Haury M, Grandien A, Sundblad A, Coutinho A, Nobrega A
Unite d'Immunobiologie, CNRS URA 359, Institut Pasteur, Paris, France.
Scand J Immunol. 1994 Jan;39(1):79-87. doi: 10.1111/j.1365-3083.1994.tb03343.x.
This paper describes a procedure for analysing multiple antibody reactivities that explores a commercially available immunoblot system, and is based on a double staining of nitrocellulose membranes, revealing both antibody reactivities and the migration position of the blotted proteins in the membrane. Quantification of both stainings by densitometry allowed the accurate superposition of the immunoreactivity and total protein profiles of each lane. Moreover, the protein stainings of the different lanes could be adjusted with a simple-scale transformation algorithm, correcting for possible distortions during electrophoretic migration, and allowing for the precise comparison of the immunoreactivity profiles in different lanes. The procedure is discriminatory enough to identify unique reactivity patterns in random pools of 10(4) activated B cells, and to define strain-specific natural antibody repertoires. The utility of this immunoblot method as an assay for simultaneously scoring multiple reactivities to hundreds of antigens in complex mixtures of antibodies, and thus defining antibody repertoires in a global manner, is discussed.
本文描述了一种分析多种抗体反应性的方法，该方法探索了一种市售免疫印迹系统，基于硝酸纤维素膜的双重染色，揭示抗体反应性以及印迹蛋白在膜中的迁移位置。通过密度测定法对两种染色进行定量，可实现每条泳道免疫反应性和总蛋白图谱的精确叠加。此外，不同泳道的蛋白染色可用简单的比例变换算法进行调整，校正电泳迁移过程中可能出现的失真，从而能够精确比较不同泳道的免疫反应性图谱。该方法具有足够的区分能力，能够识别10⁴个活化B细胞随机库中的独特反应模式，并定义品系特异性天然抗体库。本文还讨论了这种免疫印迹方法作为一种检测复杂抗体混合物中针对数百种抗原的多种反应性、从而以全局方式定义抗体库的检测方法的实用性。
