Competitive Indirect ELISA for Ceftiofur Sodium and the Effect of Different Immunizing and Coating Antigen Conjugates

Ceftiofur sodium is a broad spectrum, beta-lactamase-resistant cephalosporin. Ceftiofur and desfuroylceftiofur were used to develop competitive indirect enzyme-linked immunosorbent assays (CI-ELISA) for the determination of ceftiofur sodium. Hapten-protein conjugates were made using three different carrier proteins and three methods of conjugation. The first two methods use the free amine of ceftiofur as the site of conjugation, and coupling to bovine serum albumin and ovalbumin was achieved by using two different cross-linking reagents. The third conjugation procedure joins the hydrolyzed form of ceftiofur, desfuroylceftiofur, to the maleimide-activated carrier proteins, bovine serum albumin and keyhole limpet hemocyanin. A variety of immunization schedules is presented to show the effect of repeated immunizations on antibody maturation. Serum antibody levels were evaluated for each conjugation method using both homologous and heterologous conjugates as antigens. All of the immunogens resulted in the generation of anticeftiofur antibodies. The heterologous assay systems on average yielded more sensitive assays, but antisera obtained from all three immunogens were used successfully in developing enzyme-linked immunosorbent assays (ELISA's) for ceftiofur. Ceftiofur was detected in mouse sera in a concentration range of 3-500 ppb. The results illustrate that the method used to couple the hapten to a carrier protein as well as the site of coupling significantly influence the resulting enzyme-linked immunosorbent assays (ELISA's).