CN1757653A - Anti--HBeAg monoclonal antibody and cell strain, preparation method and purposes - Google Patents
Anti--HBeAg monoclonal antibody and cell strain, preparation method and purposes Download PDFInfo
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Abstract
The present invention is anti--and HBeAg monoclonal antibody and cell strain, preparation method and purposes relate to virological immunology detection technique field.Technical problem to be solved by this invention provide a kind of anti--the HBeAg monoclonal antibody is with the technological deficiency of the monoclonal antibody that solves the different epi-positions of shortage identification HBeAg that exist in the prior art and hybridoma cell strain and the anti--HBeAg MONOCLONAL ANTIBODIES SPECIFIC FOR method that produces anti--HBeAg monoclonal antibody is provided.Epitope outside the b epi-position of of the present invention resisting-HBeAg monoclonal antibody identification HBeAg, screening has obtained to produce the cell strain of this antibody, and preserving number is CCTCC-C200517, has obtained anti--HBeAg monoclonal antibody by this cellular segregation that increases.Antibody of the present invention can be used to detect HBeAg.
Description
Technical field
The present invention relates to virological immunology detection technique field, be specifically related to a kind of resisting-hepatitis B virus e antigen (HBeAg) monoclonal antibody, the cell strain of secreting this monoclonal antibody and this MONOCLONAL ANTIBODIES SPECIFIC FOR method and purposes.
Background technology
In recent years, hepatopathy has more and more become one of principal disease that threatens human health, wherein in the viral hepatitis especially with hepatitis B to anthropogenic influence's maximum.It is a serious major global public health problem that hepatitis B virus (HBV) infects, and becomes current a kind of worldwide disease.Whole world HBV persistent infection person reaches 3.5 hundred million at present, and wherein 75% in Asia and West Pacific region.China is the high popular district of hepatitis B, and HBV the infected reaches 1.2 hundred million, and it is about about 1,400,000 that average year is fallen ill, and occupies the 3rd of Notifiable disease.Along with China to the understanding in depth of hepatitis B, the progressively reinforcement of self health consciousness is to the prevention and the important topic that is detected as for medical profession of hepatitis B.
Because the popularity of hepatitis B range of influence, the seriousness of harm, diagnosis of hepatitis b reagent is with a wide range of applications and social demand as the biology and the chemical reagent of the diagnosis and the therapeutic process monitoring of the detection that is exclusively used in the various physical signs of hepatitis B patient, hepatitis B.No matter be the exploitation of product innovation or the marketing of existing procucts, all have great potential.
Hepatitis B virus e antigen (HBeAg) is the antigen of a relevant secretor type of hepatitis B virus, and at preceding C district coding, and cAg (C antigen) has portion homologous, but owing to hold at N a signal peptide has been arranged, so be the antigen of a secretor type.HBeAg and HBV DNA, DNAP and preceding-S height correlation, common virus replication (Matsuo M., Clinical Evaluation orHBeAg/Anti-Hbe system in HBeAg Positive Hapatitis, the Acta Hepatol Spn. of representing, 1985,26 (7): 819).HBeAg is most widely used, assesses the most reliably hepatitis B virus (HBV) to duplicate and one of communicable index in the blood serum designated object of hepatitis B virus at present.The HBeAg positive help to judge acute hepatitis prognosis, represent blood-borne strong, also can be used as one of performance assessment criteria of antiviral curative effect (Peng Wenwei chief editor, " viral hepatitis research ", the scientific and technological press in Guangdong, version in 1998,19-20; Ni Yuxing, Hong Xiuhua, Lou Changbin chief editor, " modern etiology check and clinical practice ", Shanghai: Shanghai scientific and technical literature press, 1999: 146).At present, in two double diagnosis of hepatitis B, the HBeAg diagnosis also is a wherein very important part, so the development of the antibody of HBeAg has important clinical use value.
