Development of IC-ELISA and immunochromatographic strip assay for the detection of flunixin meglumine in milk

Abstract

Flunixin meglumine (FM) is a novel nonsteroidal anti-inflammatory drug for animals, which has antipyretic, analgesic, and antiinflammatory effects. The drug, which was originally used to relieve inflammation in horses, musculoskeletal disorders, and pain, has been approved for use in endotoxemia, infectious diseases in swine, etc. A sensitive anti-FM monoclonal antibody 2H4 was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic strip assay for the detection of FM in milk. The complete antigen and coating antigen were conjugated with bovine serum albumin and ovalbumin, respectively. The monoclonal antibody 2H4, with a half inhibition concentration of 0.29 ng/mL, had a limit of detection of 0.432 ng/mL and a linear range of detection of 0.08664-0.97226 ng/ mL. A sensitive and convenient immunochromatographic strip assay was developed with an FM cutoff value of 0.29 ng/mL. The developed methods were suitable for the detection of FM in milk.

Development of an indirect competitive enzyme-linked immunosorbent assay for detecting flunixin and 5-hydroxyflunixin residues in bovine muscle and milk

“…In the sample pretreatment procedures, acid or enzyme hydrolysis is a critical part for the release of tissue-bound residues into solution because of the high (99%) protein binding of flunixin (Horii, Ikenaga, Shimoda, & Kokue, 2004). In previous studies (Lin et al, 2018), the samples were directly diluted or extracted for the analysis of flunixin residues in foods of animal origin. This method may be used for satisfactorily detecting artificially spiked samples; however, high protein binding increases the potential risk of false negative rates.…”

“…In 2018, Lin et al successfully produced a sensitive anti-flunixin meglumine monoclonal antibody 2H4, and the produced antibody had an IC 50 of 0.29 ng/mL, a LOD (IC 10 ) of 0.1 ng/mL, and a linear range of 0.08664 ng/mL -0.97226 ng/mL. Based on the developed antibody and icELISA, Lin et al developed an effective and portable immunochromatographic strip assay with a cutoff value of 0.29 ng/mL flunixin meglumine in milk (Lin et al, 2018). Compared with previous research (Lin et al, 2018), we used 5-hydroxyflunixin to synthesise immunogens, and the produced antibody showed a satisfying cross-reactivity (20.3%) with is parent drug flunixin.…”

“…Based on the developed antibody and icELISA, Lin et al developed an effective and portable immunochromatographic strip assay with a cutoff value of 0.29 ng/mL flunixin meglumine in milk (Lin et al, 2018). Compared with previous research (Lin et al, 2018), we used 5-hydroxyflunixin to synthesise immunogens, and the produced antibody showed a satisfying cross-reactivity (20.3%) with is parent drug flunixin. Based on the produced antibody, we established an icELISA for the simultaneous screening of flunixin residue in bovine muscle and 5-hydroxyflunixin residue in milk before confirmation by a mass spectrometry method.…”

“…Flunixin is easily metabolised and eliminated in vivo, and approximately 90% of the original drug is excreted via urine and feces 24 h after drug treatment (Levionnois et al, 2018). The main metabolites of flunixin are 4-hydroxyflunixin, 5-hydroxyflunixin and 2-methylhydroxyl (Kissell et al, 2012;Lin et al, 2018). Among these compounds, 5-hydroxyflunixin ( Figure 1) is identified as the marker residue in bovine milk, while flunixin is identified in bovine muscle (Jedziniak et al, 2013;Ngoh et al, 2003;Smith et al, 2015).…”

“…Immunoassays are superior to chromatographic methods in screening tests for their simplicity, sensitivity, and high-throughput capabilities (Chen et al, 2017; Kong et al, 2017;Wang, Luo, Chen, Huang, & Jiang, 2016). Recently, some immunoassays for detecting residues of flunixin and other NSAIDs have been reported (Lin et al, 2018). However, to the best of our knowledge, enzyme-linked immunosorbent assay (ELISA) methods for simultaneously detecting flunixin and 5-hydroxyflunixin residues have not been reported.…”