Purpose: Using ELISA and lateral flow immunoassays to determine flunixin residues in tissues and ante-mortem matrices
Methods: After IACUC approval, 20 cows were given the labeled dose of flunixin by IV or IM administration (10 each) for 3 consecutive days. One-half of the cows were challenged with IV LPS. Milk, saliva, and urine were collected at timed intervals and cows were slaughtered with a 4-day withdrawal period (WP). USDA, FSIS methods (CLG-FLX3.01 and Bulletin 4246) were used to screen tissues for flunixin residues. Saliva flunixin was determined using a matrix matched calibration standards on a 96-well ELISA format. Lateral flow analyses were utilized to determine flunixin in milk, saliva, and urine. Milk was assayed directly while saliva and urine samples were diluted.
Results: At a WP of 96 hours, no animals exceeded the minimum applicable levels for muscle (≥ 10 ppb) while livers of two animals exceeded minimum applicable levels (≥ 50 ppb). Saliva did not produce a predictable flunixin elimination pattern using ELISA determination. Using lateral flow analysis, 16 of 20 urine samples were flunixin positive; 6 of 20 saliva samples were positive at 96-h tissue WP. Milk of 12 cows were positive at the 36-h milk WP.
Significance: The FSIS’ ELISA screening method detected liver flunixin violative residues (confirmed by FSIS’ LC-MS method) with one false-positive result. Based on lateral flow screening assay results, urine and saliva are not good ante-mortem predictors for tissue flunixin violations; rather, they potentially can be used to test flunixin exposure in off-label species.