DEVELOPMENT AND EVALUATION OF AN ENZYME-IMMUNOASSAY FOR SULFAMERAZINE IN MILK

Competitor conjugates for sulfamerazine enzyme immunoassays (SMR EIAs) were prepared using azo and hemisuccinate linking of SMR to BSG. Hapten amounts for the ate-linked conjugates were best estimated using a [C-14]sulfamethazine-BSA standard curve, and amino acid analysis indicated that SMR was predominantly linked to histidine residues. Hapten amounts for the hemisuccinate-linked conjugates were determined by quantification of sulfanilic acid after acid hydrolysis. The best azo- and hemisuccinate-linked conjugates (hapten-protein molar ratios of 4.3 and 6.4, respectively) were compared to the Charm II and CITE sulfa trio tests using milk samples from cows treated with oral doses of sulfonamide containing 3-Sulvit. Milk samples were collected over 7 days and 10 ppb cutoff dates determined. The developed SMR EIAs with detection ranges of 0.15-16.6 ppb of SMR predicted the earliest cutoff dates, closely followed by both commercial tests.