Sasano M, Burton D R, Silverman G J
Sam and Rose Stein Institute for Research on Aging, University of California, San Diego, La Jolla 92093.
J Immunol. 1993 Nov 15;151(10):5822-39.
Unconventional Ag for B cells that are comparable to known superantigens for T lymphocytes have not been well characterized. However, the bacterial membrane protein, Staphylococcal protein A (SpA), has sites that interact with the Fab of many IgM, IgA, IgG, and IgE, and in recent reports we have provided evidence of VH restriction in Fab that bind SpA. To investigate the molecular basis for this Fab binding specificity, we have selected monoclonal Fab from a phage-display combinatorial Ig library, based on the ability to bind SpA. By this approach, in an unselected human polyclonal IgG Fab library, about 17% of antibody-expressing clones were found to bind SpA. SpA binding was completely restricted to Fab with VH3 H chains, and about 60% of VH3 Fab in the unselected library had SpA binding capacity. Analysis of 21 VH sequences and 6 VL sequences demonstrated that Fab that bind SpA use diverse VH3 genes, while the L chains derive from a variety of V kappa and V lambda gene families. By creation of antibodies with differential H-L chain pairing, the global capacity to bind SpA was shown to be dictated by VH3 usage, but different L chain usage could result in up to a fourfold change in binding affinity. The apparent KD of the SpA binding by different Fab ranged from 2.5 x 10(-7) to > 10(-5) M. Furthermore, repeated rounds of in vitro panning selected for antibodies based on higher binding affinity. These data indicate that the pattern of VH family restriction of Ig reactive with SpA is comparable to known superantigens, and there is a hierarchy within the binding affinities of VH3 Fab based on V gene usage.
与已知的T淋巴细胞超抗原类似的、针对B细胞的非常规抗原尚未得到充分表征。然而，细菌膜蛋白葡萄球菌蛋白A（SpA）具有与许多IgM、IgA、IgG和IgE的Fab相互作用的位点，并且在最近的报告中我们提供了与SpA结合的Fab中VH受限的证据。为了研究这种Fab结合特异性的分子基础，我们基于结合SpA的能力从噬菌体展示组合Ig文库中筛选了单克隆Fab。通过这种方法，在一个未筛选的人多克隆IgG Fab文库中，发现约17%表达抗体的克隆能结合SpA。SpA结合完全限于具有VH3重链的Fab，并且未筛选文库中约60%的VH3 Fab具有SpA结合能力。对21个VH序列和6个VL序列的分析表明，结合SpA的Fab使用多种VH3基因，而轻链来源于多种Vκ和Vλ基因家族。通过创建具有不同重链-轻链配对的抗体，发现结合SpA的总体能力由VH3的使用决定，但不同的轻链使用可导致结合亲和力高达四倍的变化。不同Fab与SpA结合的表观KD范围为2.5×10−7至>10−5 M。此外，基于更高的结合亲和力，通过多轮体外淘选筛选出抗体。这些数据表明，与SpA反应的Ig的VH家族受限模式与已知的超抗原类似，并且基于V基因的使用，VH3 Fab的结合亲和力存在等级差异。