At present, the detection method of HBeAg has radioimmunity detection method (RIA) and enzyme-linked immuno-sorbent assay (ELISA) etc.Owing to shortcomings such as RIA method required equipment costliness, complicated operation, price height, easy pollution are very limited its use, this use that is restricted to the ELISA method provides vast market.People such as Imai M utilize monoclonal antibody technique to prepare monoclonal anti-HBeAg (a, b) behind two strain of hybridoma, make the method that detects HBeAg reach a new height (Imai M, Journal of Immunology (J.Immunol) aspect specificity and the susceptibility, 1982,128:69).Because monoclonal anti-HbeAg hybridoma cell strain is difficult to obtain, also can't be used for ELISA detects because its excretory monoclonal anti-HBeAg antibody is unpaired, therefore, when detecting HBeAg, often adopt Sheet anti-, how anti-enzyme mark is, and this can cause technological deficiencies such as false positive rate height, susceptibility are low, and therefore adopting double-antibody sandwich-ELISA method to detect HBeAg is inexorable trend.But the double-antibody sandwich elisa method needs the monoclonal antibody of two different epi-positions of identification HBeAg, and utilize the preparation of traditional method anti--often can only obtain discerning the antibody of advantage epi-position of HBeAg (called after b epi-position) during the HBeAg monoclonal antibody, this has caused considerable restraint to clinical application that sandwich method ELISA detects HBeAg.
Summary of the invention
In order to overcome the such technological deficiency of monoclonal antibody of the different epi-positions of shortage identification HBeAg that exist in the above-mentioned prior art, the contriver has carried out extensive and detailed research work, comprises improvement, preparation and the monoclonal hybridoma of the different epi-positions of screening identification HBeAg, anti--HBeAg MONOCLONAL ANTIBODIES SPECIFIC FOR and the application in detecting HBeAg thereof etc. to traditional monoclonal antibody technique.
Based on above-mentioned research, one of technical problem to be solved by this invention is: the technological deficiency that the monoclonal antibody of anti--HBeAg monoclonal antibody to solve the different epi-positions of shortage identification HBeAg that exist in the prior art is provided.
Two of technical problem to be solved by this invention is: the hybridoma cell strain that produces anti--HBeAg monoclonal antibody is provided.
Three of technical problem to be solved by this invention is: the technological deficiency that the monoclonal antibody of anti--HBeAg MONOCLONAL ANTIBODIES SPECIFIC FOR method to solve the different epi-positions of shortage identification HBeAg that exist in the prior art is provided.
The application of anti--HBeAg monoclonal antibody that the technical problem of the solution that the present invention also wants provides in detecting HBeAg.
In order to solve the problems of the technologies described above, design of the present invention is such: in order to obtain to be different from the antibody of advantage epitope antibodies when obtaining identification advantage epitope antibodies, people for a change body is reactive strong and weak relatively to antigen different table position.The monoclonal antibody of first intravenous injection identification HBeAg advantage epi-position (b epi-position) before immunity.The preceding monoclonal antibody reactive of preferably each immunity with antigen HBeAg and b epi-position, and then carry out immunity.Increased the probability that obtains the antibody of other epi-positions outside the b epi-position like this.
Rationale of the present invention is immunologic two ultimate principles: the first, can seal this epi-position with the antibody of discerning an epi-position; Second, produce antibody in the body and have a reverse feedback regulation mechanism---when the lot of antibodies that an epi-position of identification has been arranged in the body exists, injections of antigens again, body can be regulated by reverse feedback the reactivity of this epi-position, thereby suppressed the production of antibodies level of this epi-position, and the reaction level of other epi-positions has been improved relatively.
In the present invention, the contriver uses the HBeAg immune animal, the antibody of the b epi-position of HBeAg is discerned in the preceding first intravenous injection of immunity for the first time in a large number, immunity for the first time mixes back emulsification according to method immune animal well known in the art with the Fu Shi Freund's complete adjuvant with HBeAg, for the second time, immunity for the third time mixes back emulsification immune animal with freund 's incomplete adjuvant with HBeAg, recalling with HBeAg after the immunity for the third time stimulates animal again, gets serum and detects antibody titer.Preferably before each immunity with immune animal again after the resisting of HBeAg and advantage epi-position (b epi-position)-the HBeAg monoclonal antibody reacts in advance.Antigen of immune animal or commercial hepatitis B virus e albumen (HBeAg) or the HBeAg (aminoacid sequence of HBeAg is known, Sequence ID No.1) for preparing according to known genetic engineering technique with those of ordinary skills.Immune animal can be this area animal such as rabbit, mouse etc. commonly used.Preferably use BALB/C mice.Immunization method, immunizing dose, immune pitch time etc. are because of using different animal meetings different, but those skilled in the art can be according to technique known (as editing according to Jin Baiquan, " cell and molecular immunology experimental technique ", Xi'an: press of The Fourth Military Medical University, 2002) implement it easily.Serum antibody titer detects with two-way agar diffusion method or indirect elisa method.
