Duhamel R C, Schur P H, Brendel K, Meezan E
J Immunol Methods. 1979;31(3-4):211-7. doi: 10.1016/0022-1759(79)90133-9.
Pooled human serum having a normal IgG subclass content was chromatographed on a column of protein A-Sepharose. The immunoglobulins that bound to the column at pH 7.0 were eluted with a pH gradient generated by 3 equal volumes of citrate/phosphate buffer at pH 5.0, 4.5 and 2.2. The elution pattern consisted of two major overlapping peaks centered 0.4 pH units apart in the pH gradient; on average the first peak centered at pH 4.7, and the second centered at pH 4.3. Upon second passage of each component, single major peaks centered at the appropriate pH were seen. The subclass distributions of the re-chromatographed peaks were as follows: the high pH-eluting IgG contained less than 1% IgG1, 95% IgG2 and 5% IgG4; the low pH-eluting IgG contained 90% IgG1, 6% Ig2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. Chromatography on protein A-Sepharose provides a means for separating normal human IgG1 from IgG2 and may therefore prove useful as an additional tool for studying the relative biological role of these IgG subclasses.
将具有正常IgG亚类含量的混合人血清在蛋白A-琼脂糖柱上进行层析。在pH 7.0时与柱结合的免疫球蛋白用由等体积的pH 5.0、4.5和2.2的柠檬酸盐/磷酸盐缓冲液产生的pH梯度洗脱。洗脱模式由两个主要的重叠峰组成，在pH梯度中相隔0.4个pH单位；平均而言，第一个峰位于pH 4.7，第二个峰位于pH 4.3。每种组分再次通过时，在适当的pH处出现单一的主要峰。重新层析峰的亚类分布如下：高pH洗脱的IgG含有不到1%的IgG1、95%的IgG2和5%的IgG4；低pH洗脱的IgG含有90%的IgG1、6%的Ig2和5%的IgG4。IgG3不与蛋白A结合，因此在pH梯度级分中不存在。在蛋白A-琼脂糖上进行层析为从IgG2中分离正常人IgG1提供了一种方法，因此可能被证明是研究这些IgG亚类相对生物学作用的一种有用的额外工具。
