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United States Department of Agriculture

Agricultural Research Service

Title: Development of An Immunoassay for Ceftiofur: Monoclonal Antibody Development, Cross Reactivity and Molecular Modeling Studies

Authors
item Stanker, Larry
item Rose, Beate
item Buckley, Sandra
item Beier, Ross

Submitted to: Food Safety and Quality Assurance International Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: November 1, 1995
Publication Date: N/A

Technical Abstract: Ceftiofur sodium is a broad-spectrum, beta-lactamase-resistant cephalosporin. Ceftiofur exhibits excellent antimicrobial activity by virtue of its 2-(2-aminothiazol-4-yl)2-methoxy-aminoacetamide substituent at the C-7 position of the cephem nucleus. It would therefore be advantageous to raise antibodies which would recognize this group. Monoclonal antibodies were prepared against ceftiofur using its hydrolyzed form, desfuroyl ceftiofur, as the hapten. Desfuroyl ceftiofur was prepared in situ and conjugated directly to maleimide-activated carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Balb/c mice were immunized with the desfuroylceftiofur-KLH conjugate (KLH-SMCC- desCef). Two stable monoclonal antibodies (MAbs), Cef-68 and Cef-116, of subclass IgG2a and IgG1, respectively, were isolated. A competitive inhibition enzyme-linked immunosorbent assay (cELISA) was developed for the quantitative detection of ceftiofur in liquid samples. Detection limits for ceftiofur were 32 and 0.3 ppb for the two clones Cef-68 and Cef-116, respectively. Cross-reactivity studies with a variety of related cephalosporins and penicillins indicated that only a limited number of the cephalosporins and none of the penicillins that were tested competed in the cELISA. Energy minimized models of these compounds and cross reactivity studies suggest both the thiazol and oxime portions are important for antibody binding. Preliminary studies using both fortified samples and incurred residue samples suggest that the cELISA developed is capable of detecting as little as 1 ppb of ceftiofur equivalents in raw milk with no sample preparation except for dilution. Studies are underway to adapt this assay for measurement of ceftiofur in tissue samples.

Last Modified: 11/28/2014
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