Sensitive, fast, and specific immunoassays for methyltestosterone detection

Abstract

An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%-100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed.

Figure 1

Schemes of icELISA (A) and immunochromatographic strip (B) for MT detection, respectively.

Figure 2

Optimized inhibition standard curve for MT analysis by icELISA. Error bar was calculated according to three repeats at each concentration.

Figure 3

Photograph of the immunochromatographic strip for MT detection in PBS (A); fish feed (B) and pig feed (C). The concentrations of MT from left to right were 0, 0.63, 1.25, 2.5, and 5 ng/g.

Figure 4

Correlation studies between icELISA and GC-MS detection in fish (A) and pig (B) feed. These samples were obtained from MT spiked at levels of 1.25, 2.5 and 5 ng/g (n = 3).