Preparation Of Anti-Agkistrodon Acutus Venom Monoclonal Antibodies And Determination Of Their Antivenom Activities

Objective:Mouse-derived anti-Agkistrodon acutus venom（AAV）monoclonal antibodies（m Abs）were prepared by hybridoma technology with the aim to produce high-therapeutic anti-AAV m Abs,which provides some experimental data and technical support for preparating antivenom.Methods:（1）Preparation and purification of the Monoclnoal Antibodies against Agkistrodon acutus venom.1.Immunizination of the mice with Agkistrodon acutus venom:BALB/C mice were immunized with a quantitative Agkistrodon acutus venom as antigen by subcutaneous multipoint injection,which was completely mixed with adjuvant and emulsified.2.Fusion of myeloma cells with spleen cells:Immunodiffusion method and indirect ELISA method were used to obtain the high potency serum antibody from the immunized mice.The spleen cells of the immunized mice were isolated,which were fused with SP2/0 myeloma cells by PEG to produce hybridoma cells.3.Screen of the antibody-producing cell:The hybridoma cells which could grow successfully in the medium of HAT.The hybridomas cells those strong positivereactivity were selected by Indirect ELISA.Finally,the antibody-producing cell line was obtained by subcloning.4.Preparation and purification of anti-AAV m Abs:After sensitizing the BALB/C mice with fluid-wax or freund incomplete adjuvant（FIA）,the positive hybridoma cells with 10⁵cells were injected into the mice abdominal cavity to preparate the monoclonal antibodies.The monoclonal antibody in ascites was purified by protein G chromatography assay.5.Identification of the purified anti AAV monoclonal antibody:SDS-PAGE was used to investigate the purity of the monoclonal antibodies before-and-after purification.（2）Biological characteristic research of anti AAV monoclonal antibody1.The antibody titer was determined by indirect ELISA.2.The antibody isoforms were detected by indirect ELISA.3.The ability of monoclonal antibody against AAV to bind to snake venom,whichwas detected by Western Blotting（WB）and in vitro agglutination test.4.The effects of anti-AAV m Abs on acute toxicity of AAV were determined tounderstand its neutralization and detoxification activity against the snake venom.5.In vivo,the effect of anti-AAV m Abs on the 100%minimum lethal dose of thesnake venom and different doses of AAV was observed to understand its therapeuticeffect on mice poisoned by the snake venom.Results:1.The serum titer of immunized mice was higher than 500000 after three timesimmunization plus one booster with AAV.The precipitation line of the serum andAAV was observed in double immunodiffusion experiment.2.Three monoclonal antibodies（G2B5、4G5、2H10）against AAV were obtained.3.The three monoclonal antibodies were all Ig G1 subtype.4.The monoclonal antibody（G2B5）was purified successfully.5.By detecting,the titer of the monoclonal antibody（G2B5）was 1:1280000.6.Neutralization experiments showed that anti Agkistrodon acutus venom of ascites（AAAV）can neutralize the AAV and the LD₅₀of AAV increased from 5.60mg/kg to6.47mg/kg.7.In vivo,the protection experiment showed that AAAV could reduce the toxicity ofAAV.Compared with the group of PBS,AAAVcan can significantly prolong thesurvival time of mice that were administration 100%minimum lethal dose of AAVand the survival time of all mice in the AAAV group was more than 3 hours.Two ofthem survived more than 72 hours,while all mice in the PBS group died within 2hours.In addition,AAAV protected the mice taking different doses of AAV fromdeath for 2 hours.Conclusion:Three cell lines producing m Abs of anti-venom of Agkistrodon acutus were successfully obtained by using hybridoma technology.The monoclonal antibodies with neutralizing and detoxifying effects on AAV were prepared.These antibody subtypes are all Ig G1.