Molinaro G A, Maron E, Dray S
Proc Natl Acad Sci U S A. 1974 Apr;71(4):1229-33. doi: 10.1073/pnas.71.4.1229.
A reverse hemolytic plaque-forming cell (PFC) assay for the enumeration of immunoglobulin-(Ig)secreting cells in nonimmunized rabbits was developed by using erythrocytes coated with anti-Ig antibody. These indicator cells lysed after reacting with the secreted Ig antigen and complement. The anti-Ig antibodies used were specific for the b4 or b5 allotypic specificities, which are antigenic determinants on the kappa chain of rabbit Ig and controlled by alleles at the b locus. We coated erythrocytes with anti-b4 or anti-b5 antibody, using hybrid antibody with dual specificity to sheep red blood cells and to the b4 or b5 kappa chain. The coated erythrocytes were plated with cells from various lymphoid organs of nonimmunized rabbits. These lymphoid cells formed allotype-specific plaques, i.e., the cells of b4 homozygous rabbits formed hemolytic plaques with anti-b4 but not with anti-b5-coated erythrocytes and vice versa. In eight nonimmunized rabbits, 0.06-0.3% of the spleen cells (590-2900 PFC per 10(6) cells) secreted the b locus Ig allotypes. In three nonimmunized rabbits, 320-590 PFC per 10(6) cells were counted in the lymph node or bone marrow; 26-40 PFC per 10(6) cells were detected in the thymus. Since the b4 and b5 specificities are present on approximately 90% of the serum Ig, our results indicate that less than 1% of the lymphoid cells in nonimmunized rabbits secrete Ig at any one time. By this reverse PFC assay, Ig-secreting cells were for the first time detected as antigen-secreting cells. This assay can be applied to the detection of cells secreting other antigens.
通过使用包被有抗免疫球蛋白（Ig）抗体的红细胞，建立了一种用于计数未免疫兔中分泌Ig细胞的反向溶血空斑形成细胞（PFC）检测法。这些指示细胞在与分泌的Ig抗原及补体反应后会发生裂解。所使用的抗Ig抗体对b4或b5同种异型特异性具有特异性，b4和b5是兔Ig κ链上的抗原决定簇，由b位点的等位基因控制。我们使用对绵羊红细胞和b4或b5 κ链具有双重特异性的杂交抗体，将抗b4或抗b5抗体包被在红细胞上。将包被的红细胞与来自未免疫兔各种淋巴器官的细胞一起铺板。这些淋巴细胞形成了同种异型特异性空斑，即b4纯合兔的细胞与抗b4包被的红细胞形成溶血空斑，而不与抗b5包被的红细胞形成溶血空斑，反之亦然。在8只未免疫的兔中，0.06 - 0.3%的脾细胞（每10⁶个细胞中有590 - 2900个PFC）分泌b位点Ig同种异型。在3只未免疫的兔中，每10⁶个细胞中在淋巴结或骨髓中计数到320 - 590个PFC；在胸腺中检测到每10⁶个细胞中有26 - 40个PFC。由于b4和b5特异性存在于约90%的血清Ig中，我们的结果表明，未免疫兔中任何时候分泌Ig的淋巴细胞不到1%。通过这种反向PFC检测法，首次将分泌Ig的细胞检测为抗原分泌细胞。该检测法可应用于检测分泌其他抗原的细胞。
