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Anti-lymphocyte antibodies have been demonstrated in patients with SLE and RA by immunofluorescence. They differ from those arising through iso-immunization by being cold reactive and chiefly of the IgM class. Separate anti specificities were shown for determinants on either T or B cells.

Antilymphocyte antibodies in serum from patients with systemic lupus erythematosus (SLE), as detected by microcytotoxicity and indirect immunofluorescence, were predominantly cold reactive and of the IgM class. These IgM antibodies were most active at 4 degrees C. IgG antibodies were infrequent, and were only minimally lymphocytotoxic. Most sera were cytotoxic for autologous lymphocytes and were equally reactive with normal and SLE lymphocytes, as well as with B- and T-cell preparations. Separate T- and B-cell specificities, which appeared not to be related to HL-A determinants, were identified by differential absorption experiments. The functional significance of these antilymphocyte antibodies is discussed.

High molecular weight (19S) gamma-globulin produces passive cutaneous anaphylaxis (PCA) reactions with its specific antiserum only when it is used as intravenous antigen. Reversed PCA reactions cannot be produced when the 19S protein is injected intradermally in contradistinction to the results with 7S gamma-globulin. Antibodies of the high molecular weight class, when injected intradermally, also failed to give PCA reactions following antigen injection. Heterophile antibodies from the sera of patients with infectious mononucleosis, demonstrated to be entirely of the 19S type, gave negative reactions, while 7S heterophile antibodies from guinea pig did give reactions following intravenous injection of sheep cells. Anti-B isoagglutinins of the 19S class failed to react, while those of the 7S class of similar titer gave clear reactions following the injection of B substance. Evidence was obtained that 19S antibodies were capable of inhibiting the PCA reactions obtained with the 7S type, indicating that interaction with antigen occurred. The failure to elicit PCA reactions appeared to be due to an inability to fix to guinea pig tissues in a manner similar to that known for 7S antibodies and gamma-globulin.

The sera of certain patients with systemic lupus erythematosus contain an antibody-like substance capable of reacting with highly purified DNA preparations from widely divergent sources. Precipitin reactions have been demonstrated by double diffusion in agar and quantitative precipitin curves have been obtained. Complement was observed to be fixed in the reaction. Evidence was obtained that the serum factor possessed antigenic properties similar to those of gamma-globulins and migrated with this fraction on zone electrophoresis. The interaction of this factor with DNA exhibited certain specific characteristics which differ considerably from non-specific reactions between DNA and proteins in general. The DNA-precipitating factor appeared to be one of a number of related factors reacting with nuclear constituents of many different cells. It differed in certain respects from the "LE factor" which is responsible for the formation of "LE cells." The accumulated evidence, although not yet conclusive, favors the concept that the precipitating factor represents an antibody to DNA, and that it is one of a number of autoantibodies elicited in this disease.

Human antibodies against dextran, teichoic acid, blood group A substance, levan, tetanus toxoid, and nuclei were isolated and analyzed for their content of Gm(a), Gm(b), and Inv(a) gamma-globulin genetic factors. The majority of these antibodies contained all the genetic factors determined in the donor's whole gamma-globulin, but in many antibodies at very different concentrations. In a few instances specific factors could not be detected despite their presence in the individual's whole gamma-globulin. Different antibodies isolated from the serum of the same individual showed different relative concentrations of genetic factors. The distribution of genetic factors seen in certain isolated human antibodies appeared to approach the selective occurrence of these factors in myeloma proteins.

Seventeen of 24 sera from children who had received multiple transfusions contained agglutinating antibodies against a Gm factor absent in the individual's serum gamma globulin. Each of these agglutinators was highly specific for a single Gm factor, and all proved useful as reagents for genetic typing. The accumulated evidence indicates that they resulted from genetically foreign gamma globulin introduced by transfusion.

A wide assortment of antibodies has been analysed by qualitative and quantitative techniques for their content of group I- and group II-type gamma-globulin molecules. These included Rh antibodies, isoagglutinins, thyroglobulin antibodies, dextran antibodies, and teichoic acid antibodies. All the antibodies studied showed the presence of both groups. However, the ratio of the two varied widely in different individuals and diverged markedly from the ratio in the total gamma-globulin of the individual. Two antibodies isolated from the serum of the same individual showed different ratios for the two groups.

Certain antiserums prepared against isolated cold agglutinins demonstrate specificity for gamma M globulins with this activity and not for similar gamma M globulins lacking such activity. The cold agglutinin specific antigens fall into several major and minor groups. Their exact relation to the presumed antibody-combining sites remains to be determined.

The sera of 8 patients with acquired hemolytic anemia associated with elevated levels of cold agglutinins were studied by various procedures of zone electrophoresis. The agglutinating activity was found associated with proteins of variable mobility in the different cases. The majority represented "fast" gamma-globulins. The 4 sera with the highest titers of cold agglutinins showed distinguishable abnormal electrophoretic components. The titers correlated with the height of the abnormal components. Ultracentrifugal analysis of the electrophoretic fractions indicated that the cold agglutinins were associated with proteins having a sedimentation coefficient of approximately 19 S. The abnormal component from the serum with the highest biological activity showed almost no contamination with lower molecular weight proteins. The amount of 19 S material found correlated with the titer of agglutinating activity. The high molecular weight character of the cold agglutinins was confirmed by procedures of density gradient zone centrifugation. The biological activity sedimented with proteins of the 19 S class in all the sera including those of relatively low titer with which no abnormal electrophoretic components were observed. Dissociation of the abnormal high molecular weight components was possible by means of certain sulfhydryl compounds. This resulted in disappearance of cold agglutinin activity. Some of the cases could be classified as macroglobulinemias because of the very large content of high molecular weight components. However, the same disease picture occurred without recognizable elevation of these components. The sera of 3 patients with severe acquired hemolytic anemia of the warm type associated with warm incomplete antibodies failed to show similar abnormal electrophoretic components and the antibody activity sedimented with proteins of the 7 S class.

