Polyclonal Antibody Production against Mouse Purified IgG۲a towards Use in Basic Research

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 IgG۲a and production of polyclonal antibody against purified mouse IgG۲a subclass. Materials and Methods: Mouse IgG۲a 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 IgG۲a. The production of antibody in rabbit was investigated by direct ELISA method. Rabbit serum was collected and precipitated at the final concentration of ۵۰% 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 ۵۰-KDa, ۲۵-۳۰ KDa MW and a distinct band with ۱۵۰ KDa MW. Isotype determination showed the presence of mouse IgG۲a in related fraction. The titer of Anti-mouse polyclonal antibody was ۲۰۰۰۰۰. The optimum titer of prepared HRP conjugated IgG was ۴۰۰۰. Conjugated rabbit IgG has more cross reactivity with mouse IgG۲b. Conclusion: Taking together, affinity chromatography and ion-exchange chromatography are appropriate techniques for purification of mouse IgG subclasses and rabbit IgG, respectively. .

کلیدواژه ها:

Affinity chromatography ، purification ، polyclonal antibody ، IgG۲a ، ion-exchange chromatography