Before the immune mouse earlier the antibody of injection 0.1mg～5.0mg identification HBeAg advantage epi-position, preferably inject the antibody of 0.1～2.0mg identification HBeAg advantage epi-position.Make injecting immune mouse behind the antibody mixing and emulsifying of 0.05mg～0.2mgHBeAg and 0.1～1.0mg identification HBeAg advantage epi-position before each immunity.
The antibody of injection 0.5mg～1.0mg identification HBeAg advantage epi-position earlier before the immune mouse preferably makes injecting immune mouse behind the antibody mixing and emulsifying of 0.1mg～0.2mg HBeAg and 0.5～1.0mg identification HBeAg advantage epi-position before each then immunity.
Separating spleen bone-marrow-derived lymphocyte from recall post-stimulatory animal spleen, under normal condition, make fusogen spleen bone-marrow-derived lymphocyte and murine myeloma cell are merged, select to cultivate with the nutrient solution that contains 1 * HAT (xanthoglobulin (H), aminopterin (A) and thymidine (T)) and 10% calf serum with polyoxyethylene glycol (PEG).50 * HAT has commercial prod, and it contains the A of H, 0.02mM of 5mM and the T of 0.8mM.Method according to this area routine is carried out cytogamy, and nutrient solution is RPMI1640.Merge the limiting dilution assay that hybridoma cloning adopts this area routine, obtained the cell strain S-72-3 that a strain produces anti--HBeAg monoclonal antibody at last, this cell strain is submitted Chinese typical culture collection center (the being called for short CCTCC) preservation that is positioned at Chinese Wuhan University on September 23rd, 2005, and preserving number is: CCTCC-C200517.
Be that the hybridoma cell strain of CCTCC-C200517 carries out cultured and amplified in vitro according to the conventional animal cell culture processes or the injection mouse peritoneal prepares ascites with above-mentioned preserving number.Antibody titer with in indirect elisa method detection nutrient solution or the ascites reclaims anti--HBeAg monoclonal antibody with method well known in the art from culture or ascites.
With HBeAg and other proteantigens such as bovine serum albumin as antigen, carry out antibodies specific with indirect elisa method and detect.Identify the Ig class of antibody and make the subclass that double diffusion or sandwich ELISA method are identified above-mentioned resisting-HBeAg monoclonal antibody with method well known in the art such as enzyme mark or fluorescein-labeled second antibody with the anti-subclass serum system of standard, above-mentioned resisting-the HBeAg monoclonal antibody is IgG2b, and light chain is the κ chain.
Examine and determine the identification epi-position of above-mentioned resisting-HBeAg monoclonal antibody in conjunction with test and/or biosensor (Biosensor) with competition.Synthetic the peptide (LN18) (Sequence IDNo.2) of one section 18 amino-acid residue, it is the aminoacid sequence of the 119-136 of HBeAg, indirect ELISA detects and shows that above-mentioned resisting-HBeAg monoclonal antibody combines with the LN18 specificity with the monoclonal antibody ewb of the associativity of peptide LN18 very weak and commercial identification HBeAg advantage epi-position (b epi-position), show that above-mentioned resisting of the present invention-HBeAg monoclonal antibody institute bonded epi-position is the aminoacid sequence outside the 119-136 of HBeAg, different with the epi-position of ewb.Further having expressed another polypeptide Ec (Sequence IDNo.3) with engineered means, is the polypeptide that has removed HBeAg antigens c terminal amino acid residue, that is comprises the peptide section of HBeAg1-118.Of the present invention anti--HBeAg monoclonal antibody and Ec height in conjunction with and ewb combines hardly with Ec, thereby the epi-position of of the present invention resisting-HbeAg monoclonal antibody is positioned at the 1-118 sequence of HBeAg.
