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目的优选并确定蛋白质芯片检测牛乳中乳铁蛋白(lactoferrin,Lf)的检测条件。方法利用蛋白质芯片技术,采用双抗夹心的方式,通过点样探针选择试验、点样一致性试验、棋盘滴定试验、浓度倍比试验、生物检测限试验及检测下限试验最终优化出针对目标蛋白的最佳检测条件。结果最终确定以Lf鼠单抗75#为点样探针,预点样46点,46点至90点为点样一致性区段;点样探针浓度为0.5 mg/ml,检测抗体效价为1∶2 000,在0.6~612ng/ml之间剂量-反应关系呈现S型曲线,其线性范围在9.56~306 ng/ml之间;检测下限为1.68 ng/ml,生物检测限为3.59 ng/ml;建立了对Lf具有最佳回归系数(r=0.998)的回归方程与标准曲线。结论此研究优化了利用蛋白质芯片技术检测Lf的试验条件,为建立定量检测Lf的蛋白质芯片平台奠定了基础。
Objective To determine and determine the detection conditions of lactoferrin (Lf) in milk by protein chip. Methods Using protein chip technology, double-antibody sandwich method was adopted to optimize target protein by target-like probe selection test, sample consistency test, checkerboard titration, concentration ratio test, biological detection limit test and detection limit test The best detection conditions. The results showed that the Lf mouse monoclonal antibody 75 # was used as the spot-like probe, pre-spotted 46, and spot 46-point 90 for spot-like consistency; the spotted probe concentration was 0.5 mg / ml, and the antibody titer The linear range was 9.56 ~ 306 ng / ml. The detection limit was 1.68 ng / ml. The limit of detection was 3.59 ng / ml. The regression equation and standard curve with the best regression coefficient (r = 0.998) for Lf were established. Conclusion This study has optimized the experimental conditions for the detection of Lf by using protein chip technology and laid the foundation for the establishment of a protein chip platform for quantitatively detecting Lf.