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目的优化蛋白芯片检测β-乳球蛋白(β-Lg)的技术条件。方法用芯片点样仪将β-乳球蛋白的一种抗体点样于三维基片上,用其另一种抗体作为检测抗体,以Cy3标记的羊抗作为二抗进行检测。以双抗夹心法对β-Lg抗原进行检测。结果β-Lg单克隆鼠抗66#被确定为点样探针,选择接触式点样进行芯片点样,点样数在42~92点之间可出现较好的点样一致性;β-Lg固定探针的浓度选择0.5 mg/mL,其检测抗体的稀释度为1∶2000;1%无蛋白封闭液被优选为封闭剂;β-Lg检测下限与生物检测限分别为17.54和55.31 ng/mL。根据β-Lg的S型曲线确定了其线性范围并建立了对β-Lg具有最佳决定系数(R~2=0.9993)的回归方程与标准曲线。结论本研究优化了牛乳中β-Lg检测的蛋白芯片检测条件,建立了定量检测牛乳β-Lg的蛋白芯片平台。
Objective To optimize the technique of protein chip for β-lactoglobulin (β-Lg) detection. Methods An antibody of β-lactoglobulin was spotted on a three-dimensional substrate with a chip spotter, another antibody was used as a detection antibody, and a Cy3-labeled goat anti-rabbit antibody was used as a secondary antibody. Β-Lg antigen was detected by double-antibody sandwich method. Results β-Lg monoclonal mouse anti-66 # was identified as a spot-like probe, the choice of contact spot chip-like spots, the number of spots in the 42 ~ 92 points can be better point-like consistency; β- Lg fixed probe concentration of 0.5 mg / mL, the detection antibody dilution of 1: 2000; 1% protein-free blocking solution is preferably a blocking agent; β-Lg detection limit and biological detection limits were 17.54 and 55.31 ng / mL. Based on the sigmoid curve of β-Lg, the linear range was established and the regression equation and standard curve with the best determination coefficient (R ~ 2 = 0.9993) for β-Lg were established. Conclusion This study optimized the detection conditions of β-Lg protein in milk and established the protein chip platform for the quantitative determination of β-Lg in milk.