Two major antigenic fragments were obtained from various purified gamma-globulin preparations after papain treatment. One, the F component, had a mobility faster than the original gamma-globulin and the second, the S component, had a slower mobility. Similar F and S components were also obtained with certain homogeneous myeloma proteins which were closely related to gamma-globulin immunologically. Additional minor antigenic components were detected with certain antisera. The technique of immunoelectrophoresis was particularly useful for bringing out the different antigenic constituents obtained after papain treatment. The F and S components as well as a midfraction were isolated by chromatography on DEAE-cellulose. These were immunologically homogeneous and could be utilized to absorb F and S antibodies from various antisera. The relative amount of F and S antibodies varied in different antisera from individual rabbits immunized with whole gamma-globulin. Whole gamma-globulin was separated by zone electrophoresis into a fast migrating and a more slowly migrating fraction. Each of these gave rise to F and S components after splitting with papain. The F components of the two gamma-globulins were similar in mobility, while the S components showed marked mobility differences although antigenically they were very similar. The mobility differences of the parent gamma-globulin appeared to be primarily related to the differences in the S component. Certain antisera against pathological gamma-globulins were found to give double lines with a wide variety of gamma-globulin preparations in agar diffusion. These were shown to be related to the F and S antigenic determinants of gamma-glubulin. This relationship was evident by a number of procedures utilizing both Ouchterlony plate techniques and immunoelectrophoresis. The question of whether these findings indicate heterogeneity of gamma-globulin in relation to the F and S antigenic components, or whether different antigenic groups on one molecule can give rise to separate lines in certain instances, is discussed.

The amino-terminal amino-acid sequence has been determined for the kappa light chains of nine IgM cold agglutinins with specificity for blood group-related carbohydrate antigens. Six of the nine sequences corresponded to the prototype VK(III) subgroup pattern, two to that of subgroup VK(II), and only one to the VK(I) subgroup. This distribution of kappa subgroups differs markedly from that of normal and myeloma proteins sequenced to date, where the VK(I) subgroup is more prevalent than either VK(II) or VK(III). Evidence is presented that supports the conclusion that the unusual subgroup distribution relates to the antibody property itself and not to some other attribute of these molecules.

The composition of various isolated antibodies was determined by quantitative analyses for heavy chain subgroups and light chain types. Certain antibodies such as anti-tetanus toxoid and anti-A isoagglutinins were predominantly of the major gammaG1-type. However, a high preponderance of molecules of the minor gammaG2-subgroup was found for antibodies to dextran, levan, and teichoic acid. These findings explain some unusual features previously noted for anti-dextrans such as weak PCA reactions and lack of Gm antigens. Studies of several isolated antibodies from single heterozygous individuals showed a selective absence of genetic markers in certain antibodies and their presence in others. The "allelic exclusion" principle was clearly evident in the isolated antibodies of two different individuals. Large differences in the ratio of kappa to lambda light chains were observed for the same type of antibody from different individuals. Subfractionation of dextran antibodies by affinity for specific glycosidic linkage or combining site size produced marked changes in the ratios. The isomaltohexaose eluates of the dextran antibodies from two subjects were primarily kappa and the isomaltotriose eluates were predominantly lambda. The one anti-levan antibody studied was uniquely homogeneous, consisting exclusively of gammaG2-heavy chains and kappa light chains. By these criteria as well as others, it closely resembled myeloma proteins.

The effect of highly purified rheumatoid factor on the precipitin reactions of various antigen-antibody systems was determined. The amount of nitrogen precipitated was increased over a broad range when the factor was added to ovalbumin, human albumin, or human gamma globulin, and the corresponding rabbit antibodies. In the zone of antigen excess, soluble antigen-antibody complexes were precipitated by rheumatoid factor. Soluble aggregates of human and rabbit gamma globulin, produced by heating at 63 degrees C., treatment with urea plus mercaptoethanol or treatment with guanidine, also precipitated with rheumatoid factor. Ultracentrifugal analysis of dissolved specific precipitates showed the presence of aggregated gamma globulin. The sedimentation rate of reactive aggregates was greater than 20 S, and concentrated preparations free of the non-reactive 7 S gamma globulin could be prepared by various procedures of zone centrifugation. These aggregates showed a high inhibitory capacity in the sensitized sheep cell agglutination reaction. Solid gamma globulin, prepared by heat denaturation, also selectively adsorbed the rheumatoid factor, and removed or decreased the activity in the various precipitation and agglutination reactions. Elution of highly purified active preparations from the solid gamma globulin could be carried out with urea or acid buffers. Evidence for interaction between rheumatoid factor and low molecular weight gamma globulin without precipitation, was also obtained. This interaction appears to occur in the circulation of patients with rheumatoid arthritis. The question of whether the rheumatoid factor represents an antibody to gamma globulin was discussed. Points of similarity to the behavior of complement also were cited.

Certain individuals with hypergammaglobulinemic purpura contain in their serum anti gamma globulins of restricted heterogeneity. These antibodies, characteristically of the gammaG type, were isolated from two unrelated individuals and their light polypeptide chains were subjected to amino acid sequence analysis. Although these anti gamma globulin antibodies conform to the basic VK(1) sequence, substitutions not previously encountered were found in five positions. The two antibodies were identical with each other up to residue 40 from the N-terminus of the light chains.