Synthesis of furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) and novel haptens for the development of a sensitive enzyme-linked immunosorbent assay (ELISA)

Analytical Methods Pub Date: 2014-01-09 DOI: 10.1039/C3AY41145A

Abstract

A sensitive and specific monoclonal enzyme-linked immunosorbent assay (ELISA) for the determination of tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. Three different haptens of AMOZ were synthesized. The haptens (I, III) were derived from 4-carboxybenzaldehyde and ethyl 4-bromobutyrate, respectively. Hapten II was based on hapten I but a C[double bond, length as m-dash]N bond in the parent structure was reduced. Corresponding immunogens and coating antigens were prepared to improve the assay sensitivity. The hybridomas 4 × 108 secreting antibodies against CPAMOZ were obtained from immunogens I-BSA by monoclonal antibody (mAb) technology. The antibody indicated that: (1) the small molecular AMOZ cannot induce the specific response against AMOZ; (2) the weakened conjugation effect improved the recognition of AMOZ. Assessment of twelve coating antigen/antibody combinations consequently resulted in the development of an indirect competitive enzyme-linked immunosorbent assay (icELISA). The results showed that the sensitivity was highly improved about fifteen-fold for the novel heterologous coating antigen at low hapten density. The IC50 value of the ELISA for CPAMOZ was 0.13 ng mL?1. The cross reactivity values of the assay with NPAMOZ, AMOZ and FTD were 118%, 2.3% and 16.3%. Recoveries from AMOZ fortified animal tissues were in a range of 82.6–108.4% and the CV values were less than 12.5%. The immunoassay was further validated by a LC-MS/MS method and the two methods showed good correlation (r2 = 0.9908). The proposed icELISA could be used for an accurate monitoring of AMOZ in animal tissues.

Graphical abstract: Synthesis of furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) and novel haptens for the development of a sensitive enzyme-linked immunosorbent assay (ELISA)
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