Thereby, the invention provides a kind of resisting-the HBeAg monoclonal antibody, it is in conjunction with advantage epi-position (b epi-position) epi-position in addition of HBeAg, preferably in conjunction with the epi-position outside the 119-136 of HBeAg, most preferably in conjunction with the epi-position that is positioned at the 1-118 aminoacid sequence of HBeAg.
The present invention also provides a strain to produce the cell strain of above-mentioned resisting-HBeAg monoclonal antibody.
In addition, the invention provides preparation above-mentioned anti--the HBeAg monoclonal antibody method, comprise the steps:
(a) amplification produces the cell strain of above-mentioned resisting-HBeAg monoclonal antibody;
(b) from amplified production, reclaim anti--HBeAg monoclonal antibody.
The method of amplifying cells strain is being known in the art, and as with external animal cell culture method, for example rolls bottle, square vase or zooblast fermentor cultivation amplified hybridization oncocyte; Perhaps,, gather ascites after 7-10 days as the above-mentioned hybridoma cell strain abdominal injection BABL/C mouse then of increasing earlier with preparation ascites method in the body.
The method that reclaims anti--HBeAg monoclonal anti from nutrient solution or ascites is also used always in this area, as ammonium sulfate precipitation method, anion exchange chromatography or staphylococcal protein A,SPA affinity chromatography.
The different epi-positions of the monoclonal antibody ewb identification HBeAg of of the present invention resisting-HBeAg monoclonal antibody and commercial identification HBeAg advantage epi-position can be used, and are used for the HBeAg that sandwich method ELISA detects serum.With horseradish peroxidase or fluorescein isothiocyanate or biotin labeling of the present invention anti--the HBeAg monoclonal antibody, cooperate the HBeAg that is used for detecting serum with ewb.In advance ewb is fixed on the solid phase carrier, add sample to be checked then, positive control and negative control are set, antibody-antigen adds the of the present invention anti--HBeAg monoclonal antibody of mark in conjunction with the back, reaction, adding enzyme reaction substrate colour developing or luminous, the HBeAg in the test sample.Certainly, mark ewb and predetermined fixed antibody of the present invention also can reach testing goal.Method of the present invention has very high specificity and higher accuracy rate.
Should be clear and definite, monoclonal antibody and monoclonal antibody are synonym in the present invention, can exchange use.Of the present invention resisting-HBeAg monoclonal antibody is also represented with antibody S-72-3 sometimes, (preserving number: what CCTCC-C200517) produce resists-the HBeAg monoclonal antibody, thereby antibody S-72-3 can not use with anti--HBeAg monoclonal antibody exchange but antibody S-72-3 censures hybridoma cell strain S-72-3.
Description of drawings
Fig. 1 is the mensuration of serum antibody titer after the BALB/C mice immunity, and method is an indirect elisa method, ◆ for mice serum, the ■ of immune HBeAg is normal mouse serum.
Fig. 2 is that indirect elisa method detection monoclonal antibody ascites is tired, ◆ be hybridoma cell strain S-72-3 ascites, ■ pastern bone myeloma clone SP2/0 ascites, ▲ be normal mouse serum.
Fig. 3 is that indirect elisa method detects monoclonal antibody ascites and HBeAg bonded specificity, and 1 is reorganization HBeAg antigen (5 μ g/mL), and 2 is reference protein (bovine serum albumin, BSA, 10 μ g/mL).
Fig. 4 is that indirect elisa method detects combining of antibody and peptide LN18, and LN18+S-72 represents LN18 and antibody S-72-3, LN18+ewb representative peptide LN18 and commercialization antibody ewb combination.
Fig. 5 is that indirect elisa method detects combining of antibody S-72-3 and ewb and Ec, and wherein: S-72 represents that antibody S-72-3 combines with Ec, and ewb represents that antibody ewb combines with Ec.
Fig. 6 is that the sandwich ELISA method is identified antibody identification meter position, and wherein E represents reorganization HBeAg antigen, the irrelevant antigen bovine serum albumin (BSA) of BLANK representative.
Fig. 7 is the affinity costant that Biocore detects antibody S-72-3.
