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Development of Amoxicillin Enzyme-Linked Immunosorbent Assay and Measurements of Tissue Amoxicillin Concentrations in a Pigeon Microdialysis Model

L.-C. Yeh^*,, W.-M. Lee^*, B.-W. Koh^*, J. P. Chan^*, C.-H. Liu, J.-P. Kao^* and C.-C. Chou^*,1

^* Veterinary Medicine Teaching Hospital and Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, 402; and The Branch Institute of Animal Drug Inspection, Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan, 350

¹ Corresponding author: ccchou{at}nchu.edu.tw

A sensitive ELISA was developed for the detection of amoxicillin (AMX) in serum, urine, and milk. The ELISA used an indirect competitive method produced by coating the plate with ovalbumin conjugated with AMX hapten. Antibodies against AMX-BSA were detected by a goat-antirabbit antibody conjugated with peroxidase. Calibration standard curves ranged from 1.28 ng/mL to 20 µ g/mL [IC₅₀ (inhibition concentration 50%) = 100 ng/mL], and the limits of detection were 1.3, 2.7, and 4.8 ng/mL for urine, milk, and serum, respectively. The intra- and interassay variations were less than 4 and 9.6%. The antibody produced against AMX cross-reacted highly with penicillin G (77%); cross-reacted moderately with ampicillin, oxacillin, and cloxacillin (56.9, 51.4, and 48.8%, respectively); but was considered non-cross-reactive with dicloxacillin (7.4%), cefadroxil (<1%), and cefazolin (<1%). Concentrations of AMX were measured simultaneously in venous blood and muscles by using the developed AMX ELISA in an in vivo microdialysis model designed for pigeons. Following i.m. injection (25 mg/kg), AMX attained a peak blood level of 4.74 ±0.30 µ g/mL and decreased with a half-life of 2.38 ±0.16 h. In contrast, measurements in pectoral and femoral muscles exhibited delayed appearances, reduced peak concentrations, and prolonged half-lives of 4.07 ±0.48 (pectoral) and 3.01 ±0.26 (femoral) that were significantly different from each other and those in the blood (P < 0.05). Blood protein binding was calculated to be 27.9 ±5.7%. This study demonstrated the semiquantitative application of a selective AMX ELISA in the first microdialysis procedure for continuous monitoring of drug levels in specific tissues of pigeons and maybe useful for related studies in other poultry species.

Key Words: pigeon microdialysis • amoxicillin • enzyme-linked immunosorbent assay