Idiotypy has been studied in antibodies against a protein antigen: hen ovalbumin. Various fractions of antisera to hen ovalbumin have been compared from the standpoint of the idiotypic specificities found on the immunoglobulins that they contain. Idiotypic specificities were found to be comnion to (a) antibodies that were not eluted from the same immunoadsorbent by the same MgC12 concentration, but by four different concentrations. (b) Antibodies that were precipitable only by hen ovalbumin, only by hen and turkey ovalbumin, or also by duck ovalbumin. (c) Antibodies that were precipitated or not precipitated by the homologous ovalbumin, and proteins apparently devoid of antiovalbumin antibody function. These findings are discussed from the standpoint of (i) the differences in function between the various fractions with the same idiotypic specificities, and ( Idiotypy of antibodies has been defined as their property of possessing different antigenic specificities, both among antibodies of one individual against distinct antigens and among antibodies of different individuals (or -perhaps of different groups of individuals) against the same antigen (1, 2). Since the time when the first observations of this property were made, almost simultaneously for human (3) and rabbit (4) antibodies, further observations have been made in rabbits, concerning antibodies against antigens that were found to be carbohydrate in nature, when this nature was known, and also against haptenic groups (5-16).It might be inferred from the structures of proteins and polysaccharides that the variety of antigenic determinants would be greater in proteins than in polysaccharides; this might explain the frequent crossreactivity observed among phylogenetically unrelated polysaccharides, but not among proteins. It therefore seemed of interest to study also idiotypy among antiprotein antibodies. Ovalbumin was chosen as the first protein antigen to be studied, and this paper describes work designed to compare idiotypy among antibodies that have properties different from each other. MATERIALS AND METHODSPreparation of Ovalbumins and Antisera to Ovalbumin. Hen ovalbumin (hen Ov) and turkey ovalbumin (turkey Ov) were crystallized five times and three times, respectively, by salting out with (NH4)2SO4, and each crystal preparation was washed twice. Duck ovalbumin (duck Ov) was precipitated twice by 60%-saturated (NH4)2S04 and washed three times after each precipitation. The reactions of each of the three ovalbumin preparations (concentration 1-2 mg/ml) with the antisera to hen Ov in gels resulted in a single precipitation zone.Antisera to hen Ov were prepared in the rabbit by intravenous injections (4 per week during several months) of 1-6 mg of hen Ov with alum.Antiidiotypic Immunizations. For the purpose of antiidiotypic immunizations, antibodies to hen Ov were prepared from antisera to hen Ov of two rabbits (No. 663 and 765). The precipitates, in proportions close to equivalence of each of these two antisera with hen Ov, were washed three ti...

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The injection into one rabbit (with Freund's adjuvants) of a specific precipitate made with antibodies from the serum of another rabbit is usually followed by the appearance in the serum of the first rabbit of antibodies which precipitate the serum of certain rabbits but not of others. It was found that the antigen (or one of the antigens) concerned in the reaction of these anti rabbit serum antibodies with rabbit sera had an antibody function, and was therefore a protein. It was concluded that at least one serum protein antigen, the specificity of which so far has been considered to be uniform throughout the animal species, can instead be present in different individuals as different forms or allotypes with somewhat different antigenic specificities. A large number of rabbit sera were allowed to react with a large number of rabbit immune sera. The gel method of immunochemical analysis made it possible to enumerate the allotypes that took part in each reaction. In addition, the technique mainly used (simple diffusion in separate tubes) made it possible to recognize the presence of one given allotype by the mere aspect of the precipitation zone in the reaction of one suitable immune serum with any serum in which the concerned allotype occurred. Neighboring reactions of sera, in contact with each other and with the suitable immune serum, in suitable cells easily constructed in the laboratory, were carried out occasionally and, each time, their results agreed with the previous identification. The analysis of the reactions in tubes lead to a list of seven allotypes designated by a, b, c, d, e, f, and g, of which two or more (e not included) were contained in almost every serum. The specific conditions necessary for antibody formation against an allotype are its absence from the serum of the immunized animal and, except in the case of cross-reactivity, its presence in the immunizing material. When these necessary conditions are fulfilled for several allotypes at the same time, their competition in the immunization seems to favor the allotype present at the highest concentration. The individuality of six of the listed allotypes has been discussed independently of the part of their specificity that may be common to all the allotypes of one given protein antigen in all the individuals of the same animal species. A cross-reaction of the anti f rabbit antibodies with allotype g has been observed. When two allotypic specificities were detected in one serum, attempts were made to find whether they were carried by two allotypes, i.e. by two distinct kinds of molecules, instead of being the manifestation of two "allotypic patterns" present on the same molecules. The presence of several allotypes in the immune sera made it often impossible to find definitive answers in this regard. However, for a limited number of cases of two allotypic specificities present in one serum, it could be demonstrated that at least a large proportion (if not the totality) of the two allotypes were independent of each other. No sign of a systematic coexistence of two allotypic patterns on the same molecules has been observed to date.

