Abstract. Previous work indicated that the light chains of a monotypic immunoglobulins G2-K and M-K from a single patient (Til) are identical. Our present data show that the monotypic immunoglobulins G and M share idiotypic determinants not present in their isolated light chains or in any of a large number of other immunoglobulins tested, and that amino acid sequences of the first 27 residues from the NH2-terminal end of the ay-and u-chains are identical. These results support the hypothesis that at least two genes control the synthesis of each heavy and light chain and suggest that the monotypic immunoglobulin G and monotypic immunoglobulin M of this patient share three of the four genes involved. It is proposed that, during normal immunoglobulin synthesis, different cells of a single clone synthesize immunoglobulins lvi and G, and that the light chains and the variable segments of the heavy chains of the proteins of the two classes are identical within the clone. A genetic switching mechanism is suggested.A patient (Til) with multiple myeloma, having two serum paraproteins (IgG2-K and IgM-K), was recently investigated by Wang et al.1 The light chains of the two monotypic proteins appeared identical by the following criteria: amino acid composition, peptide maps, electrophoretic mobility in starch gel containing urea at pH 3 and pH 8, optical rotary dispersion, and circular dichroism measurements. We have now compared these two monotypic proteins in terms of "idiotypic"2 antigefic determinants (i.e., determinants specific for a given myeloma protein') and amino-terminal amino acid sequences.Materials and Methods. The monotypic IgG and IgM used for sequence analysis were isolated as previously described.' For ase in immunization the IgG was isolated by precipitation with sodium sulfate, passage through DEAE-cellulose in 0.04 1 phosphate, pH 6.9, and further fractionation in an NaCl concentration gradient on carboxymethyl cellulose, pH 6.7. IgM was eluted from the DEAE-cellulose with 0.04 M phosphate containing 0.5 M NaCl and further purified by gel filtration on Sephadex G-200. The proteins were characterized by immunoelectrophoresis and by their capacity to react specifically in agar gel with monospecific antisera to pooled IgG or IgM. The sedimentation coefficients (S2o,,) of the isolated IgG and IgM weie 6.6 and 15.1, respectively, at a concentration of approximately 7 mg/ml.

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Idiotypic antibodies were investigated quantitatively by a method of indirect precipitation, which utilizes labeled F(ab')2 fragments of specifically purified antibenzoate antibody from the donor, anti-antibody, and an antiglobulin reagent. The contribution of allotypic and hidden determinants to these reactions was excluded. Greater fractions of an idiotypic antibody population are precipitated by this method, as compared to direct precipitation, and in two instances large proportions of idiotypic antibodies were detected in populations which failed to form precipitates by double diffusion in agar gel. The greater sensitivity of the indirect method was attributed to its capacity to detect molecules bearing a small number of antigenic determinants. Extensive studies of cross-reactions, carried out by an inhibition technique, failed to reveal any strong reactions of anti-idiotypic antibodies with heterologous antibenzoate antibody preparations, heterologous sera, or IgG, although a few weak cross-reactions were noted. One definite cross-reaction was observed by a direct binding measurement with heterologous antiserum. Antisera prepared in more than one recipient against a single donor preparation reacted with identical or overlapping subpopulations of the donor molecules. Instances in which two recipient antisera reacted with different proportions of the molecules of a single donor provided evidence for the existence of more than one idiotypic antibody population in the antibenzoate antibody of an individual rabbit.