Describe the present invention in detail below in conjunction with concrete embodiment. Except specified otherwise is arranged, all (Jin Dongyan, Li Mengfeng etc. translate according to " molecular cloning experiment guide " (second edition) in concrete operations, J. not Ritchie, T. Manny A Disi show for Pehanorm Brooker, E.F., Beijing: Science Press, 1998) and " cell and molecular immunology experimental technique " (Jin Baiquan chief editor, Xi'an: publishing house of The Fourth Military Medical University, version in 2002) carry out. After describing these specific embodiment in detail, those of ordinary skill in the art can fully know how to design other reliable methods, thereby obtains same information by using achievement of the present invention. Therefore no matter how concrete in detail the description of these specific implementation methods is, all do not mean that any restriction to the invention overall range, and scope of the present invention only is as the criterion with the scope of claim.
Embodiment
The foundation and the MONOCLONAL ANTIBODIES SPECIFIC FOR thereof of embodiment 1 anti--HBeAg hybridoma cell strain
(1) immune animal
Three immune balb/c mices of HBeAg albumen with gene recombination.For the first time the day before yesterday of immunity, first intravenous injection 0.5mg discerns the antibody ewb (Shanghai Ke Hua company limited product) of the b epi-position of HBeAg.Immune for the first time with emulsification behind Fu Shi Freund's complete adjuvant and the HBeAg albumen equivalent mixing, intravenous injection, 0.1mg/, volume injected 0.2～0.3mL.Second, third is immune with emulsification behind freund 's incomplete adjuvant and the HBeAg albumen equivalent mixing, and immunization method and consumption are with immunity for the first time.All make before each immune mouse 0.1mgHBeAg and 0.5mg antibody ewb mix, with the emulsification of equivalent adjuvant mixing.Each immunity one month at interval.Immunity for the third time is after one month, and the HBeAg albumen of abdominal injection 0.1mg is recalled stimulates BALB/C mice.Indirect ELISA method detects the serum titer after the immunity, Fig. 1.As can be seen from Figure 1, after three immunity, the antibody titer that the antigenic indirect elisa method of reorganization HBeAg is detected serum is 64000, and normal mouse serum is to the not reaction of reorganization HBeAg antigen.The HBeAg antigen concentration of bag quilt was 1.5 μ g/ml during indirect elisa method detected.
The equal commodity in use product of freund 's incomplete adjuvant and Fu Shi Freund's complete adjuvant.
(2) preparation of hybridoma cell strain and screening
Fetch and recall the mouse spleen cell and the SP2/0 myeloma cell that stimulate after 3 days and merge with 50%PEG-4000 according to a conventional method.Cultivate with the RPMI-1640 that contains 1 * HAT and 10% calf serum.Merge and began to occur fused cell in back three days, 335 are the fusion hole in 512 holes, and fusion rate is 65.4%.Detect anti--HBeAg monoclonal antibody with the ELISA method, detect 281 holes, wherein positive hole is 12, and positive rate is 4.2%.Through three limiting dilution assay clonings, obtain at last a strain stably excreting anti--cell strain of HBeAg monoclonal antibody, be labeled as S-72-3.
(3) preparation of ascites and tiring and the evaluation of antibody subtype
With positive hybridoma cell strain S-72-3 with 1 * 10
6The BABL/C female mice in individual/only the presensitized 8-10 of amount abdominal injection age in week after 7-10 days, is gathered mouse ascites, detects tiring of S-72-3 monoclonal antibody with indirect ELISA method.With myeloma cell line SP2/0 ascites and normal mouse serum is contrast, and the HBeAg antigen concentration of bag quilt is 1.5 μ g/mL.
The result: SP2/0 ascites and normal serum to the reorganization HBeAg antigen reactionless (Fig. 2), monoclonal antibody S-72-3 tire for: 1.024 * 10
6And then make double fastener heart ELISA with the anti-subclass serum of standard system and determine that cell strain S-72-3 excretory antibody subtype is IgG2b, light chain is the κ chain.
