Preparation Of Monoclonal Antibodies Of Cobra Venom And Determination Of Its Biological Activities

ObjectiveThe hybridoma technique was used to prepare high-therapeutic anti-cobra venom antibody.The aim was to provide new production methods for anti-snake blood,to solve the shortage of antivenin serum and to provide help for snake injury.Methods1.Immune miceCobra venom was used as an antigen and as adding adjuvan.After antigen mixing and emulsification,the Balb/c mice were immunized by subcutaneous multipoint injection.2.Cell fusionIndirect ELISA was used to select the immune mice with high potency.The spleen of immunized mice was isolated to obtain the sensitized B cells.Using PEG,B cells were fused with the homologous SP2/O myeloma cells to produce hybridoma cells that have both characteristics,which can produce antibodies and grow continuously.3.Cell screeningThis step was done using HAT and HT,except for cell fusion so that go unless the fusion cell.Indirect ELISA was conducted to analyze hybridoma cells reactivity.After that,hybridoma cells with strong positive reactivity were selected.Then monoclonal cells were cultured by using limited dilution method.Hybridoma was named as BE12B5 based on the place of 96-well plates.4.Expand trainingThe single clonal cell cultured in 10⁶ cells/ml was injected into the abdominal cavity of the mouse that were gave the liquid paraffin in advance,and the ascites of the monoclonal antibody with high efficiency was named as the anti-cobra ascites（ACA）.5.Active identification1).Indirect ELISA was used to detect ACA for determining its antibody titer.2).Indirect ELISA based on the difference between different subtype antibodies and ACA was used to detect the antibody subtype of ACA.3).The affinity of monoclonal antibody was detected by competitive ELISA method.4).Indirect ELISA and immunodiffusion test were used to detect the reaction of different snake venom and ACA in order to determine the binding capacity of both.5).Indirect ELISA and immunodiffusion test were used to detect the different components of cobra venom for identifying ACA’s detoxification mechanism.6).Neutralization test was used to detect the effect of ACA on acute toxicity of cobra venom,in order to determine the neutralization activity of the cobra venom.7).In vivo protection experiment was used to detect the effect of ACA on acute toxicity of cobra venomin,in order to determine the therapeutic activity of cobra venom8).Neutralization test was used to detect the effects of ACA on the acute toxicity of king cobra venom,in order to determine the neutralization activity of the king cobra and to understanding the cross reaction of ACA to cobra venom..Results1.Establish a hybridoma cell strain BE12B5 that produces anti-cobra venom antibodyIn this study,a hybridoma cell strain BE12B5 with good stability was screened by immunizing mice,cell fusion,and cell screening,and a high-price anti-cobra ascites（ACA）was prepared.2.Partial biological characterization of anti-brason venom antibodiesAfter testing,the antibody titer of the supernatant of the BE12B5 cell line was up to 1024,the antibody subtype was lgG1,and the anti-cobra ascites fluid（ACA）titer was more than 4 million.The measured affinity constant was 2.0 x 10^-77 L/mol.It is believed that monoclonal antibodies with affinity constants of 10^-88 to 10^-1111 L/mol are high affinity and anti-cobra ascites（ACA）have high affinity.3.Species-specificity of ACAIndirect ELISA and two-way immunodiffusion experiments found that ACA not only has a specific binding reaction to NAV,but also has a strong specific binding reaction to king cobra venom,and has a statistically significant difference compared with the control group（P<0.0001）.ACA has no specific binding reaction to silver venom,venom,and venom.4.Effects of ACA mixed with NAV on cobra venom toxicityNeutralization experiments,ACA 10ul（very low dose）and different concentrations of NAV were mixed and injected intraperitoneally to reduce the mortality of mice and prolong the survival time of mice.The LD₅₀0 of NAV increased from 1.623mg/kg to 1.903 mg/kg.Using the neutralization titer ED₅₀0 as an indicator of the activity of the obtained anti-cobra venom antibody,the ACA neutralizing potency of ACA that protects 50%of the mice from death without killing venom of A.venom was 3.618mg/ml,95%confidence interval was 3.592⁴.028 mg/ml.5.Therapeutic Effect of ACA on NAV Poisoning MiceProtective experiments in vivo,ACA 10ul intraperitoneal injection reduced the mortality of NAV-intoxicated mice and prolonged the survival of mice.The LD₅₀0 of cobra venom increased from1.623mg/kg to 1.809mg/kg.The therapeutic valency of ACA for NAV was 3.618 mg/ml,with a 95%confidence interval of 3.302 to 3.958 mg/ml.6.Effects of ACA on the Toxicity of King Cobra VenomNeutralization experiments,ACA 10ul mixed with different concentrations of king cobra venom increase the LD₅₀0 value of King cobra from 0.568mg/kg to 0.695mg/kg.Therefore,the neutralization titer ED₅₀0 of ACA on king cobra venom was 1.390mg/ml,and the 95%confidence interval was 1.234².016mg/ml.conclusionIn this study,a strain of monoclonal antibody strain named BE12B5 with good stability was prepared and a high-cost anti-cobra ascites（ACA）was prepared.ACA is a specific anti-cobra venom monoclonal antibody and has a high affinity.ACA can neutralize the cobra venom,reduce its toxicity.ACA have a attenuating effect in vivo,and have a therapeutic effect on NAV poisoned mice.In addition.,ACA has a certain cross-neutralization effect on king cobra venom and also reduces its toxicity.