Polyclonal antibody production against mouse purified IgG2a towards use in basic research

Abstract

Background: The ability of polyclonal antibodies to react with many epitopes of an antigen makes them valuable reagents in research and diagnosis. The aim of this study was purification of mouse IgG2a and production of polyclonal antibody against purified mouse IgG2a subclass. Materials and Methods: Mouse IgG2a was purified by ProA affinity. Verification method of the purified antibody was SDS-PAGE and ELISA by a mouse isotyping Kit. Rabbit was immunized with purified IgG2a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of 50% ammonium sulfate. Polyclonal antibody was purified by ion-exchange chromatography and labeled with HRP. The titre and cross reactivity of product was detected by direct ELISA method. Results: The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 50-KDa, 25-30 KDa MW and a distinct band with 150 KDa MW. Isotype determination showed the presence of mouse IgG2a in related fraction. The titer of Anti-mouse polyclonal antibody was 200000. The optimum titer of prepared HRP conjugated IgG was 4000. Conjugated rabbit IgG has more cross reactivity with mouse IgG2b. Conclusion: Taking together, affinity chromatography and ion-exchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively.