Loria R C, Prange J O, Wedner H J
Washington University School of Medicine, Department of Medicine, St. Louis, MO 63110.
J Allergy Clin Immunol. 1988 Nov;82(5 Pt 1):834-41. doi: 10.1016/0091-6749(88)90086-3.
Oak-pollen extracts, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose, demonstrated extensive banding in the absence of human sera or with nonatopic sera with both a horseradish peroxidase-conjugated goat antihuman IgE and 125I-labeled rabbit antihuman IgE. When the nonionic detergent Tween 20 was removed from all incubations and washes, negative banding appeared, corresponding to bands that were clearly visible when Tween 20 had been used as a blocking agent. When Tween 20 was reintroduced, the bands reappeared. These results demonstrate that, although Tween 20 is effective in blocking unbound sites on nitrocellulose membranes, this and other nonionic detergents may cause nonspecific binding to immunoblots of oak-pollen proteins. Thus, care must be used in the interpretation of immunoblots to detect allergens when Tween 20 is used as the blocking agent.
用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离并用电泳法转移到硝酸纤维素膜上的橡树花粉提取物，在没有人类血清或使用非特应性血清时，用辣根过氧化物酶偶联的山羊抗人IgE和125I标记的兔抗人IgE均显示出广泛的条带。当从所有孵育和洗涤步骤中去除非离子去污剂吐温20时，出现了阴性条带，这与使用吐温20作为封闭剂时清晰可见的条带相对应。当重新引入吐温20时，条带重新出现。这些结果表明，尽管吐温20在封闭硝酸纤维素膜上的未结合位点方面有效，但这种和其他非离子去污剂可能会导致与橡树花粉蛋白免疫印迹的非特异性结合。因此，当使用吐温20作为封闭剂检测过敏原时，在解释免疫印迹结果时必须谨慎。
