A monoclonal antibody-based ELISA for thiabendazole in liver

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for analysis of thiabendazole [2-(4-thiazoly1)-1H-benzimidazole, TBZ] residues in bovine liver. Monoclonal antibodies which bind TBZ and 5-OH-TBZ were developed using the haptens, 2-(2-succinamido-4-thiazolyl)benzimidazole and 5-succinamido-2-(4-thiazolyl)benzimidazole. The ELISA method uses monoclonal antibody in a competitive format, following a one-step, 10-min aqueous extraction of samples. Monoclonal antibodies with various specificities permit an ELISA specific for TBZ alone or for the group of thiazolyl compounds including TBZ, 5-OH-TBZ, and cambendazole. Tissue samples of 0.1 g can be used, and the limit of detection, determined by analysis of liver samples fortified with TBZ, is 20 ppb. The extraction and assay can be completed within 2 h. The -say materials are sufficiently stable to be packaged a8 a kit with a shelf life of at least 6 months. ELISA results were validated by comparison to results obtained by high-pressure liquid chromatography with UV detection. Since samples exceeding the U.S. tolerance (100 ppb) can readily be distinguished from negative samples simply by visual inspection of the ELISA wells, the method provides a rapid screening test which could be used for regulatory monitoring of TBZ residues in meat.

Review of methodology for the determination of benzimidazole residues in biological matrices

“…Alternatively, extraction may be carried out using a more polar organic solvent without pH manipulation. Some researchers have extracted these drug residues using a single solvent system, such as acetonitrile [119,134], or binary water/organic solvent mixtures which would be around neutral pH [131,[135][136][137]. Extraction with acidified extractants has only been used by a small number of researchers for methods requiring hydrolysis or digestion steps [101,125,136,137] or methods including only a limited number of residues [138].…”

“…These simpler clean-up procedures (although less selective) may be more suitable to allow for isolation of a broad range of benzimidazoles, which are difficult to recover using more comprehensive clean-up procedures. Brandon et al [135] extracted TBZ residues from liver tissues using DMSO-H 2 O (10:90, v/v) and diluted extracts in PBS Tween prior to determination by ELISA. Brandon et al [156] developed a similar type of extraction procedure for determination of some 11 benzimidazole residues in liver tissues.…”

2-Morpholinoethylisocyanide as a coupling agent for hapten-protein conjugates

“…Preparation of mouse monoclonal antibodies (mAbs) elicited with the 2-succinamido-MBZ conjugate was performed as described previously (Brandon et al 1992). …”

Methods for generation of monoclonal antibodies to the very small drug hapten, 5-benzimidazolecarboxylic acid

“…Following a study of the relevant literature which indicated that protein-bound tissue residues of TBZ possess an intact benzimidazole ring structure (Coulet et al, 1998a,b), the purpose of this work was to choose a drug (hapten) that could be bound to a suitable carrier protein (to increase its immunogenicity) and allow the ring structure of benzimidazole to be exposed as a major antigenic site. Previously, Brandon et al (1992) reported the production of monoclonal antibodies (MAbs) cross-reacting with TBZ and its major metabolite 5-hydroxythiabendazole using the immunogens 2-and 5-Succinamidothiabendazole. During this study, the TBZ parent compound plus its four analogues were used as immunogens, i.e.…”