Embodiment 2 anti--HBeAg MONOCLONAL ANTIBODIES SPECIFIC FOR
With positive hybridoma cell strain S-72-3 with 1 * 10
6The BABL/C female mice in individual/only amount abdominal injection presensitization 8-10 age in week after 7-10 days, is gathered mouse ascites, detects tiring of S-72-3 monoclonal antibody with indirect ELISA method.4 ℃ of ascites, the centrifugal 15min of 12000rpm get 1 part of (volume) ascites and mix with the acetate buffer solution of 2 parts of (volume) 60mM, pH4.8, dropwise add n-caprylic acid 33 μ L/mL ascites under the stirring at room, mixed at room temperature 30min.4 ℃ leave standstill more than 2 hours it are fully precipitated, and 4 ℃ then, the centrifugal 30min of 12000rpm discard precipitation, and supernatant filters the PBS that the back adds the 0.1MpH7.4 of 1/10 volume through sand core funnel, with 2M NaOH adjusting pH to 7.4.The ammonium sulfate that adds 0.277g/mL under the ice bath in 30min makes saturation ratio reach 45%, 4 ℃ and leaves standstill more than 1 hour 4 ℃ then, the centrifugal 30min of 10000rpm, supernatant discarded.Precipitation is with the PBS dissolving of the pH7.4 that contains 137mM NaCl, 2.6mM KCl, 0.2mM EDTA, and in the above-mentioned PBS of 50-100 times of volume 4 ℃ of dialysed overnight.Be further purified with Q SepharoseFast Flow exchange column chromatographic technique then.Level pad is that 20mMpH7.5Tris-HCl damping fluid, elution buffer are the Tris-HCl damping fluid of the 20mM, the pH7.5 that contain 1.0M NaCl, chromatography column XK 16/20, Pharmacia AKTA FPLC chromatographic system.According to the specification sheets dress post that manufacturer provides, level pad balance chromatography column, flow velocity 5mL/min.Not conjugated protein with the level pad flush away of 3 times of column volumes after last sample finishes, with the linear gradient elution bonded antibody protein that NaCl increases progressively, linear gradient is a 0-1.0MNaCl/10 times of cylinder.Detect A
280, the antibody protein of collection wash-out.SDS-PAGE detects purity, and with this area determined by ultraviolet spectrophotometry anti-body contg commonly used, indirect elisa method detects antibody titer.
The specificity of embodiment 3 anti--HBeAg monoclonal antibodies
Detect the specificity of anti--HBeAg monoclonal antibody with indirect ELISA method.The antigen of bag quilt is reorganization HBeAg antigen (5 μ g/ml), contrasts to be bovine serum albumin (10 μ g/ml).The concentration of antibody S-72-3 is 5 μ g/ml (embodiment 2).Test-results is seen Fig. 3.
Antibody S-72-3 and recombinant protein HBeAg have very strong reactivity as can be seen from Figure 3, and very low with the reactivity of reference protein.Show: antibody S-72-3 antigen-specific is good.
Evaluation and the property analysis of embodiment 4 antibody S-72-3 in conjunction with epi-position
For the epi-position that the monoclonal antibody of institute's preparation is discerned, polypeptide, called after LN18 (seeing sequence table Sequence No.2) have been synthesized in design.The sequence of LN18 peptide is according to documents and materials and computer simulation design, its representative be the epi-position that the aminoacid sequence of HBeAg antigen 1 19-136 is formed.Indirect elisa method detects the associativity of antibody S-72-3 and ewb and LN18, result such as Fig. 4.Ewb and LN18 have associativity preferably, and the combination of antibody S-72-3 and LN18 does not almost have, show that ewb institute bonded epi-position probably is on this section sequence of HBeAg at LN18 place, and antibody S-72-3 bonded epi-position is the zone outside the 119-136.The result can illustrate very clearly that also these two antibody institute bonded positions are different simultaneously, is positioned on the different zones of HBeAg.
In order further to study antibody S-72-3 institute bonded particular location, expressed a new antigen, the peptide section of a HBeAg, be labeled as Ec (Sequence No.3), 31 amino-acid residues of HBeAgC end are removed in not being both of this antigen and HBeAg maximum, this that removes section zone has comprised the sequence at LN18 place, also is that Ec is the amino acid whose sequence of HBeAg1-118.Indirect ELISA method detection antibody S-72-3 and ewb combine with Ec's, and antibody concentration is 5 μ g/mL, the results are shown in Figure 5.As can be seen, S-72-3 has preferably with Ec and combines, and ewb does not have with combining almost of Ec.The experimental result of front has shown that ewb's is likely the sequence at LN18 place in conjunction with epi-position, so that has removed albumen Ec after the sequence at LN18 place and ewb does not react the conclusion of also further having verified the front, and the reaction of S-72-3 and Ec shows that S-72-3 institute bonded epi-position is the zone at Ec place, is different from ewb.
