Immunoassay direct for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), the metabolite of furaltadone, based on a specific antibody was usually unavailable due to its small molecule weight. In this work, a hapten with a double bond and an active carboxyl group was designed and then conjugated to peptide dendrimer to obtain novel multiple haptens. The haptens and multiple haptens were coupled to bovine serum albumin as immunogens. Polyclonal antibodies showed binding to free AMOZ in a heterologous competitive indirect enzyme-linked immunosorbent assay were obtained. The assay showed IC50 (half maximal inhibitory concentration) of 4.1 μg/kg and limit of detection of 0.2 μg/kg for AMOZ. Recoveries of AMOZ from grass carp were tested from 83.1% to 117.0%, with the coefficient of variation below 12%. As no laborious derivatisation procedure was employed, the proposed assay should be useful for the screening of AMOZ for a large number of samples.
Jianhua Shu,Liming He,Huanzhong Ding,Liqi Wang,Hongyan Guo,Yan Gao,Emmanuel E. Dzakah,Zhenling Zeng
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 CN 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.
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