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)

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]N bond in the parent structure was reduced. Corresponding immunogens and coating antigens were prepared to improve the assay sensitivity. The hybridomas 4 Â 10 8 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 enzymelinked 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 IC 50 value of the ELISA for CPAMOZ was 0.13 ng mL À1 . 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 (r 2 ¼ 0.9908). The proposed icELISA could be used for an accurate monitoring of AMOZ in animal tissues.

Scientific Opinion on nitrofurans and their metabolites in food

“…A number of ELISA methods have been reported for the determination of NPAMOZ in shrimps and fish samples with reported LOD values of 0.1-0.3 µg/kg and LOQ or CCβ values of 0.3-0.36 µg/kg (Pimpitak et al, 2009;Shen et al, 2012;Sheu et al, 2012;Yang et al, 2012;Liu et al, 2013). Other ELISA methods were developed and applied to the determination of NPAMOZ in chicken, pork, fish and shrimp samples, reporting LOD values ranging from < 0.1 to 0.34 µg/kg (Luo et al, 2012;Xu et al, 2013;Shu et al, 2014). A number of ELISA methods have also been reported for direct determination of AMOZ, without derivatisation, with reported LOD values of 0.4 µg/kg (Song et al, 2012;Yan et al, 2012).…”

“…Other ELISA methods were developed and applied to the determination of NPAMOZ in chicken, pork, fish and shrimp samples, reporting LOD values ranging from < 0.1 to 0.34 µg/kg (Luo et al, 2012;Xu et al, 2013;Shu et al, 2014). A number of ELISA methods have also been reported for direct determination of AMOZ, without derivatisation, with reported LOD values of 0.4 µg/kg (Song et al, 2012;Yan et al, 2012).…”

Development of Antibodies and Immunoassays for Monitoring of Nitrofuran Antibiotics in the Food Chain

“…Monoclonal and polyclonal antibodies were produced, the best sensitivity to NPAMOZ being a polyclonal with IC 50 = 0.16 µg/L. The same derivatising agent was used by Liu et al [42] and Shu et al [43]. Liu et al also performed the reaction at room temperature but in a water/DMF mix for 6 h. A water wash was used to purify the product which was confirmed by LC-MS. A monoclonal antibody was produced again with better sensitivity to NPAMOZ than CPAMOZ or AMOZ.…”

Substrate-Assisted Carbon Dioxide Activation as a Versatile Approach for Heterocyclic Synthesis

“…An interesting application for this method was found in the synthesis of 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ, Scheme 16). AMOZ is a metabolite of several larger drug molecules (for instance of furaltadone) 71 which has been used as an antibiotic. 72 A simple three-step process transforms allyl amine (50) into AMOZ (53), through initial coupling with CO2 in the presence of tBuOI to form the iodinated oxazolidinone 51.…”