In order further to analyze the antibodies epi-position, the contriver has carried out preliminary qualitative analysis to their institute's bonded epitopes, and what determine that they discern is linearity or configuration epi-position.If identification is the configuration epi-position, after the antigen sex change, reactive behavior will descend greatly so, in general＞50%; If identification is linear epitope, so active fall can be smaller, in general＜50%.
At first with the HBeAg sex change of reorganization, detect antibody S-72-3 and ewb to the associativity before and after the HBeAg sex change, result such as table 1 with indirect elisa method.
Table 1: monoclonal antibody is to reaction result before and after the antigen sex change
| Antibody | OD 450(HBeAg before the sex change) | OD 450(HBeAg after the sex change) | Reactive behavior reduces (%) |
| S-72-3 | 2.425 | 0.269 | 89 |
| ewb | 2.924 | 1.652 | 43 |
The result finds out from table, and antibody S-72-3 has descended 89% respectively to the reactive behavior of the HBeAg before and after the sex change, and this shows that the epi-position that antibody S-72-3 is discerned may be the configuration epi-position; And ewb has only descended 43%, and this shows that the epi-position that it is discerned may be a linear epitope.In conjunction with the experimental result of front, what can judge tentatively that antibody S-72-3 discerned is the configuration epi-position that the 1-118 amino acid of HBeAg is formed, and this epi-position may have been crossed over whole Ec.
Embodiment 5 sandwich assays detect reorganization HBeAg
Detect the HBeAg that recombinates with the sandwich ELISA method, wherein antibody ewb is coated on the enzyme plate, with the antibody horseradish peroxidase-labeled of sodium periodate method with embodiment 2, the antigen of detection is reorganization HBeAg, contrasts to be BSA.The results are shown in Figure 6.The result shows, horseradish peroxidase-labeled antibody S-72-3 can match special detection recombinant E protein with ewb, shows that further it is different with the ewb identified epitope.This result shows that monoclonal antibody S-72-3 is the monoclonal antibody of other epi-positions outside the identification HBeAg advantage epi-position b epi-position, and can match with known antibodies ewb and detect HBeAg.
The mensuration of embodiment 6 monoclonal antibody S-72-3 affinity costants
Select the extensive at present biocore technical measurement affinity costant that adopts.
System information: BIAcore 3000 (Shanghai life science institute institute of materia medica)
CM5sensorchip
Amine coupling(Directly immobilization)
System buffer liquid: 20mM HEPEs
150mM NaCl
5mM EDTA
0.1％P20
Experimental result is seen Fig. 7.Wherein the antibody concentration gradient is as follows: 4 μ M, 2 μ M, 1 μ M, 0.5 μ M, 0.25 μ M, 0.125 μ M and 0 μ M.According to the divalence combination model, the result is as follows for the reaction power mathematic(al) constant:
k
a1＝1.14±0.129×10
31/Ms k
d1＝2.49±0.168×10
-51/s
k
a2＝1.46±0.187×10
-31/Ms k
d2＝0.0651±0.002 1/s
Affinity costant K=k
a/ k
d=4.57 * 10
10M
Ka wherein: binding constant; Kd: dissociation constant.As seen antibody S-72-3 has the avidity of height to HBeAg.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉anti--HBeAg monoclonal antibody and cell strain, preparation method and purposes
<160>3
<170>PatentIn version 3.3
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＜213〉hepatitis B virus (Hepatitis B virus)
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<222>(1)..(149)
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-10 -5 -1 5
Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp
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Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr
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Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala
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Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr
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Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg Asp Leu Val Val
75 80 85
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln Leu Leu Trp
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Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu Tyr
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115
Claims (12)
1. anti--the HBeAg monoclonal antibody, it is characterized in that discerning the epitope outside the b epi-position of HbeAg.
2. according to the anti--HBeAg monoclonal antibody of claim 1, it is characterized in that discerning the epitope that is positioned at HBeAg aminoacid sequence 1-118.
By preserving number be the hybridoma cell strain excretory of CCTCC-C200517 anti--identification of HBeAg monoclonal antibody is positioned at the epitope of HBeAg aminoacid sequence 1-118.
