CN100387709C - Anti HBeAg monoclone antibody cell strain, preparation method and anti HBeAg monoclone antibody - Google Patents
Anti HBeAg monoclone antibody cell strain, preparation method and anti HBeAg monoclone antibody Download PDFInfo
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Abstract
The present invention relates to an anti-HBeAg monoclonal antibody cell strain, a preparation method thereof and an anti-HBeAg monoclonal antibody thereof, which belongs to the virus immunology detecting technical field. The present invention aims to solve the technical defects of lacking a monoclonal antibody for recognizing different epitopes of HBeAg and difficultly preparing a monoclonal antibody for recognizing epitopes except for predominant epitopes of HBeAg in the prior art. The present invention discloses two hybridoma cell strains for producing anti-HBeAg monoclonal antibody. Animals are pre-injected HBeAg before immunity and/ or after the reaction of HBeAg and ewb to obtain antigen epitope monoclonal antibody for recognizing epitopes except for b epitopes. The present invention also discloses two kinds of anti-HBeAg monoclonal antibodies. The antibody of the present invention has favorable specificity and favorable affinity, can recognize different epitopes, can respectively be matched with ewb or mutually matched for using a double antibody sandwich method ELISA to detect HBeAg.
Description
Technical field
The present invention relates to virological immunology detection technique field, be specifically related to resist-hepatitis B virus e antigen (anti--HBeAg) cell strain of monoclonal antibody and preparation method thereof and anti--HBeAg monoclonal antibody.
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 or HBeAg/Anti-Hbesystem 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 EL1SA 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 one HBe hybridoma cell strain is difficult to obtain, also since its excretory anti--the HBeAg monoclonal antibody is unpaired and can't be used for ELISA and detect, therefore, when detecting HBeAg, often adopt that Sheet is anti-, how anti-the enzyme mark is, 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 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: provide the hybridoma cell strain that produces anti--HBeAg monoclonal antibody to discern the technological deficiency of the cell strain of monoclonal antibody of the different epi-positions of HBeAg to solve the shortage that exists in the prior art.
Two of technical problem to be solved by this invention is: the preparation method that the hybridoma cell strain that produces anti--HBeAg monoclonal antibody is provided is to solve the such technological deficiency of monoclonal antibody hybridoma cell strain that is difficult to prepare the b epi-position epitope in addition of discerning HBeAg that exists in the prior art.
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.In addition can also be before each immunity with the monoclonal antibody reactive of antigen HBeAg and b epi-position, and then carry out injecting immune.Thereby increased the probability of the antibody that obtains other epi-positions outside the b epi-position.
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, promptly when a large amount of antibody 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 prepare resulting HBeAg (aminoacid sequence of HBeAg is known, Sequence ID No.1) with the known genetic engineering technique of 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～0.2mg HBeAg 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 cell culture fluid 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, cell strain S-29-3 and S-72-3 that two strains produce anti--HBeAg monoclonal antibody have been obtained at last, wherein cell strain S-29-3 submits the Chinese typical culture collection center preservation that is positioned at Chinese Wuhan University on September 23rd, 2005, and preserving number is: CCTCC-C200516; Cell strain S-72-3 submits to the People's Republic of China (PRC) to be positioned at the Chinese typical culture collection center preservation of Chinese Wuhan University on September 9th, 2005, and preserving number is: CCTCC-C200517.
Be that the hybridoma cell strain of CCTCC-C200516 and 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.Detect antibody titer in cultivation or the ascites with indirect elisa method, from culture or ascites, reclaim anti--HBeAg monoclonal antibody with method well known in the art, promptly obtain anti--HBeAg monoclonal antibody, be expressed as antibody S-29-3 and antibody S-72-3 for convenience of explanation respectively.
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, antibody S-29-3 is IgG1 in above-mentioned resisting-HBeAg monoclonal antibody, and light chain is the κ chain; Antibody S-72-3 is the IgG2b hypotype, 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 the monoclonal antibody (ewb) that shows antibody S-72-3 and the associativity of peptide LN18 very weak and commercial identification HBeAg advantage epi-position (b epi-position) and combines with the LN18 specificity, show that antibody S-72-3 of the present invention 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 ID No.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.Antibody S-72-3 of the present invention and Ec height in conjunction with and ewb combines hardly with Ec, thereby the epi-position of antibody S-72-3 of the present invention is positioned at the 1-118 sequence of HBeAg.
With the associativity of indirect elisa method detection antibody S-29-3 and peptide section LN18 and irrelevant peptide, antibody S-29-3 highly combines with LN18, shows that the epi-position of antibody S-29-3 identification may be exactly the epitope of LN18 representative, i.e. the HBeAg119-136 aminoacid sequence.This result shows that immunization method of the present invention has obtained the antibody of identification with the akin epi-position of b epi-position of HBeAg equally.
