| Surimi is composed of stabilized myofibrillar proteins obtained from mechanically deboned fish flesh. It is an intermediate product used in a variety of products ranging from traditional Japanese kamaboko to various surimi-based seafood products. Some manufacturers use microbial transglutaminase(MTGase) as a texture modi?er to improve the gelation properties of surimi, enabling it to be sold at a higher price. The addition of MTGase to surimi defrauds surimi product manufacturers and undercuts legitimate industry prices, as well as violates the national food safety standards. However, studies on how to detect MTGase concentration are very few, cannot fulfill the demand for an accurate, rapid, convenient detection of the presence of MTGase in surimi. Based on the theory of enzyme-linked immunosorbent assay and immunochromatographic strip assay, a sandwich-ELISA detection method and gold immunochromatographic test strip detection method were developed for detection of MTGase in foods.An accurate, sensitive and quantitative sandwich-ELISA detection method using MTGase mouse monoclonal antibody and MTGase rabbit polyclonal antibody was established for quantitative detection of MTGase. The detection limit was determined as 2 ng/m L, the range of quantitative detection of MTGase was from 0.6 ug/m L to 10 ug/m L, intar- and inter–assay coefficients of variation were less than 4% and 7%, respectively.. In surimi products, the detection limit was determined as 20 ng/m L, the average recovery value was 95.89% and the standard deviation of recoveries was 1.09%. Analytical validity, including accuracy, specificity, sensitivity suggested that the sandwich-ELISA detection method was suitable for quantitative detection of MTGase in foods.A rapid, sensitive, and qualitative gold immunochromatographic test strip detection method was developed based on anti-MTGase antibody conjugated gold particle. Colloidal gold were prepared through reducing HAu Cl4?3H2O by sodium citrate and its average diameter was 20.87 nm. Colloidal gold were characterized by dynamic light scattering using a particle size/zeta potential analyser, ultraviolet-visible spectrophotometer and transmission electron microscope. The suitable p H of MTGase mouse monoclonal antibody labeling with colloidal gold was 9.0, and the suitable concentration of monoclonal antibody was 11 ug/m L. The gold labeled antibody and colloidal gold were analyzed by ultraviolet-visible spectrophotometer and fluorescence spectroscopy, and the results showed that the conjugation was successful. Gold immunochromatographic test strip detection method using gold labeled antibody, MTGase rabbit polyclonal antibody and rat anti-mouse Ig G1 antibody was established for qualtitative detection of MTGase in foods. The detection process could be finished within 10 min. The detection limit with visual observation of this strip was determined as 1 ug/m L with great reproducibility and stability. As being stored at 4℃ in a vacuum packed silver paper bag for more than 4 months, the gold immunochromatographic test strip still kept stable properties. The results obtained using the immunochromatographic strip correlated well with the results of the sandwich-ELISA and analytical validity included rapidness and convenience, indicating that immunochromatographic strips offer a rapid, effective and convenient way for detecting MTGase in foods.We presented here the establishment of quantitative sandwich-ELISA and qualitative immunochromatographic strip methods for detection of MTGase in food materials. Both methods are sensitive and have potential application for authorities and manufactures. |
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