Development of an icELISA and Immunochromatographic Assay for Methyl-3-Quinoxaline-2-Carboxylic Acid Residues in Fish
- 250 Downloads
To evaluate residues of olaquindox (OLA) and its marker compound methyl-3-quinoxaline-2-carboxylic acid (MQCA) in aquatic products in the field, five types of hapten and corresponding antigens with exposure of different antigenic determinants were designed and synthesized. The optimal immunogen-coating combinations were obtained by antiserum detection and heterologous coating screening, successfully achieving a highly specific monoclonal antibody (mAb) against MQCA. Based on the highly specific mAb 1A11 showing no cross-reactivity with other analogs, an indirect competitive ELISA and an immunochromatographic strip for MQCA detection in fish were developed using the MQCA derivative MQCA-(NH2)4 coupled to KLH as the immunogen and MQCA-NH2 coupled to OVA as the heterologous coating antigen. The IC50 value of the indirect competitive enzyme-linked immunosorbent assay (icELISA) was 3.17 ng/mL, and the linear range was 0.58–17.29 ng/mL for MQCA under optimized concentrations of coating antigen and antibody of 0.03 and 0.3 μg/mL, respectively. The recoveries of MQCA by the developed icELISA in fish samples ranged from 81.6 to 90.7%. The cutoff value of the strip was 5 ng/mL, and the limit of detection was 0.25 ng/mL under optimal working conditions of 4 μL 0.1 M K2CO3 and 50 μL 0.2 mg/mL per 1 mL colloidal gold solution. The developed icELISA and immunochromatographic strip will be very useful tools for the primary screening of MQCA in fish.
KeywordsMQCA Monoclonal antibody icELISA Immunochromatographic strip
This work is financially supported by the National Key R & D Program (2016YFD0401101) and grants from Natural Science Foundation of Jiangsu Province, MOF, and MOE (BE2016307, BK20150145, BX20151038, CMB21S1614, CLE02N1515, 201513006).
Compliance with Ethical Standards
Conflict of Interest
Juan Peng declares that she has no conflict of interests. Shanshan Song declares that she got grant from Jiangsu Province (BK20150145). Liqiang Liu declares that he got grant from Jiangsu Province (BE2016307) and MOF (201513006). Hua Kuang declares that she got grant from MOST (2016YFD0401101). Chuanlai Xu declares that he got grant from grant from Jiangsu Province and Wuxi City (BK20150145, BX20151038, CMB21S1614, CLE02N1515).
This article does not contain any studies with human subjects. All animal studies were carried out under the guidance of animal welfare committee of Jiangnan University.
- Merou A, Kaklamanos G, Theodoridis G (2012) Determination of Carbadox and metabolites of Carbadox and Olaquindox in muscle tissue using high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 881-82:90–95CrossRefGoogle Scholar
- Peng, J., Liu, L., Xu, L., Song, S., Kuang, H., Cui, G., & Xu, C. (2016b) Gold nanoparticle-based paper sensor for ultrasensitive and multiple detection of 32 (fluoro)quinolones by one monoclonal antibody. Nano. Res.1–11Google Scholar
- Segato G, Biancotto G, Agnoletti F, Berto G, Montesissa C, Benetti C (2015) In vivo studies to highlight possible illegal treatments of rabbits with carbadox and olaquindox. Food Additives and Contaminants Part a-Chemistry Analysis Control Exposure & Risk Assessment 32(12):1976–1991Google Scholar
- Suryoprabowo, S., Liu, L., Peng, J., Kuang, H., & Xu, C. (2014) Antibody for the development of specific immunoassays to detect nadifloxacin in chicken muscles. Food Agric. Immunol 1–8Google Scholar
- Yong Y, Liu YH, He LM, Xu LX, Zhang YP, Fang BH (2013) Simultaneous determination of quinocetone and its major metabolites in chicken tissues by high-performance liquid chromatography tandem mass spectrometry. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 919:30–37CrossRefGoogle Scholar
- Zhao DY, Shi QY, He X, Zhang J (2014) A novel sorbent for solid-phase extraction coupling to high performance liquid chromatography for the determination of Olaquindox in fish feed. Czech J Food Sci 32(5):449–455Google Scholar