冷冻鱼糜中微生物谷氨酰胺转胺酶ELISA检测方法的建立及应用

ELISA-based Method to Detect Microbial Transglutaminase in Frozen Surimi

摘要:

本文研究了冷冻鱼糜中微生物谷氨酰胺转胺酶（MTG）的双抗体夹心酶联免疫检测方法（sandwich-ELISA法），以抗MTG兔多抗为包被抗体，抗MTG鼠单抗为检测抗体，再结合HRP标记的抗IgG1二抗，构成sandwich-ELISA检测方法，对冷冻鱼糜中是否添加MTG进行定量检测，防止冷冻鱼糜原料掺假。实验结果表明，包被抗体的最佳工作浓度是2.00 g/mL，检测抗体的最佳工作浓度为0.10 g/mL，酶标二抗的最佳稀释倍数为1:5000。方法的检测限为20 ng/mL，该检测方法在0.6 mg/mL~10 mg/mL范围内OD450值与MTG浓度的log值呈线性关系。建立掺假冷冻鱼糜样品模拟体系，测定其中MTG的添加量：MTG的添加回收率为94%以上，测定过程中回收率结果的板内变异系数为1.12%~4.02%，板间变异系数为5.43%~6.87%。经试验证明该方法灵敏度高，样品前处理简便，适用于冷冻鱼糜中MTG的定量检测。

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.