Thereby, the invention provides the hybridoma cell strain that produces anti--HBeAg monoclonal antibody, preserving number is respectively CCTCC-C200516 and CCTCC-C200517.
The invention provides the method for preparing the hybridoma cell strain that produces anti--HBeAg monoclonal antibody, promptly before with the HBeAg immune animal, inject the antibody of identification HBeAg advantage epi-position earlier, and then use the HBeAg immune animal.Preferably before each immune animal with immune animal again behind the antibody response of HBeAg and identification HBeAg advantage epi-position.Immune animal is rabbit or mouse, preferably BALB/C mice.From immune animal, separate the spleen bone-marrow-derived lymphocyte that produces anti--HBeAg monoclonal antibody, then the myeloma cell of spleen bone-marrow-derived lymphocyte and corresponding strain is merged the acquisition hybridoma, use the general technology screening hybridoma in this area then; Separate monoclonal cell with technology such as limited dilution method that this area is commonly used, identify monoclonal hybridoma, obtain to produce the hybridoma cell strain of anti--HBeAg monoclonal antibody with indirect elisa method, frozen with the technology that this area is commonly used.
The present invention also provides anti--HBeAg monoclonal antibody, wherein antibody S-72-3 discerns advantage epi-position (b epi-position) epi-position in addition of HBeAg, preferably discern the epi-position outside the 119-136 of HBeAg, most preferably identification is positioned at the epi-position of the 1-118 aminoacid sequence of HBeAg.The 119-136 epi-position of antibody S-29-3 identification HBeAg, it is the IgG1 type.
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 amplified production is also used always in this area, as ammonium sulfate precipitation method, anion exchange chromatography or staphylococcal protein A,SPA affinity chromatography.
Antibody S-72-3 of the present invention and the commercial different epitopes of discerning monoclonal antibody (ewb) the identification HBeAg of HBeAg advantage epi-position can be used the HBeAg that is used for sandwich method ELISA detection 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, and antibody-antigen adds the of the present invention of mark in conjunction with the back and resists-the HBeAg monoclonal antibody, the HBeAg in reaction, the colour developing of adding enzyme reaction substrate or the luminous detection 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.
Two anti--HBeAg monoclonal antibodies of the present invention have different antigen recognition epi-positions, thereby can cooperate and be used for sandwich method ELISA and detect HBeAg, are used to detect hepatitis B virus clinically.
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 and antibody S-29-3 sometimes, but antibody S-72-3 censures hybridoma cell strain S-72-3 (preserving number: the anti--HBeAg monoclonal antibody that CCTCC-C200517) produces; Antibody S-29-3 censures hybridoma cell strain S-29-3, and (preserving number: CCTCC-C200516) anti--HBeAg monoclonal antibody of generation, thereby antibody S-72-3 and antibody S-29-3 can not exchange with anti--HBeAg monoclonal antibody and use.
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-29-3 ascites, ■ pastern bone myeloma clone SP2/0 ascites.
Fig. 3 is that indirect elisa method detects monoclonal antibody S-29-3 ascites and HBeAg bonded specificity, and is reorganization HBeAg antigen (5 μ g/mL), and ■ is reference protein (bovine serum albumin, BSA, 10 μ g/mL).
Fig. 4 is that indirect elisa method detects combining of antibody S-29-3 and peptide LN18, and represent LN18 and antibody S-29-3 combination, and ■ represents have nothing to do peptide and antibody S-29-3.
Fig. 5 is that indirect elisa method detects combining of antibody S-72-3 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. 6 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. 7 sandwich method ELISA detects HBeAg, and wherein E represent to recombinate HBeAg, BLANK represents irrelevant protein B SA.
Fig. 8 is the affinity costant that Biocore detects antibody S-29-3.
Fig. 9 is the affinity costant that Biocore detects antibody S-72-3.
Describe the present invention in detail below in conjunction with concrete embodiment.Except that 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 Sa nurse Brooker, E.F., Beijing: Science Press, 1998) and " cell and molecular immunology experimental technique " (Jin Baiquan chief editor, Xi'an: press of The Fourth Military Medical University, version in 2002) carry out.After describing these embodiments 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/ only injects 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.Each immunity one month at interval.All make before each immune mouse 0.1mg HBeAg and 0.5mg antibody ewb mix, with the emulsification of equivalent adjuvant mixing.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, tire 64000 to the antigenic indirect elisa method detection of reorganization HBeAg is sero-fast, 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 limited dilution clonings, obtain at last a strain stably excreting anti--cell strain of HBeAg monoclonal antibody, be labeled as S-29-3 and S-72-3.