4. hybridoma cell strain, preserving number is: CCTCC-C200517.
5. according to the preparation method of the hybridoma cell strain of claim 4, comprise with antigen HBeAg immune animal, separating spleen bone-marrow-derived lymphocyte and and the myeloma cell is merged, screening hybridoma and hybridoma detect step, it is characterized in that before with the HBeAg immune animal first intravenous injection discerns the antibody of the b epi-position of HBeAg in a large number.
6. according to the preparation method of the hybridoma cell strain of claim 5, it is characterized in that before with the HBeAg immune animal, making earlier behind the antibody response of b epi-position of HBeAg and identification HBeAg immune animal again.
7. according to the preparation method of the hybridoma cell strain of claim 5 or 6, it is characterized in that immune animal is that BALB/C mice, myeloma cell are SP2/0.
8. according to the preparation method of the hybridoma cell strain of claim 7, the antibody that it is characterized in that the b epi-position of the identification HBeAg of intravenous injection 0.1mg～2.0mg before the immune balb/c mice, make antibody mixing, emulsification, the immune mouse then of the b epi-position of 0.05mg～0.2mg HbeAg and 0.1mg～1.0mg identification HBeAg before each immune mouse.
9. MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 3 comprises that the vitro culture preserving number is the cell of CCTCC-C200517 and the step that reclaims anti--HBeAg monoclonal antibody from nutrient solution.
10. MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 3, comprise with preserving number be CCTCC-C200517 hybridoma abdominal injection animal, reclaim ascites and from ascites, separate anti--HBeAg monoclonal antibody.
11. MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 10 is characterized in that reclaiming monoclonal antibody with sad-ammonium sulfate precipitation method from ascites, uses the anion exchange chromatography purified monoclonal antibody then.
12. anti--HBeAg monoclonal antibody the purposes during detecting HBeAg arbitrary according to claim 1-3.
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| CNB2005100308494A CN100398559C (en) | 2005-10-28 | 2005-10-28 | Anti-HBeAg monoclonal antibody and its cell strain, prepn. method and use thereof |
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| CNB2005100308494A CN100398559C (en) | 2005-10-28 | 2005-10-28 | Anti-HBeAg monoclonal antibody and its cell strain, prepn. method and use thereof |
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| CN100398559C CN100398559C (en) | 2008-07-02 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043049A (en) * | 2009-10-23 | 2011-05-04 | 上海荣盛生物药业有限公司 | In vitro detection method and application of antibody |
| CN111187349A (en) * | 2019-10-30 | 2020-05-22 | 四川迈克生物新材料技术有限公司 | Method for preparing monoclonal antibody |
| CN112979787A (en) * | 2019-12-13 | 2021-06-18 | 东莞市朋志生物科技有限公司 | Binding protein for binding HBeAg and application thereof |
| CN115677853A (en) * | 2021-07-29 | 2023-02-03 | 东莞市朋志生物科技有限公司 | anti-HBeAg antibody or antigen binding fragment thereof and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102327611B (en) * | 2011-09-14 | 2017-04-26 | 西安交通大学 | Vaccine taking HBeAg egg white and/or HBeAg epitope polypeptide as immunogen |
-
2005
- 2005-10-28 CN CNB2005100308494A patent/CN100398559C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043049A (en) * | 2009-10-23 | 2011-05-04 | 上海荣盛生物药业有限公司 | In vitro detection method and application of antibody |
| CN111187349A (en) * | 2019-10-30 | 2020-05-22 | 四川迈克生物新材料技术有限公司 | Method for preparing monoclonal antibody |
| CN112979787A (en) * | 2019-12-13 | 2021-06-18 | 东莞市朋志生物科技有限公司 | Binding protein for binding HBeAg and application thereof |
| CN115677853A (en) * | 2021-07-29 | 2023-02-03 | 东莞市朋志生物科技有限公司 | anti-HBeAg antibody or antigen binding fragment thereof and application thereof |
| CN115677853B (en) * | 2021-07-29 | 2023-10-31 | 东莞市朋志生物科技有限公司 | anti-HBeAg antibody or antigen binding fragment thereof and application thereof |
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| CN100398559C (en) | 2008-07-02 |
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