Synthesis of novel haptens against ciprofloxacin and production of generic monoclonal antibodies for immunoscreening of fluoroquinolones in meat.

BACKGROUND: The residues of fluoroquinolone drugs in foods of animal origin are dangerous to the consumers. The objective of this study was to produce a generic monoclonal antibody for determination of fluoroquinolone residues in meat. RESULTS: Two novel haptens of ciprofloxacin containing a free amidogen group on the piperazinyl ring were synthesised that were used to produce the monoclonal antibodies. The antibodies obtained simultaneously recognised 12 fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, sarafloxacin, diflocaxin, danofloxacin, ofloxacin, marbofloxacin, pefloxacin, lomefloxacin, amifloxacin and enofloxacin). After evaluation of different coating antigen-antibody combinations, a heterologous competitive indirect ELISA was used to determine the 12 drugs. The cross-reactivities were in the range of 23-120% and the limits of detection were in the range of 1.0-4.5 ng mL(-1). Eight fluoroquinolone drugs licensed as veterinary drugs in China were fortified into blank chicken for analysis. The recoveries were in the range of 61.5-82.5% with coefficients of variation in the range of 7.5-15.2%. CONCLUSION: This method could be used as a rapid screening tool for routine monitoring the residues of these fluoroquinolone drugs in animal-derived foods.

Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers.

The introduction of spacers in coating steroid protein complexes and/or enzyme conjugates or immunogens is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We investigated the impact of different homobifunctional spacers, ranging in atomic length from 3 to 10, on the sensitivity and specificity of prednisolone (PSL) enzyme immunoassays. In this study, four homo-bifunctional spacers, namely, carbohydrazide (CH), adipic acid dihydrazide (ADH), ethylene diamine (EDA), and urea (U), were incorporated between PSL and horseradish peroxidase (HRP) for preparing the enzyme conjugate with an aim to improve the sensitivity of the assay without compromising assay specificity. The assays were developed using these enzymes conjugated with antibodies raised against the PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of a bridge in the enzyme conjugate was 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL, and 0.018 ng/mL with ADH, CH, EDA, and urea as a spacer, respectively. Among the four combinations, the PSL-21-HS-BSA-antibody with PSL-21-HS-U-HRP-enzyme conjugate gave better sensitivity and less cross-reaction. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32%-102.50%. The intra and inter-assay CV% was< 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r2 = 0.98). The present study suggests that the nature of the spacer is related to assay sensitivity and not the spacer length.