ELISA-based Method to Detect Microbial Transglutaminase in Frozen Surimi
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Abstract:
In this study, a sandwich-type enzyme-linked immunosorbent assay (ELISA) was developed to detect microbial transglutaminase (MTG) in frozen surimi, using rabbit anti-MTG polyclonal antibody as the capture antibody and mouse anti-MTG monoclonal antibody as the detection antibody. Combined with anti-IgG1 secondary antibody horseradish peroxidase (HRP) conjugate, the sandwich ELISA was developed to quantitatively detect MTG in frozen surimi samples, thus providing a means to prevent adulteration of frozen surimi. The results showed that the optimum working concentration of the capture antibody was 2 μg/mL whereas that of the detection antibody was 0.1 μg/mL, and the optimum dilution of HRP-conjugated secondary antibody was 1:5,000. The visual detection limit of the assay was 20 ng/mL, and alinear relationship between OD450 values and the log values of MTG concentration was observed in the range of 0.6~10 mg/mL. A simulation model of adulteration in frozen surimi was established to determine the amount of added MTG. The recovery rate of MTG was over 94%, whereas the intra- and inter-plate coefficients of variation during the measurement were 1.12%~4.02% and 5.43%~6.87%, respectively. These results proved that this method was sensitive, required simple sample pretreatment, and was suitable for the quantitative detection of MTG added to frozen surimi.
