Guidry A J, Squiggins K E, Vann W F, Westhoff D C
USDA-ARS, Beltsville, MD 20705.
J Immunol Methods. 1991 Oct 25;143(2):159-65. doi: 10.1016/0022-1759(91)90041-d.
The Fc region of IgG of most mammals binds protein A on S. aureus resulting in high backgrounds when measuring specific antibodies to S. aureus in the ELISA. Removal of protein A from S. aureus or modification of the Ig Fc to prevent binding to protein A could affect specific antibody binding. We compared effects of blockage of Fc binding to protein A with purified protein A to trypsin removal of protein A from S. aureus, on specific antibody binding. When NMS was incubated without and with protein A (0 microgram, 50 micrograms, 200 micrograms and 400 micrograms) and high protein A Cowan I was the bound S. aureus antigen in the ELISA, absorbance OD405 was 0.769, 0.240, 0.224 and 0.210 +/- SE 0.026. When mouse Mab (IgG1, kappa) to bovine IgA was incubated without and with protein A (400 micrograms) prior to reaction with bovine IgA in the ELISA, absorbance was 0.645 and 0.639, indicating protein A had no effect on specific antibody binding. To determine the effect of trypsin on specific binding, Becker S. aureus was trypsin treated before linking it to microtiter wells. When Mab (IgM) to Becker (Nelles et al., Infect. Immun. (1985) 49, 14) was incubated with protein A (400 micrograms) before use in the ELISA, trypsin treatment of Becker resulted in reduced specific antibody activity (untreated Becker = 1.306, trypsin treated Becker = 0.331). These results suggest that purified protein A can be used to block nonspecific binding via Fc of Ig to S. aureus, thus avoiding trypsin denaturation of surface antigens.
大多数哺乳动物的IgG的Fc区域与金黄色葡萄球菌上的蛋白A结合，这在ELISA中检测针对金黄色葡萄球菌的特异性抗体时会导致高背景。从金黄色葡萄球菌中去除蛋白A或修饰Ig Fc以防止与蛋白A结合可能会影响特异性抗体结合。我们比较了用纯化的蛋白A阻断Fc与蛋白A的结合和用胰蛋白酶从金黄色葡萄球菌中去除蛋白A对特异性抗体结合的影响。当将正常小鼠血清（NMS）分别与0微克、50微克、200微克和400微克蛋白A一起孵育，且在ELISA中使用高蛋白A的考恩I株作为结合的金黄色葡萄球菌抗原时，吸光度OD405分别为0.769、0.240、0.224和0.210±标准误0.026。当在ELISA中与牛IgA反应之前，将抗牛IgA的小鼠单克隆抗体（IgG1，κ）分别与0微克和400微克蛋白A一起孵育时，吸光度分别为0.645和0.639，表明蛋白A对特异性抗体结合没有影响。为了确定胰蛋白酶对特异性结合的影响，在将贝克尔金黄色葡萄球菌连接到微量滴定板之前对其进行胰蛋白酶处理。当在ELISA中使用之前，将抗贝克尔金黄色葡萄球菌的单克隆抗体（IgM）（内尔斯等人，《感染与免疫》（1985年）49卷，第14页）与400微克蛋白A一起孵育时，对贝克尔金黄色葡萄球菌进行胰蛋白酶处理导致特异性抗体活性降低（未处理的贝克尔金黄色葡萄球菌=1.306，胰蛋白酶处理的贝克尔金黄色葡萄球菌=0.331）。这些结果表明，纯化的蛋白A可用于阻断Ig的Fc与金黄色葡萄球菌的非特异性结合，从而避免表面抗原的胰蛋白酶变性。
