VH gene segments represent the products of the repeated duplication and subsequent diversification of a primordial V gene element. It is widely assumed that natural selection, operating via pathogens, has played the dominant role in this process. Here, we screen some 3.7 x 10(4) C mu+ colonies of mitogen-activated B cells for the production of antibodies specific for phosphorylcholine or hen egg lysozyme and expression of the VH X-24, S107, Q52, or J558 gene families. These gene families were expressed at frequencies proportional to their genomic complexity among both unselected and antigen-specific C mu+ colonies. Thus, the capacity to encode equivalent antibody-combining sites is dispersed uniformly among VH families. This result suggests that individual VH genes have not evolved to address specific antigens.

No abstract

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two‐stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag™ octapeptide epitope system. We have analyzed two peptides: Ac‐Asp‐Tyr‐Lys‐Leu‐Gly‐Asp‐Asp‐Leu‐NH2 (peptide l), which contains several non‐core positions randomized, and Ac‐Asp‐Tyr‐Lys‐Asp‐Asp‐Asp‐Asp‐Leu‐NH2 (peptide 2), which closely corresponds to the original Flag™ sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab‐bound peptide were found to be largest for the amide groups of Asp‐1 and Asp‐6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp‐1 and Asp‐4, with the other aspartic acid residues also showing significant but smaller changes. © Munksgaard 1995.

LPS-stimulated B cells were used to generate a panel of mAb that were a random sample of the preimmune repertoire of C57BL/6 and highly autoimmune, viable motheaten mice. These mAb were tested for reactivity to a number of "self" and foreign Ag. Binding that could be detected only at nM mAb concentrations or less was considered significant. We found that a surprisingly high number of the mAb bound one or more of the Ag tested, and many mAb bound more than a single Ag. Ag-induced mAb were likewise tested and found to have greatly reduced cross-reactivities. We found no significant differences, either in frequency of Ag binding or degree of cross-reactivity, between normal and autoimmune mice. Furthermore, the frequency with which a given Ag was bound by our panel of mAb was found to be proportional to the size of the Ag. The frequency with which individual VH gene families were expressed by our panel was consistent with a stochastic usage of VH genes in the preimmune repertoire. We interpret these data as showing that the preimmune repertoire is highly cross-reactive and that the activation of autoreactive clones in autoimmune animals is due to a defect in cellular regulation rather than a difference in repertoire.

No abstract