Production and characterisation of monoclonal antibodies for the detection of AOZ, a tissue bound metabolite of furazolidone

Abstract

3-amino-2-oxazolidinone (AOZ) is a tissue bound toxic metabolite derived from the nitrofuran antibiotic, furazolidone. AOZ is detected in the derivatised form of 3-{[(2-nitrophenyl) methylene] amino}-2-oxa-zolidinone (NP AOZ). 3-{[(3-carboxyphenyl)-methylene] amino-2-oxazolidinone (CP AOZ) was used as the immunising hapten for the production of monoclonal antibodies against NP AOZ. Monoclonal antibodies were produced using hybridomas from the fusion of murine myeloma cells and spleen cells isolated from BALB/c mice immunised with CP AOZ-ethylenediamine-human serum albumin (CP AOZ-ed-HSA). The antibody production in ascitic fluids from clones 3B8/2B9 and 2D11/A4 was monitored during a 16 month period. Repeated cultures of these hybridomas, followed by injection into mice and cloning did not change the assay parameters. Clone 2D11/A4 exhibited long term stability in antibody production throughout the experiment whereas clone 3B8/2B9 demonstrated variability in particular antibody yields whilst retaining assay sensitivity. Reasons for this production variability in clones are discussed. In an optimised direct ELISA format, the antibodies exhibited a 50% binding inhibition in the range of 0.52–1.15 ng/ml with NP AOZ (0.22 -0.50 ng/ml, respective AOZ equivalents) and showed high specificity towards this analyte. The sensitivity of monoclonal antibodies incorporated into the ELISA is compatible with the European Union MRLP and is currently in use for routine analysis.

Development of an enzyme-linked immunosorbent assay for the detection of nitrofurantoin metabolite, 1-amino-hydantoin, in animal tissues

“…The amino group moiety may be a key part of antigenic determinant and is unsuitable for conjugation with carboxyl group. Inspired by previous reports about AOZ and SEM (Cooper et al, 2004;Cooper et al, 2007;Vass et al, 2005), CPAHD was used for the conjugation with the carrier protein.…”

“…In previous reports (Cooper et al, 2004;Cooper et al, 2007;Vass et al, 2005), most of the successful hapten designs used 3-carboxybenzaldehyde for modification. In this study, 4-carboxybenzaldehyde was used for the modification of hapten.…”

“…However, these methods are time-consuming and expensive, and there is now an urgent need for a rapid, low cost, high-capacity and sensitive method to screen AHD residue in animal tissues. Some immunoassays that detect AOZ residue (Cheng et al, 2009;Cooper, Caddell, Elliott, & Kennedy, 2004;Vass et al, 2005), SEM (Cooper et al, 2007;Gao, Chen, Cheng, Lei, & Zeng, 2007) and AMOZ (Pimpitak et al, 2009) residues have already been reported. In addition, the detection of the nitrofuran parent drugs has also been reported (Li, Liu, & Wang, 2009;Liu et al, 2007;Xu et al, 2009).…”

Validation of a monoclonal antibody-based ELISA for the quantification of the furazolidone metabolite (AOZ) in eggs using various sample preparation

“…3([(3-carboxyphenyl)methylene]-amino)-2-oxazolidinone (CPAOZ) and CPAOZ-ethylene diamine-human serum albumin (CPAOZ-ed-HSA) were provided by the Veterinary Science Division, Department of Agriculture and Rural Development (Belfast, Northern Ireland) and previously described (Cooper et al, 2004a). Monoclonal antibody against CP-AOZ-ed-HSA (clone 3B8/2B9) was produced by this laboratory (Vass et al, 2005). All other chemicals were of analytical grade and purchased from Dorapis (Brno, Czech Republic).…”

Ultrasensitive indirect competitive ELISA and strip sensor for detection of furazolidone metabolite in animal tissues

“…High accuracy and precision were achieved for sensitive detection of PAOZ derivative from the AOZ (Chang, Peng, Wu, Wang, & Yuan, 2008). M. Vass has prepared a monoclonal antibody against CPAOZ hapten and established the ELISA method to detect AOZ in shrimp, poultry, pork and beef (Vass et al, 2005). Tzong-fu Kuo established an immunological detection method based on polyclonal antibody produced from a new immunogen containing hapten 2-NP-HXA-AOZ.…”