Nobrega A, Grandien A, Haury M, Hecker L, Malanchère E, Coutinho A
Unité d'Immunobiologie, CNRS URA 1961, Institut Pasteur, Paris, France.
Eur J Immunol. 1998 Apr;28(4):1204-15. doi: 10.1002/(SICI)1521-4141(199804)28:04<1204::AID-IMMU1204>3.0.CO;2-G.
The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM-containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi-modal distribution of specific frequencies between 1/220 and 1/100,000. Cross-correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross-reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen-antibody interactions of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non-degenerate. Furthermore, reactivity patterns obtained with complex antibody mixtures correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.
本实验研究了小鼠可用抗体库中的功能性抗体多样性和克隆分布。通过对C57BL/6小鼠静止脾B细胞进行无偏倚的多克隆刺激来制备含IgM的上清液，以确保每个培养物中反应性克隆的数量相似，且所有克隆的生长和成熟程度相当。通过对同源肝脏提取物的十二烷基硫酸钠聚丙烯酰胺凝胶电泳免疫印迹进行有限稀释分析（LDA），对克隆和克隆混合物的抗体库进行定量分析，从而能够确定针对数百种印迹抗原的特异性克隆频率。结果显示，B细胞与提取物反应的克隆频率在1/220至1/100,000之间呈双峰分布。对各个上清液中不同条带反应性的交叉相关分析显示交叉反应性水平较低，这表明印迹提取物提供了一组非常多样化的抗原。对我们检测抗原-抗体相互作用的亲和力/浓度阈值的研究支持了这样一种观点，即免疫印迹上的整体抗体库分析具有高度的区分性且不会退化。此外，用复杂抗体混合物获得的反应模式与通过LDA确定的克隆反应频率相关。结果表明静止B淋巴细胞具有很大的功能多样性，这表明最小抗体库大小比先前理论预测的要高出几个数量级，并且克隆特异性大小存在广泛的异质性。