(3) preparation of ascites and tiring and the evaluation of antibody subtype
With positive hybridoma cell strain S-29-3 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-29-3 monoclonal antibody with indirect ELISA method.With myeloma cell line SP2/0 ascites is contrast, and the HBeAg antigen concentration of bag quilt is 1.5 μ g/mL.
The result: SP2/0 ascites reorganization HBeAg antigen reactionless (Fig. 2), monoclonal antibody S-29-3 tire for: 1.28 * 10
6And then make double fastener heart ELISA with the anti-subclass serum of standard system and determine that cell strain S-29-3 excretory antibody subtype is IgG1, light chain is the κ chain.
Embodiment 2 anti--HBeAg MONOCLONAL ANTIBODIES SPECIFIC FOR
With positive hybridoma cell strain S-29-3 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-29-3 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) 0.06MpH4.8, 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 pH7.4PBS dissolving 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 Sepharose Fast Flow exchange column chromatographic technique then.Level pad is that Tris-HCl damping fluid, the elution buffer of 20mM pH7.5 is the Tris-HCl damping fluid that contains the 20mM pH7.5 of 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 the content of antibody detects antibody titer with this area determined by ultraviolet spectrophotometry commonly used with indirect elisa method.
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 the antibody S-29-3 of embodiment 2 is 5 μ g/ml.Test-results is seen Fig. 3.
Antibody S-29-3 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-29-3 has good antigen-specific.
Evaluation and the property analysis of embodiment 4 antibody S-29-3 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 ID 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-29-3 and LN18, and contrast is one section irrelevant peptide section, result such as Fig. 4.Antibody S-29-3 and LN18 have associativity preferably, show: antibody S-29-3 institute bonded epi-position probably is on this section sequence of HBeAg at LN18 place.
For further analyze antibody S-29-3 in conjunction with epi-position, the contriver has carried out preliminary qualitative analysis to their institute's bonded epitopes, what determine that they discern is linear epitope 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-29-3 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
The result finds out from table, and antibody S-29-3 has descended 80% respectively to the reactive behavior of the HBeAg before and after the sex change, and this shows that the epi-position that antibody S-29-3 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.Discover that further antibody ewb has certain associativity to LN18, the epi-position that shows antibody S-29-3 identification is the configuration epi-position of the sequence of LN18 representative.
Evaluation and the property analysis of embodiment 5 antibody S-72-3 in conjunction with epi-position
Prepare antibody S-72-3 with hybridoma cell strain S-72-3 according to the method for embodiment 2.
Indirect elisa method detects the associativity of antibody S-72-3 and ewb and LN18, result such as Fig. 5.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 ID No.3), the amino-acid residue of HBeAgC end is removed in not being both of this antigen and HBeAg maximum, this that removes section zone has comprised the sequence at LN18 place, and just 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 6.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, also further verified the conclusion of testing previously so removed the sequence albumen Ec and not the reacting of ewb afterwards at LN18 place, 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.
Further analyze the antibodies epi-position:, detect antibody S-72-3 and ewb to the associativity before and after the HBeAg sex change, result such as table 2 with indirect elisa method at first with the HBeAg sex change of reorganization.
Result from table 2 finds out that 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.
Table 2: monoclonal antibody is to reaction result before and after the antigen sex change
Embodiment 6 sandwich assays detect reorganization HBeAg
Detect the HBeAg that recombinates with the sandwich ELISA method, wherein use 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 7.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 b epi-position, and can match with known antibodies ewb and detect HBeAg.
The mensuration of embodiment 7 monoclonal antibody S-29-3 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)
CM5?sensorchip
Amine?coupling(Directly?immobilization)
System buffer liquid: 20mM HEPEs
150mM?NaCl
5mM?EDTA
0.1％P20
Experimental result is seen Fig. 8.Wherein the antibody concentration gradient is as follows: 4,2,1,0.5,0.25,0.125,0 μ M.According to the divalence combination model, the result is as follows for the reaction power mathematic(al) constant:
k
a1＝1.24±0.187×10
4 1/Ms k
d1＝7.78±1.68×10
-51/s
k
a2＝2.34±0.334×10
-3?1/Ms k
d2＝0.0836±0.0011/s
Affinity costant: K=Ka/Kd=1.59 * 10
9M
Ka wherein: binding constant; Kd: dissociation constant.As seen antibody S-29-3 has the avidity of height to HBeAg.
