同时检测多种营养标记蛋白的液相蛋白芯片检测平台条件的优化

Optimization of platform conditions for simultaneous detection of multiple nutritional marker proteins by liquid protein microarray

目的优化同时检测血清铁蛋白(serum ferritin,SF)、可溶性转铁蛋白受体(soluble transferrin receptor,sTfR)、C反应蛋白(C-reactive protein,CRP)、视黄醇结合蛋白4(retinol-binding protein4,RBP4)4种目标蛋白的液相蛋白芯片的检测条件。方法将4种蛋白的捕获抗体偶联到不同编码的磁珠上,加样于96孔板中,采用双抗夹心法对抗原抗体进行检测;对5例血清分别用商用稀释液、1%牛血清白蛋白(albumin from bovine serum,BSA)、磷酸缓冲盐溶液(phosphate buffer saline,PBS)进行稀释后检测4种目标蛋白,并对结果进行比较;采用抗体特异性验证实验对抗体特异性结合能力进行检验;采用单独反应体系和混合反应体系信号值的配对t检验对蛋白之间有无干扰进行检验;采用倍比稀释法对目标蛋白进行稀释,找出每种蛋白检测下限和生物检测限;根据倍比稀释结果建立标准曲线和回归方程。结果针对本实验4种蛋白的检测实验,优选1%BSA和PBS代替商用稀释液作为稀释液;4种抗原与其他捕获抗体和检测抗体交叉反应率在2%以下;每种蛋白在单独反应体系与混合反应体系的信号值差异无统计学意义;混合体系中SF的检测下限为1.155 ng/mL,生物检测限为1.625 ng/mL;sTfR的检测下限为2.682 ng/mL,生物检测限为5.208 ng/mL;CRP的检测下限为0.302 ng/mL,生物检测限为0.391 ng/mL;RBP4的检测下限为1.814 ng/mL,生物检测限为3.540 ng/mL。4种蛋白的标准曲线和回归方程分别是:SF y=172.5x-39.65,R^(2)=0.9968;sTfR y=60.10x+77.38,R^(2)=0.9972;CRP y=-6.000x^(2)+210.3x+246.1,R^(2)=0.9063;RBP4 y=-0.6998x^(2)+64.31x+134.8,R^(2)=0.9748。结论该研究较好地实现了利用液相蛋白芯片技术同时检测SF、sTfR、CRP和RBP4蛋白的检测平台的条件优化。

OBJECTIVE To optimize the technical conditions for simultaneous detection of serum ferritin(SF),soluble transferrin receptor(sTfR),C-reactive protein(CRP)and retinol-binding protein four(RBP4)by liquid protein microarray.METHODS The trapping antibodies of the four proteins were coupled to magnetic beads with different codes,and the samples were added to the 96-well plate.The antibodies were detected by double antibody sandwich method.The serum of 5 patients were diluted with commercial diluent,1%albumin from bovine serum(BSA)and phosphate buffer saline(PBS)to detect 4 target proteins,and the results were compared.The antibody specific binding ability was tested by antibody specific validation test.The interference between proteins was verified by the paired t test of the signal values of the single reaction system and the mixed reaction system.The lower limit of detection and the limit of biological detection of each protein were found by using multiple dilution method.The standard curve and regression equation were established.RESULTS 1%BSA and PBS were selected to replace commercial diluent as diluents for the detection of 4 proteins in this experiment.The cross-reaction rate of the four antigens with other capture antibodies and detection antibodies was less than 2%.There was no significant difference in the signal value of each protein in the single reaction system and the mixed reaction system.The limit of detection and the limit of biological detection of SF were 1.155 and 1.625 ng/mL,respectively.The lower limit of detection and the limit of biological detection of sTfR were 2.682 and 5.208 ng/mL,respectively.The detection limit and biological detection limit of CRP were 0.302 and 0.391 ng/mL,respectively.The lower limit of detection and the limit of biological detection for RBP4 were 1.814 and 3.540 ng/mL,respectively.The standard curve and regression equation of the four proteins within the common linear range were as follows:SF y=172.5x-39.65,R^(2)=0.9968;sTfR y=60.10x+77.38,R^(2)=0.9972;CRP y=-6.000x^(2)+210.3x+246.1,R^(2)=0.9063;RBP4 y=-0.6998x^(2)+64.31x+134.8,R^(2)=0.9748.CONCLUSION The conditions of the detection platform for four proteins such as SF,sTfR,CRP and RBP4 were optimized by using liquid protein chip technology.