The Preparation Of Anti-Insulin Monoclonal Antibody And Polyclonal Antibody

Insulin is a very important hypoglycemic hormone in the body.It is a critical measurement index and research target in the process of diabetes diagnosis,diabetes research and anti-diabetes drug development.Therefore,the detection of insulin level is an essential technical requirement.At present,methods for detecting the insulin levels are based on the principle of specific antigen-antibody reactions.Therefore the preparation of high efficient and specific anti-insulin monoclonal antibodies and polyclonal antibodies are the essential foundation for establishing the method to detect insulin level.In this study,we prepared and characterized the mouse monoclonal antibodies and guinea pig polyclonal antibodies against insulin.The obtained results were shown as follows:1.Optimization of indirect ELISA method for detecting the level of antibodies against insulin.We optimized the indirect ELISA method to establish stable system for measuring the titer of prepared antibodies before the positive clones were screened.We optimized these conditions,including antigen coating concentration,serum dilution ratio,blocking buffer and its incubation time,incubation time of serum,dilution ratio of HRP-labeled antibodies and its incubation time,respectively.The optimal conditions were identified as follows: the best antigen concentration for coating was 5 ?g/mL and the optimal dilution of serum was 1:100;the best blocking solution was 1% BSA and its optimal incubation time was 2 h;the best sample incubation time was 1.5 h;the optimal dilution ratio of HRP-labeled antibodies was 1:6000 and its best incubation time was 30 min.2.Preparation of monoclonal antibodies against insulin.Firstly,we used commercial porcine insulin as antigen,BALB/c mice were immunized through multi-point back injection and intraperitoneal injection in order to stimulate effective immune responses in mice.The antibody titer in mouse serum was tested after 3 times of immunization and the mouse with highest antibody titer was selected for the final booster immunization.Then,three days after booster immunization,the spleen cells were obtained from the immunized mouse,and fused with the SP2/0 cells or NS-1 cells which were in the logarithmic phase in the sterile environment by using chemical reagent Polyethyleneglycol(PEG).We performed four times of cell fusion totally.After screening and subcloning,4D4,2D7 and 5D12 clones were obtained as positive hybridoma cell clones.The subtypes of the three kinds of monoclonal antibodies were all IgM with light chain Kappa.The monoclonal antibodies from 2D7,4D4 and 5D12 clones were expanded in vivo with titers as 1:3200,1:51200 and 1:12800,respectively.Finally,we characterized the specificity and cross-reaction of monoclonal antibodies by using ELISA and Western blot.ELISA detection results showed that three monoclonal antibodies were cross-reactive with bovine insulin.The results of western blot showed that the three antibodies might recognize the conformational epitopes but not the linear epitopes of insulin.3.Preparation of polyclonal antibodies against insulin.We used commercial porcine insulin and bovine insulin as antigen,guinea pigs were immunized by multi-point back injection and intraperitoneal injection.Totally 4 times of immunization were carried out and 5 days after the last immunization,as much as possible sera were collected by intracardiac puncture.The antiserum titer was tested by indirect ELISA method.Results showed that the titer of anti-porcine insulin polyclonal antibodies in the sera was 1:25600 and the highest titer of anti-bovine insulin polyclonal antibodies in the sera was 1:51200.Antibodies were successfully purified by saturated ammonium sulfate precipitation.We characterized the specificity and cross-reaction of polyclonal antibodies by using ELISA and Western blot.ELISA detection results showed that anti-porcine insulin polyclonal antibodies were cross-reactive with bovine insulin and anti-bovine insulin polyclonal antibodies could cross react with porcine insulin.Western blot results showed that polyclonal antibodies could recognize porcine insulin and bovine insulin specifically.To sum up,we successfully obtained monoclonal antibodies against insulin from three 3 strains of hybridoma cells as well as the polyclonal antibodies against insulin from guinea pigs,and characterized the mouse monoclonal antibodies and guinea pig polyclonal antibodies against insulin preliminary,which will set a foundation to establish our own method to detect insulin.