Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V‐genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V‐genes were shuffled at random and cloned for display as single‐chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V‐genes, with high specificities of binding to human self‐antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)‐10(6) M‐1s‐1, k(off) = 10(−2)s‐1 and Ka = 10(7) M‐1). The kinetics of association are typical of known Ab‐protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.

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To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.

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The agglutination patterns have been established for the reaction between 29 monoclonal antibodies with specificity for the Rh antigen D and red cells of D categories IIIa, IIIc, IVa, IVb, Va, Vc, VI and VII, which are known to lack certain epitopes on the D polypeptide. Six different agglutination patterns were recognized and interpreted to indicate the recognition of seven different epitopes. These epitopes are termed epD1 through to epD7. The separate existence of epD6 and epD7 is deduced from previous observations in inhibition studies using purified 125I-labelled antibodies; they cannot yet be distinguished in agglutination tests. The number of epitopes lacking from cells of each category varied between two and five. As all the antibodies agglutinated cells of categories IIIa, IIIc and VII and cells of categories II, IIIb and Vb were not available, it is probable that there are epitopes other than the seven presently recognized. Eighteen out of the 29 antibodies which were examined recognized epitopes epD6/7 and it is suggested either that antibodies recognizing these epitopes predominate in polyclonal anti-D sera, or that the lymphocytes producing these antibodies are preferentially selected during establishment of cell lines.