the present invention is the antibody for hepatitis A virus (HAV) high specific, has higher Neutralizing titer to hepatitis A virus (HAV) simultaneously.
(2) 6# monoclonal antibody of the present invention corresponds to hepatitis A virus (HAV) neutralizing epitope, can be used for hepatitis A virus (HAV) IgG antibody competition law and detects, thus the immune effect of effectively evaluating vaccine more.
(3) 6# monoclonal antibody of the present invention has stronger neutralization to hepatitis A virus (HAV), and prompting can be transform as curative monoclonal antibody.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. hepatitis A virus monoclonal antibody hybridoma cell strain HAVmAb-6, its deposit number is CGMCCNo.7859.
2. the monoclonal antibody produced by hybridoma cell strain described in claim 1.
3. for detecting HAV-Ag, or the test kit of anti-hepatitis A virus (HAV) IgG or IgM, reagent, it comprises monoclonal antibody according to claim 2.
4. test kit according to claim 3, reagent, is characterized in that, described monoclonal antibody is through biomarker or chemical labeling.
5. test kit according to claim 4, reagent, is characterized in that, described in be labeled as enzyme labelling or fluorescent mark.
6. test kit according to claim 5, reagent, is characterized in that, described in be labeled as horseradish peroxidase, pyruvate kinase or glucose oxidase enzyme labelling, or fluorescein-labelled.
7. monoclonal antibody described in claim 2 is for the preparation of detection HAV-Ag, or the test kit of anti-hepatitis A virus (HAV) IgG or IgM, application in reagent.
8. application according to claim 7, is characterized in that, described monoclonal antibody is through biomarker or chemical labeling.
9. monoclonal antibody described in claim 2 for the preparation of prevention or treatment hepatitis A virus (HAV) infect humanized antibody in application.
10. by the humanized antibody for preventing or treat hepatitis A virus (HAV) to infect of monoclonal antibody preparation described in claim 2.
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| CN108517315B (en) * | 2018-03-30 | 2019-06-04 | 四川迈克生物新材料技术有限公司 | Anti-human IgM monoclonal antibody, its hybridoma cell strain and application |
| CN111138533B (en) * | 2019-12-30 | 2022-06-10 | 南京融捷康生物科技有限公司 | Single domain antibody against hepatitis A virus and derived protein thereof |
| CN111138532B (en) * | 2019-12-30 | 2022-05-20 | 南京融捷康生物科技有限公司 | Use of single domain antibodies against hepatitis a virus |
| CN116514965B (en) * | 2023-06-15 | 2023-11-10 | 上海精翰生物科技有限公司 | Hepatitis A virus antibody and application thereof |
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