The genetics of immunoglobulins (Ig) has been discussed from the standpoint of the determinism of the antigenic specificities of different kinds that they carry: isotypic specificities, which are uniform in all indviduals of the same animal species; allotypic specificities, which are not the same in all individuals, but are uniform within groups of individuals; idiotypic specificities, each of which not only is particular to the antibody against one given antigen but is, in addition, variable with individuals.Allotypic variants of the Ig are mainly these: in the rabbit, of the L polypeptide chains ( a b group) and of the IgG H chains (aa group); in man, of the L chains (Inv system) and of the IgG H chains (Gm system); in the mouse, of the IgG and IgA H chains. The control of the allotypic variants of the IgG H chains is mediated in man and mouse by alleles at several closely linked loci which were distinguished, with the help of myeloma proteins, because of differences in isotypic speci6cities. In the mouse, these loci are closely linked also to the locus which controls the IgA H chains. Several linked loci are also responsible for the IgG H chains of rabbits, and nearly certainly for their L chains.The cellular aspect of the genetics of immunoglobulins has been reviewed with the main conclusion that each cell seems to be specialized in the synthesis of Ig made of H and L chains of only one allotypic kind. The number of cells specialized in the synthesis of one kind of molecules seems to be proportional to the concentration of these molecules in serum.

In the preceding paper (1), an answer has been given to a few of the questions raised by the idiotypy of rabbit immunoglobulins (2) with the following conclusions: that each idiotypic pattern detected in antisera against Salmonella typhi was carried by antibodies; that the antibodies of one rabbit against antigenic materials other than S. typhi did not carry the idiotypic patterns of the anti-S, typhi antibodies, or even carried definitely different idiotypic patterns; that the idiotypic specificities of the antibodies of different individuals against S. typhi were definitely different without any sign of hereditary transmission that could be comparable to that of allotypic specificifies.In this paper, the comparison of the idiotypic specificities will be restricted to antibodies elaborated against S. typhi by the same individuals. The antibodies to be compared will differ from each other: (a) by the stage of the anti-S. typhi immunization at which they had been collected; (3) by the immunoglobulin class to which they belong in one given serum sample; (c) by their ability or inability to be precipitated by the polysaccharide of S. typhi.The discussion of the results of the preceding paper, on the basis of fairly simple postulates, led us to consider comparatively the antibody heterogeneity involving differences in antibody functions or idiotypic specificities, and the cellular heterogeneity to which the antibody heterogeneity may be logically assumed to be correlated. This discussion will be resumed in the present paper and applied to the results obtained as answers to the questions mentioned above. Materials and MethodsThe antibacterial and anti-idiotypic immunizations have been described in the preceding paper (1) in which the immunizing and anti-idiotypic rabbits are listed in Table I. The techniques of immunochemical analysis (double diffusion in cells or in tubes) and of immunoelectrophoretic analysis in agarose were used as described in references (3 and 4).