Novel hapten synthesis for antibody production and development of an enzyme-linked immunosorbent assay for determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

Abstract

Publisher rights This is the author's version of a work that was accepted for publication in Talanta. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Talanta, VOL 103, 01/2012 General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Cite this article as: Zhen-Lin Xu, Yu-Dong Shen, Yuan-Ming Sun, Katrina Campbell, Yuan-Xin Tian, Shi-Wei Zhang, Hong-Tao Lei and Yue-Ming Jiang, Novel hapten synthesis for antibody production and development of an enzyme-linked immunosorbent assay for determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), Talanta, http://dx.doi.org/10. 1016/j.talanta.2012.10.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractA heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) was developed. AMOZ was derivatised with 2-(4-formylphenoxy)acetic acid or 2-(3-formylphenoxy)acetic acid to obtain two novel immunizing haptens. The ability of these haptens in producing specific polyclonal antibodies against the nitrophenyl derivative of AMOZ (NPAMOZ) was compared with that of traditional immunizing haptens (derivatised AMOZ with 3-carboxybenzaldehyle or 4-carboxybenzaldehyle). The results indicated that the novel immunizing haptens were able to produce antibodies with almost a two-fold improvement in sensitivity of the ciELISA for NPAMOZ in comparison with the existing antibody based ELISAs. The differences in sensitivity were explained by the molecular modeling of the lowest energy conformations of NPAMOZ and the haptens. Another novel hapten, derivatised AMOZ with 2-oxoacetic acid, was synthesized and used as a heterologous coating hapten. The results showed that this strategy of using only a partial structure of the target molecule as the coating hapten was able to obtain a two to three-fold improvement in sensitivity. This study provided a modern approach for the development of an immunoassay with improved sensitivity for the metabolites of nitrofuran antibiotics.

A novel chemiluminescent ELISA for detecting furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) in fish, egg, honey and shrimp samples

“…The formed metabolite derivatives were then typically chemically coupled to carrier proteins, via the carboxylic group, for the production of immunogens and coating antigens. In other studies, the metabolite AMOZ was also synthesized with 2-(4-formylphenoxy) acetic acid, 2-(3-formylphenoxy) acetic acid or 2-oxoacetic acid (Song et al, 2012;Xu et al, 2013). In the present study, AMOZ was synthesized with 4-CBA and the CPAMOZ-BSA conjugate was prepared using the active ester method.…”

“…Based on different monoclonal antibodies, the IC 50 values for NPAMOZ ranged from 1.59 to 72.3 μg/l in the homologous ciELISA (Pimpitak et al, 2009). AMOZ has also been derivatized with 2-(4-formylphenoxy) acetic acid or 2-(3-formylphenoxy) acetic acid, with an IC 50 value for NPAMOZ at 2.1 μg/l (Xu et al, 2013). Moreover the results from the CLELISA method developed in this study correlate well with the results achieved using commercial ELISA methods, with R 2 values ≥ 0.99.…”

“…After dissolving completely, 49 mg of AMOZ was added to the solution, and stirred at room temperature for 6 h. After precipitate appeared in the solution, the suspension solution was centrifuged and washed three times in deionized water. The synthesized compound was confirmed by LC-MS. Then, CPAMOZ was coupled to BSA and OVA for immunogen and coating antigen, respectively according to Xu reported method (Xu et al, 2013). And the detailed synthesis method as follows, firstly, CPAMOZ (4.5 mM), NHS (4.5 mM) and DCC (4.5 mM) were dissolved in 50 μL of DMF, respectively.…”

Hapten synthesis and production of specific antibody against 3-amino-5-morpholinomethyl-2-oxazolidone for immunoassay without derivatisation

“…For example, in the first immunoassay for AMOZ by Pimpitak et al, a monoclonal antibody was produced using the 3-carboxybenzaldehyde derivative of AMOZ (CPAMOZ) as the immunogen hapten and competitive indirect enzyme-linked immunosorbent assay (ciELISA) was performed for the 2-nitrobenzaldehyde derivative of AMOZ rather than AMOZ. The derivative procedure for AMOZ is always time-consuming and cumbersome (Jester et al, 2014;Pimpitak et al, 2009;Xu et al, 2013). Although antibodies produced against CPAMOZ sometimes showed good binding affinity to free AMOZ (cross-reactivity (CR)), the binding ability varied significantly for different reports, and sometimes it is hard to screen.…”

Determination of 3-Amino-2-oxazolidinone in Animal Tissue by an Enzyme-Linked Immunosorbent Assay and a Time-Resolved Fluoroimmunoassay

“…However, the metabolite, AOZ, may bind to tissue proteins and may persist for considerable periods of time in animal tissues. Therefore, the administration of furazolidone may be monitored through the analytical measurement of AOZ (Xu et al 2013). As AOZ is covalently bound to proteins, the analysis usually entails the release of the metabolite under acidic conditions and a derivatization step with nitrobenzaldehyde or benzaldehyde prior to detection .…”