The mensuration of embodiment 8 monoclonal antibody S-72-3 affinity costants
Measuring method and parameter condition the results are shown in Figure 9 with embodiment 7.Calculation result is as follows:
k
a1＝1.14±0.129×10
3 1/Ms k
d1＝2.49±0.168×10
-51/s
k
a2＝1.46±0.187×10
-3?1/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 cell strain of monoclonal antibody, preparation method and anti--HBeAg monoclonal antibody
<160>3
<170>PatentIn?version?3.3
<210>-10
<211>149
<212>PRT
＜213〉hepatitis B virus (Hepatitis B virus)
<220>
<221>SIGNAL
<222>(-10)..(-1)
<220>
<221>CHAIN
<222>(1)..(149)
<400>1
Ser?Lys?Leu?Cys?Leu?Gly?Trp?Leu?Trp?Gly?Met?Asp?Ile?Asp?Pro?Tyr
-10 -5 -1 5
Lys?Glu?Phe?Gly?Ala?Thr?Val?Glu?Leu?Leu?Ser?Phe?Leu?Pro?Ser?Asp
10 15 20
Phe?Phe?Pro?Ser?Val?Arg?Asp?Leu?Leu?Asp?Thr?Ala?Ser?Ala?Leu?Tyr
25 30 35
Arg?Glu?Ala?Leu?Glu?Ser?Pro?Glu?His?Cys?Ser?Pro?His?His?Thr?Ala
40 45 50
Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu?Leu?Met?Thr?Leu?Ala?Thr
55 60 65 70
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
90 95 100
Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Gly?Arg?Glu?Thr?Val?Ile?Glu?Tyr
105 110 115
Leu?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg?Pro
120 125 130
Pro?Asn?Ala?Pro?Ile?Leu?Ser?Thr?Leu?Pro?Glu?Thr?Thr?Val?Val
135 140 145
<210>2
<211>18
<212>PRT
＜213〉artificial sequence (Artificial)
<400>2
Leu?Val?Ser?Phe?Gly?Val?Trp?Ile?Arg?Thr?Pro?Pro?Ala?Tyr?Arg?Pro
1 5 10 15
Pro?Asn
<210>3
<211>118
<212>PRT
＜213〉artificial sequence (Artificial)
<400>3
Met?Asp?Ile?Asp?Pro?Tyr?Lys?Glu?Phe?Gly?Ala?Thr?Val?Glu?Leu?Leu
1 5 10 15
Ser?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Arg?Asp?Leu?Leu?Asp
20 25 30
Thr?Ala?Ser?Ala?Leu?Tyr?Arg?Glu?Ala?Leu?Glu?Ser?Pro?Glu?His?Cys
35 40 45
Ser?Pro?His?His?Thr?Ala?Leu?Arg?Gln?Ala?Ile?Leu?Cys?Trp?Gly?Glu
50 55 60
Leu?Met?Thr?Leu?Ala?Thr?Trp?Val?Gly?Gly?Asn?Leu?Glu?Asp?Pro?Ile
65 70 75 80
Ser?Arg?Asp?Leu?Val?Val?Ser?Tyr?Val?Asn?Thr?Asn?Met?Gly?Leu?Lys
85 90 95
Phe?Arg?Gln?Leu?Leu?Trp?Phe?His?Ile?Ser?Cys?Leu?Thr?Phe?Gly?Arg
100 105 110
Glu?Thr?Val?Ile?Glu?Tyr
115
Claims (4)
1. hybridoma cell strain, its preserving number is CCTCC-C200516.
2. anti--the HBeAg monoclonal antibody, it is characterized in that this monoclonal antibody is is that the hybridoma cell strain of CCTCC-C200516 produces by preserving number.
3. MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 2 comprises the steps: that (a) amplification produces the hybridoma cell strain of this anti--HBeAg monoclonal antibody; (b) from amplified production, reclaim anti--HBeAg monoclonal antibody.
4. MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 3 is characterized in that, comprises that the amplification preserving number is hybridoma cell strain, abdominal injection animal, the recovery ascites of CCTCC-C200516 and separates anti--HBeAg monoclonal antibody from ascites.
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| CN102062778A (en) * | 2009-11-17 | 2011-05-18 | 上海荣盛生物药业有限公司 | Composition for in-vitro detection 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1105703A (en) * | 1994-08-25 | 1995-07-26 | 中国药品生物制品检定所 | Building of anti-type-C hepatitis virus antigen monoclonal antibody cell strain and monoclonal antibody thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1105703A (en) * | 1994-08-25 | 1995-07-26 | 中国药品生物制品检定所 | Building of anti-type-C hepatitis virus antigen monoclonal antibody cell strain and monoclonal antibody thereof |
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