Abstract
In this study novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive indirect ELISA method to directly detect residues of 3,5-dinitrosalicyclic acid hydrazide (DNSH), a toxic metabolite of nifursol present in chicken tissues. The hapten DNSHA was first designed and used to covalently couple to BSA to form an immunogen which was immunized to rabbits to produce a polyclonal antibody against DNSH. Furthermore, a novel 3,5-dinitrosalicylic acid-ovalbumin (DNSA-OVA) immunoconjugate structurally different from DNSHA-OVA was designed and used as a “substructural coating antigen” to improve the sensitivity of an indirect ELISA analysis for a direct DNSH detection. Based on the “substructural coating antigen” concept, an optimized indirect ELISA method was established that exhibited good specificity and high sensitivity for detecting DNSH, with a cross-reactivity of less than 0.1% (excluding the parent compound nifursol), IC50 of 0.217 nmol/mL and detection limit of 0.018 nmol/mL. Finally, a simple and efficient analysis of DNSH samples in chicken tissues showed that the average recovery rate of the indirect ELISA analysis was 82.3%, with the average coefficient of variation 15.9%. Thus, the developed indirect ELISA method exhibited the potential for a rapid detection of DNSH residues in tissue.
Highlights
Nifursol is a member of the nitrofuran antibiotics family (Figure 1), which is used extensively as a feed additive for the prevention of histomoniasis
As nifursol was rapidly metabolized to form the metabolic marker 3,5-dinitrosalicyclic acid hydrazide (DNSH, Figure 1) which can persist for a long time in vivo, several laboratories have focused on development of DNSH detection methods [2,3,4]
A short unsaturated arm based on glyoxalic acid was directly introduced into the target analyte to form a DNSHA hapten which was covalently linked to the carrier protein bovine serum albumin (BSA) to produce a DNSHA-BSA
Summary
Nifursol is a member of the nitrofuran antibiotics family (Figure 1), which is used extensively as a feed additive for the prevention of histomoniasis. The detection of DNSH is mainly at the μg/kg level by HPLC-MS methods, which are based on the analysis of the DNSH derivative 2-hydroxy-3,5-dinitro-N'-(2-nitrobenzylidene)benzohydrazide (NPDNSH, Figure 1) [2,3,4]. These analytical methods require a time-consuming and troublesome derivatization step and expensive apparatus and have low sample throughput. To the best of our knowledge, there have been no reports on the development of a specific antibody to DNSH and an immunoassay for a direct detection of DNSH. This study reports for the first tme the synthesis of immunoconjugates for DNSH, the preparation of a polyclonal antibody against
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
