Detection of flunixin in greyhound urine by a kinetic enzyme-linked immunosorbent assay.
A two-step kinetic enzyme-linked immunosorbent assay was developed to detect the presence of flunixin in the urine of greyhound dogs. The assay system was developed using polyclonal antiflunixin antisera, a rabbit albumin-flunixin conjugate adsorbed onto polystyrene microtiter strips, and flunixin reference standards for calibration. The assay parameters were optimized and the performance characteristics were determined. The quantitative intra- and inter-run precisions (%CV) of the analysis of replicate (n = 10) flunixin-spiked urine samples were 9.9-12.5% and 10.2-13.6%, respectively. The linear dynamic range was 1-100 ng/mL, and the quantitative accuracy, as determined by calculation of percent error of measured flunixin in flunixin-spiked drug-free greyhound urine, was -16% to +14% over this range. The I50 of the ELISA was 17.3 ng/mL. The limit of detection was 25 ng/mL in greyhound urine. The reactivity in the assay system relative to flunixin (100%) was 147% for flunixin glucuronide, 25% for clonixin, and 5% for niflumic acid. The ELISA was capable of detecting total flunixin for up to 72 h in dogs administered flunixin at 0.55 mg/kg orally and up to 96 h in a dog that was administered flunixin at 1.0 mg/kg orally.
Brady
T C
TC
Pathobiology Department, University of Connecticut, Storrs 06269-4193, USA.
Yang
T J
TJ
Hyde
W G
WG
Kind
A J
AJ
Hill
D W
DW
D000276:Adjuvants, Immunologic / Q000008:administration & dosage; D000284:Administration, Oral; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000008:administration & dosage / Q000652:urine*; D000917:Antibody Formation; D000941:Antigens / Q000008:administration & dosage / Q000276:immunology; D002851:Chromatography, High Pressure Liquid / Q000662:veterinary; D003002:Clonixin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000652:urine; D003429:Cross Reactions / Q000276:immunology; D004285:Dogs / Q000652:urine*; D004300:Doping in Sports; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D005260:Female; D006433:Hemocyanins / Q000008:administration & dosage / Q000276:immunology; D011817:Rabbits; D012015:Reference Standards; D015203:Reproducibility of Results; D013329:Structure-Activity Relationship
D000276:Adjuvants, Immunologic; D000894:Anti-Inflammatory Agents, Non-Steroidal; D000941:Antigens; C014557:flunixin; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D003002:Clonixin