Feng S H, Rubinstein L J, Stein K E
Laboratory of Bacterial Polysaccharide, Food and Drug Administration, Bethesda, MD 20892.
J Immunol Methods. 1991 Mar 21;137(2):261-6. doi: 10.1016/0022-1759(91)90032-b.
A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accommodate NC of different sizes and shapes. PS-coated NC disks were used to localize antibody secreting hybridoma cells cultured on filter paper disks. These were analyzed by blotting with size-matched PS-coated NC disks and specific antibodies secreted by individual colonies were detected by enzyme-linked immunoblot. In another application of this method, immune sera were separated by isoelectric focusing and the gels were blotted with PS-coated NC sheets. The spectrotype and isotype of antibodies that bound to the NC were examined using isotype specific enzyme-linked antibody. These immunoblots showed high resolution and specificity. The advantages of this method are that the PS used for coating does not need to be derivatized in order to bind the NC, and that smaller quantities of PS may be utilized by this coating method when compared to other techniques. This provides a useful tool to ask many questions regarding the immune response to PS.
已开发出一种将天然、未衍生化的多糖（PS）包被到硝酸纤维素（NC）上以鉴定PS特异性抗体的方法。该方法的新特点是，PS分子通过能够容纳不同大小和形状NC的装置以其天然状态真空过滤到NC上。PS包被的NC圆盘用于定位在滤纸圆盘上培养的分泌抗体的杂交瘤细胞。用大小匹配的PS包被的NC圆盘进行印迹分析，并通过酶联免疫印迹检测各个菌落分泌的特异性抗体。在该方法的另一个应用中，通过等电聚焦分离免疫血清，并用PS包被的NC片对凝胶进行印迹。使用同型特异性酶联抗体检查与NC结合的抗体的光谱型和同型。这些免疫印迹显示出高分辨率和特异性。该方法的优点是用于包被的PS无需衍生化即可与NC结合，并且与其他技术相比，这种包被方法可以使用较少量的PS。这为回答许多关于对PS免疫反应的问题提供了一个有用的工具。
