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Title: COMPETITIVE INDIRECT ELISA FOR CEFTIOFUR SODIUM AND THE EFFECT OF DIFFERENTIMMUNIZING AND COATING ANTIGEN CONJUGATES

Author
item Stanker, Larry
item ROSE, BEATE - 6202-30-10
item Holtzapple, Carol

Submitted to: Bioconjugate Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Antibody-based immunoassays are used to measure antibiotic and pesticide residues in biological fluids and tissues. Antibodies are substances which are produced by the immune system in response to foreign substances which enter the body. Once the antibodies are isolated they can be used to detect the foreign substances against which they were generated. Antibiotics or pesticides are not recognized by the immune system since they are too small in size; therefore, they need to be linked to a larger molecule such as a protein. Different cross-linking reagents are available for this purpose. The conjugated antibiotic should preserve the chemical and physical properties of the original compound so that the antibodies produced are specific for this compound. In this paper, a comparison is made by using three crosslinkers with different spacer arm lengths to analyze the nature and degree of conjugation of ceftiofur (a veterinary antibiotic used for respiratory disease in cattle and swine) to various carrier proteins. The ceftiofur-protein conjugate, which gives the best analytical values for the detection of ceftiofur, is presented. This information is important for the optimum design of antibody-based immunoassay methods.

Technical Abstract: Ceftiofur sodium is a broad spectrum, B-lactamase-resistant cephalosporin. Ceftiofur and desfuroyl ceftiofur were used to develop competitive inhibition 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 BSA and OVA was achieved by using two different cross-linking reagents. The third conjugation procedure joins the hydrolyzed form of ceftiofur, desfuroyl ceftiofur, to the maleimide-activated carrier proteins, BSA and KLH. 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 anti-ceftiofur antibodies. However, the results presented here clearly demonstrate 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). The antisera obtained from all immunogens were used successfully in developing a CI-ELISA for ceftiofur.