Bg. Rose et al., COMPETITIVE INDIRECT ELISA FOR CEFTIOFUR SODIUM AND THE EFFECT OF DIFFERENT IMMUNIZING AND COATING ANTIGEN CONJUGATES, Bioconjugate chemistry, 6(5), 1995, pp. 529-535
Ceftiofur sodium is a broad spectrum, beta-lactamase-resistant cephalo
sporin. Ceftiofur and desfuroylceftiofur were used to develop competit
ive indirect enzyme-linked immunosorbent assays (CI-ELISA) for the det
ermination of ceftiofur sodium. Hapten-protein conjugates were made us
ing 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 ac
hieved by using two different cross-linking reagents. The third conjug
ation procedure joins the hydrolyzed form of ceftiofur, desfuroylcefti
ofur, to the maleimide-activated carrier proteins, bovine serum albumi
n and keyhole limpet hemocyanin. A variety of immunization schedules i
s presented to show the effect of repeated immunizations on antibody m
aturation. Serum antibody levels were evaluated for each conjugation m
ethod using both homologous and heterologous conjugates as antigens. A
ll of the immunogens resulted in the generation of anticeftiofur antib
odies. The heterologous assay systems on average yielded more sensitiv
e assays, but antisera obtained from all three immunogens were used su
ccessfully 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 co
uple the hapten to a carrier protein as well as the site of coupling s
ignificantly influence the resulting enzyme-linked immunosorbent assay
s (ELISA's).