通过半制备等电聚焦进行基于等电点的抗体亚型分离

pI-dependent isolation of antibody isoforms by semipreparative isoelectric focusing.

作者信息

Gangopadhyay A, Saravis C A, van den Abbeele A D, Kassis A I

机构信息

Department of Radiology (Division of Nuclear Medicine), Harvard Medical School, Boston, MA 02115, USA.

出版信息

Q J Nucl Med. 1995 Jun;39(2):129-33.

PMID:8574807

Abstract

A procedure based on isoelectric point (pI) was developed to separate immunoreactive antibody isoforms. A polyclonal IgG, rabbit anti-human serum albumin (R-HSA), was subjected to free-flow isoelectrophoresis using a semipreparative isoelectric focusing apparatus that fractionates proteins by pI. Twenty fractions were collected and their pH, protein content, and immunoreactivity determined. The development of a pH gradient and separation of proteins took place within 3 hours with about 93% protein recovery. The protein concentration of the individual fractions varied. Isoelectric focusing of fractions in agarose slab gels confirmed the clear separation of antibody isoforms. Enzyme-linked immunosorbent assay demonstrated significantly higher immunoreactivity (P < or = 0.03) of the majority of the antibody isoform fractions compared with native R-HSA IgG. The procedure is capable of isolating immunoreactive antibody isoform fractions from immunologically irrelevant and low-affinity antibodies.

摘要

开发了一种基于等电点（pI）的方法来分离免疫反应性抗体亚型。使用通过pI对蛋白质进行分级分离的半制备等电聚焦装置，对多克隆IgG兔抗人血清白蛋白（R-HSA）进行自由流动等电聚焦。收集了20个级分，并测定了它们的pH值、蛋白质含量和免疫反应性。pH梯度的形成和蛋白质的分离在3小时内完成，蛋白质回收率约为93%。各个级分的蛋白质浓度各不相同。在琼脂糖平板凝胶中对级分进行等电聚焦证实了抗体亚型的清晰分离。酶联免疫吸附测定表明，与天然R-HSA IgG相比，大多数抗体亚型级分的免疫反应性显著更高（P≤0.03）。该方法能够从免疫无关和低亲和力抗体中分离出免疫反应性抗体亚型级分。