Abstract

A heterologous indirect competitive enzyme linked immuno sorbent assay (icELISA) has been developed for the determination of enrofloxacin (ENR) residues in poultry. For this purpose, carbodiimide active ester method was employed to synthesize the artificial antigen of ENR- bovine serum albumin (BSA) while mixed-anhydride technique was used to synthesize the coating antigen of ENR- ovalbumin (OVA), to pursue the heterologous sensitivity. By square matrix titration, an icELISA method was developed, and the linear range was from 0.02 to 86.3 ng/mL, with limit of detection (LOD) and IC₅₀ value of 0.8 and 0.01 ng/mL, respectively. After optimization, 5% of NaOH was used in the assay buffer and this ELISA system can tolerate methanol not higher than 30%. The correlation coefficients (R²) between concentration spiked and concentration determined were 0.9975 in chicken muscle and 0.9959 in duck muscle, respectively. Therefore, this assay has the potential to be incorporated into a quantitative monitoring program for the rapid screening of ENR residue in poultry muscles.

Key words: Enrofloxacin, artificial antigen, polyclonal antibody, indirect competitive enzyme linked immuno sorbent assay (ELISA), heterologous, poultry.

Abbreviation

ENR, Enrofloxacin; icELISA, indirect competitive enzyme linked immuno sorbent assay; pAb, polyclonal antibody; BSA, bovine serum albumin; OVA, ovalbumin; FCA, Freund's complete adjuvant; FIA, Freund's incomplete adjuvant; GaRIgG-HRP, the peroxidase-conjugated rabbit anti-mouse IgG; A_max, maximum absorbance; IC₅₀, half-maximum inhibition concentration; LOD, limit of detection; CR, cross-reactivity; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS, N-hydroxysuccinimide; FQs, fluoroquinolones; PBS, phosphate buffer saline; TMB, 3,3,5,5-tetramethylbenzidine; HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; pAb, polyclonal antibody.