Hu G R, Harrop P, Warlow R S, Gacis M L, Walls R S
Department of Medicine, University of Sydney, Australia.
J Immunol Methods. 1997 Mar 28;202(2):113-21. doi: 10.1016/s0022-1759(96)00235-9.
We report a procedure for optimisation of Western blotting using protein G-horseradish peroxidase (protein G-HRP) which avoids the false positive reactions often caused by second antibodies and increases the detection of autoantibodies by protein G conjugate. A number of modifications were investigated. Higher concentrations of serum and protein G-HRP at 1:5 to 1:10 and 1:100, respectively, increased the detection to the same order as that obtained with second antibody systems and gold staining with silver enhancement. The role of various detergents in the procedure was established. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) in incubation with protein G-HRP increased the binding between protein G and immunoglobulin G. Addition of Tween-20 for blocking produced little background so that protein blockers could be avoided. Prolonged incubation with serum increased markedly the sensitivity of the procedure when compared with the recommended 2 h incubation period. Polyvinylidene difluoride membrane provided better transfer effect, lower background and higher mechanical strength than nitrocellulose membrane. The utilization of only one antibody-specific ligand increased the simplicity, reliability, economy, efficiency and specificity of the method. These modifications make this method significantly better for detection and screening for autoantibodies.
我们报告了一种使用蛋白G-辣根过氧化物酶（protein G-HRP）优化蛋白质印迹法的程序，该程序可避免二抗常常引起的假阳性反应，并提高蛋白G偶联物对自身抗体的检测能力。我们研究了多种改进方法。分别将血清和蛋白G-HRP的浓度提高到1:5至1:10和1:100，检测能力提高到与二抗系统及银增强金染色相同的水平。确定了该程序中各种去污剂的作用。与蛋白G-HRP一起孵育时，3-[(3-胆酰胺丙基)-二甲基铵]-1-丙烷磺酸盐（CHAPS）可增强蛋白G与免疫球蛋白G之间的结合。添加吐温20进行封闭产生的背景很少，因此可以避免使用蛋白封闭剂。与推荐的2小时孵育时间相比，血清长时间孵育可显著提高该程序的灵敏度。聚偏二氟乙烯膜比硝酸纤维素膜具有更好的转移效果、更低的背景和更高的机械强度。仅使用一种抗体特异性配体提高了该方法的简便性、可靠性、经济性、效率和特异性。这些改进使该方法在自身抗体的检测和筛选方面有显著提升。
