Production and purification of polyclonal antibody against bovine immunoglobulins in rabbits

Abstract

Antibodies are important tools in medical researches which have led to many advances in this field. Anti-bovine immunoglobulins and its conjugate with horse radish peroxidase (HRP) is used to diagnose cows' disease by ELISA or western blotting tests. In this study, the production, purification and horse radish peroxidase (HRP) conjugation of polyclonal IgG against bovine immunoglobulins in rabbits were carried out. Three 6-month-old New Zealand White rabbits were immunized by bovine immunoglobulins in combination with Freund's adjuvant. Purified antibody (using ion-exchange chromatography) was labeled to HRP. Direct enzyme linked immunosorbent assay (ELISA) was used to determine the optimum titer and cross reactivity of HRP conjugated IgG. The purity of various IgG preparations was about 98%. The optimum dilution of prepared HRP conjugated IgG was 1:12800. This conjugated IgG has no cross reactivity with sheep and goat immunoglobulins at optimized dilution. This study showed that ion-exchange chromatography could be an appropriate method for purification of IgG antibodies.