A method for recovery of native, clonally-restricted immunoglobulins from agarose gels.
A method for recovery of native, clonally-restricted immunoglobulins from agarose gels.
复制标题
一种从琼脂糖凝胶中回收天然克隆限制性免疫球蛋白的方法。
DOI：
10.1002/elps.1150100104
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Kelly,RH
中科院分区：
文献类型：
--
作者：
Janson,RW;VertosickJr,FT;Kelly,RH
临床实验室在琼脂糖上使用高分辨率区带电泳进行常规血清蛋白电泳时，经常会遇到多种低水平、克隆限制性免疫球蛋白 (Ig)。我们寻找一种从临床实验室凝胶中回收天然结构的克隆限制性 Ig 的方法，作为研究其临床意义的第一步。我们发现两阶段电泳程序始终具有良好的回收率。常规琼脂糖凝胶电泳后，将含有克隆限制性 Ig 的电泳图部分切除，并在琼脂糖中进行平板等电聚焦，以增强单个抗体克隆型的分离。可以从区带电泳图上的相邻轨迹（即加载相同样本的轨迹）上切割出包含相同克隆限制性 Ig 的多个板，并将其应用于聚焦凝胶上的单个轨迹上，以改善分离并提高产量。切割聚焦的凝胶以分离出含有单个克隆型的平板。洗涤这些板以去除载体两性电解质并在-20°C下保持过夜。通过物理破坏凝胶后进行超速离心，从解冻的凝胶中提取 Ig，回收率为 61-68%。回收的 Ig 的抗原结合活性通过速率比浊法进行验证。通过该程序成功地从免疫动物血清中分离出克隆限制性抗体，并对其进行生物素化，用作蛋白质印迹的探针。
Multiple low level, clonally‐restricted, immunoglobulins (Ig) are commonly encountered on routine serum protein electrophoresis by clinical laboratories using high resolution zone electrophoresis on agarose. We sought a method for recovering the clonally‐restricted Ig, in native configuration, from clinical laboratory gels as a first step in the investigation of its clinical significance. We found that a two‐stage electrophoretic procedure gave consistently good recoveries. After routine agarose gel electrophoresis, portions of the electropherogram, containing clonally‐restricted Ig, were excised and subjected to flatbed isoelectric focusing in agarose to enhance separation of the individual antibody clonotypes. Multiple slabs, containing the same clonally‐restricted Ig, could be cut from adjacent tracks (i. e., tracks loaded with the same specimen) on the zone electropherogram and applied to a single track on the focusing gel to improve separation and increase yields. The focused gels were cut to isolate slabs containing individual clonotypes. These slabs were washed to remove carrier ampholytes and held at −20 °C overnight. Ig was extracted from the thawed gels, with 61–68 % recovery, by ultracentrifugation following physical disruption of the gel. Antigen binding activity of the recovered Ig was verified by rate nephelometry. Clonally‐restricted antibodies were successfully isolated from an immune animal serum by this procedure and biotinylated for use as probes on Western blots.