Submitted to: ARS Food Safety and Inspection Service Research Workshop
Publication Type: Abstract Only
Publication Acceptance Date: November 15, 1995
Publication Date: N/A
Technical Abstract: Ceftiofur is an extended-spectrum, beta-lactamase, third generation cephalosporin. It exhibits excellent antimicrobial activity by virtue of its 2-(2-aminothiazol-4-yl)2-methoxy-aminoacetamide substiutent at the C-7 position of the cephem nucleus. 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 the maleimide-activated carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). Balb/c mice were immunized with the desfuroyl ceftiofur-KLH conjugate (KLH-SMCC-desCef). Splenic lymphocytes from these mice were fused with SP2/phi myeloma cells to produce hybridomas. 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 using a heterologous assay system to provide a more sensitive ELISA. Detection limits for ceftiofur were 32 and 0.3 ppb for the two monoclonals Cef-68 and Cef 116, respectively. 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. Excellent recoveries were observed in milk samples obtained from individual cows, as well as from the bulk tank, as a local daily. Ceftiofur was also administered to several cows and milk collected over 10 days. Ceftiofur levels were measured in these milk samples using the cELISA method developed. Studies are underway to adapt this assay for measurement of ceftiofur in tissues.