Biewenga J, Faber A, de Lange G, van Leeuwen F, van Eede P, Jefferis R, Haaijman J J, Vlug A
Mol Immunol. 1986 Jul;23(7):761-7. doi: 10.1016/0161-5890(86)90088-x.
The specificity of 14 monoclonal antibodies has been determined by immunoblotting (IB) and haemagglutination-inhibition (HAI) analysis using IgA1 and IgA2 myeloma proteins and eight different IgA1 fragments. Two antibodies probably recognized epitopes on the CH1 domain of IgA. They reacted with all Fab-containing fragments irrespective of whether these originated from the same or different IgA proteins. Seven antibodies were directed against epitopes on the CH2 domain. These antibodies were reactive with F(abc)2 fragments. They failed to react with Fab, Fab' and F(ab')2 fragments. Two out of these seven antibodies did not react with two-chain IgA half-molecules and Fabc fragments containing a single heavy and a single light chain. This suggests that these two antibodies recognized an epitope whose structure is dependent on disulfide linked heavy chains. Five other antibodies showed specificity for the CH3 domain. They were reactive with all CH3-containing molecules, irrespective of whether they comprised one or two alpha chains. Our study demonstrates that IB is an appropriate technique to determine domain specificity of monoclonal anti-immunoglobulin reagents. Although the IB tests were performed on denatured proteins the results agreed surprisingly well with those of the HAI analyses. Moreover, the IB technique could be used on fragments which could not be purified well enough for HAI analyses.
通过免疫印迹（IB）和血凝抑制（HAI）分析，利用IgA1和IgA2骨髓瘤蛋白以及8种不同的IgA1片段，确定了14种单克隆抗体的特异性。两种抗体可能识别IgA的CH1结构域上的表位。它们与所有含Fab的片段反应，无论这些片段来自相同还是不同的IgA蛋白。七种抗体针对CH2结构域上的表位。这些抗体与F(abc)2片段反应。它们不与Fab、Fab'和F(ab')2片段反应。这七种抗体中有两种不与双链IgA半分子和含有一条重链和一条轻链的Fabc片段反应。这表明这两种抗体识别的表位结构依赖于二硫键连接的重链。另外五种抗体对CH3结构域具有特异性。它们与所有含CH3的分子反应，无论这些分子包含一条还是两条α链。我们的研究表明，免疫印迹是确定单克隆抗免疫球蛋白试剂结构域特异性的合适技术。尽管免疫印迹试验是在变性蛋白上进行的，但结果与血凝抑制分析的结果惊人地一致。此外，免疫印迹技术可用于无法纯化到足以进行血凝抑制分析的片段。
