Production of Gentamycin and Ceftiofur Specific Polyclonal Antibodies by Conjugating them with Bovine Serum Albumin

Abstract

Polyclonal antibodies (pAbs) were produced against gentamycin (GEN) and ceftiofur (CEF) antibiotics in sprague dawley rats (aged 7-8 weeks) by conjugating them with bovine serum albumin (BSA). Total 3 groups (3 rats in each group): one control (without antibiotic) group and two test groups (GEN and CEF) of rats were maintained. Total of four blood samplings were done from each group as follows: First three samples was serially at 15 days’ time interval after 1st booster, 2nd booster, 3rd booster and the 4th sampling was one and half month after the third booster. The antibody titres in the antisera of each antibiotic in all the four immunization cycles were determined by an icELISA at various serum dilutions ranging from 1/100 to 1/6400. Analysis of antibiotic conjugates by SDS-PAGE and Coomassie blue staining revealed higher molecular weights when compared to normal BSA (68 KDa). The molecular weights of conjugates were 90 kDa, 78 kDa for GEN-BSA and CEF-BSA respectively. The GEN antisera gave positive antibody titres up to a dilution of 1/1600 in first immunization cycle, 1/6400 in 2nd and 3rd immunization cycles and 1/1600 in 4th sampling. Maximum optical density at 450 nm (OD450) value of 0.928 was obtained at 1/100 antiserum dilution in 3rd immunization cycle. The CEF antisera gave positive antibody titres up to a dilution of 1/800 in 1st immunization cycle, 1/1600 in 2nd immunization cycle, 1/6400 in 3rd immunization cycle and 1/3200 in 4th sampling. Maximum OD450 value of 2.072 was obtained at 1/100 antiserum dilution in 3rd immunization cycle.