蛋白质芯片检测可溶性转铁蛋白受体条件的优化研究

Optimizing the detection of soluble transferrin receptor with protein microarray

目的优化蛋白芯片检测可溶性转铁蛋白受体(sTfR)的技术条件。方法用芯片点样仪将血清铁蛋白的一种抗体点样于PSG芯片上,用其另一种抗体做为检测抗体,以Cy3标记的羊抗做为二抗进行检测。以双抗夹心法对sTfR抗原进行检测。结果以sTfR单克隆鼠抗为固定探针,选择接触式点样进行芯片点样,预点样在40以后可出现较好的点样一致性;sTfR的固定探针的浓度选择0.25mg/ml,其检测抗体的浓度为0.18μg/ml;3%脱脂乳粉和GE公司的羊抗兔IgG被优选为封闭剂与第二抗体。sTfR检测下限与生物检测限分别为0.253ng/ml和0.78ng/ml;建立了对sTfR具有最佳相关系数的回归方程(r=0.99699)与标准曲线。结论该研究优化了用于sTfR检测的蛋白芯片检测条件,从而建立了定量检测sTfR的蛋白芯片平台。

Objective To optimize the conditions of protein microarray assay for soluble transferrin receptor（sTfR）.Methods A microarrayer was used for printing anti-sTfR as antibody I on each protein microarray;anti-sTfR antibody was used as detection antibody II and goat antibody coupled to Cy3 was used as antibody III.The standard sTfR was detected by double antibody sandwich technique.Results Mouse monoclon sTfR antibody was chosen as a probe and contact printing as the printing method.A better homogenicity was appeared after pre-printing for 40 spots.The concentration of sTfR probes was 0.25mg /ml.The concentration of sTfR detection antibody was 0.18μg /ml.3% skim milk powder and goat anti rabbit antibody made by GE was chosen as blocking buffer and secondary antibody.The lowest detection limits and the biological detection limits of sTfR were 0.253 ng /ml and 0.78 ng /ml respectively.The best fitting models and standard curve were established for sTfR（r = 0.99699）.Conclusion Conditions of a quantitative analysis system for measurement of sTfR with protein microarray were optimized.