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Hybridomas have been produced from mice immunized with human IgG. Culture supernates were assayed for the presence of antibody-producing cells by passive haemagglutination. Hybridomas producing antibodies to human kappa (kappa) and lambda (lambda) light chains have been cloned and grown as ascitic tumours in BALB/c mice. The antigen-binding characteristics of the monoclonal antibodies, contained in the ascitic fluid, were assessed by haemagglutination inhibition, ELISA and radioimmunoassay systems and by the binding of radiolabelled antigen in analytical flat-bed iso-electric focussing gels. One monoclonal anti-kappa reacted better with free than with combined kappa chains; for another the reverse was true. Antibody fractions separated by DEAE chromatography of ascitic fluids were coupled to ox red cells with chromic chloride and compared with polyclonal antibodies for the detection of cell-surface immunoglobulins.

Complexing a human IgG lambda paraprotein with a monoclonal antibody (McAb) to a lambda-chain determinant markedly depressed the anti-lambda response of immunized mice. No anti-lambda was found in the serum after two or four injections of the complex, whereas high titres of anti-lambda chain antibody were found in the sera of mice immunized with the IgG paraprotein complexed with any single anti-gamma chain McAb or with a pool of anti-gamma chain McAbs. Responses to the highly immunogenic Fc gamma portion of the molecule were not suppressed by complexing with a single anti-gamma chain McAb and were only slightly suppressed after complexing with a pool of anti-gamma chain McAbs. Some anti-Fc gamma antibody was produced by all animals receiving a single injection of the immunogen complexed to any McAb, but free immunogen did not generate a primary response. Complexing the IgG with an anti-Fc gamma McAb enhanced the response to to the poorly immunogenic C gamma 1 domain but no anti-C-gamma 1 was produced by mice receiving the IgG complexed with an anti-C-gamma 1 McAb. In immunizations with 'free' (Bence-Jones) lambda chain, all the antibody produced was directed against 'free'-specific determinants. Complexing the Bence-Jones protein with McAbs to 'free' or 'general' determinants on the lambda chain did not enhance or suppress the response. It is concluded that the response to weakly or moderately immunogenic, but not to strongly immunogenic, regions of the molecule may be suppressed by complexing the immunogen with a McAb of appropriate specificity. Possible reasons for this result are discussed.

Following immunization with various lambda Bence-Jones proteins, over 100 hybridomas were established. A majority of the monoclonal antibodies (McAb) produced were demonstrated to recognize epitopes common to all lambda chains or unique to the immunogen. Twenty-nine McAb exhibited profiles of 'restricted' reactivity. Analysis with a panel of serologically defined antigens allowed McAb recognizing the lambda II subgroup to be identified. Other McAb recognized epitopes selectively expressed by lambda II and lambda III subgroup proteins. A further twenty-one McAb exhibited individual specificity profiles, some of which detect minor lambda chain subpopulations. Several of these McAb have potential for analysis of the clonality of specific antibody responses and for detecting monoclonal B-cell proliferations.

Immune complex formation of four different mouse monoclonal antibodies against human IgG has been studied using analytical zonal centrifugation. A theoretical model has been used to depict thermodynamic ideal immune complex formation of monoclonal antibodies. It was found that the four monoclonal antibodies differed very much with respect to immune complex formation with human IgG. One of the monoclonal antibodies formed immune complexes in agreement with the theoretical model. Another was strongly related thereto. A third monoclonal antibody formed only a single complex and might exhibit a positive co-operativity between its two sites. A fourth formed as excess of a possibly cyclic complex. Thus monoclonal antibodies differ substantially with respect to physical properties adding a new aspect to the problems of antibody heterogeneity. It is moreover found that analytical zonal centrifugation can be used to estimate the number of antigenic determinants and antibody-binding sites thereby assuring whether or not a particular IgG monoclonal antibody has two binding sites.

Immunization with intact IgG has allowed the isolation of four hybridomas producing antibodies recognizing epitopes expressed within subpopulations of human kappa light chains unrelated to known polymorphisms (Km) and previously defined V-region subgroups. The V-region-associated epitopes recognized are conformation-dependent, being expressed on intact light chain but not on isolated VK or CK fragments. The frequency of expression within paraprotein panels of different heavy chain isotypes varied between individual antibodies. An epitope recognized by B2A6, expressed by greater than 85% IgGK paraproteins, was not represented in 16 IgM paraproteins tested, suggesting that association of VK with mu chains does not result in display of the epitope recognized, or alternatively, that selective association between VK and CH gene products occurs. These data contrast with the reactivity of other McAb for CK epitopes which were reactive with isolated CK fragments, and for all kappa-bearing paraproteins, regardless of heavy chain isotypes.

Antibody-producing hybridoma clones have been isolated following immunization of mice with human IgG. Twenty-five monoclonal antibodies (nine anti-C gamma 3, fourteen anti-C gamma 2, one anit-kappa and one anti-lambda) were selected for study of their cross-reactivity with the IgG of fifteen mammalian species and chicken immunoglobulin. Each antibody exhibited a unique reaction profile suggesting that human IgG expresses a very large repertoire of immunogenic epitopes. Whilst some antibodies showed a very restricted cross-reactivity profile for others a very wide reactivity profile was observed-including two clones producing autoantibodies. Antibodies demonstrating cross-reactivity between human Fc gamma and 7S chicken immunoglobulin allow its definitive assignment as a homologue of human IgG. Four clones demonstrated specificity for bovine IgG subclass gamma 1 and gamma 2 and the degree of reactivity allows their application to qualitative and quantitative assay systems. These studies suggest new perspectives for the characterization of immunoglobulins and the standardization of anti-immunoglobulin reagents.

Thirteen monoclonal antibodies (McAbs) showing subclass-specific and subclass-restricted reactivity to human IgG have been produced by the hybridoma technique. Their specificity has been defined by passive haemagglutination (HA) and haemagglutination-inhibition (HAI) assays using a large panel pf purified IgG paraproteins of known subclass. Their use in assaying human IgG subclasses in radial immunodiffusion (RID) and in purifying human subclasses by both positive and negative immunoabsorption chromatography is illustrated.

Two monoclonal antibodies to major histocompatibility complex (MHC) class II antigens, which in combination identify beta chains encoded by the SB and DR loci, were used to investigate which of these gene products were expressed at the cell surface of unstimulated T cells and at various stages of mitogen-induced T-cell maturation. In tests on blood lymphocytes from healthy donors 12% of T cells expressed class II antigens, but only SB antigens were expressed. During activation of T lymphocytes, SB-coded antigens were expressed before DR antigens, and the kinetics of SB expression correlated with the proliferative response of T cells. These results and consideration of recent reports from other laboratories lead us to suggest that SB-coded class II antigens play a role in T-cell proliferation.

Proteolysis of lymphocyte surface membrane immunoglobulin (SIg) by trypsin, papain and pronase was investigated. Cleavage in the hinge region resulting in removal of Fab delta was distinguished from removal of the whole SIgD molecule using antisera specific for c delta l and Fc delta. Using the sensitive direct rosette assay SIgD determinants were detected after digestion under conditions previously thought to remove IgD from the membrane. By increasing digestion times is was shown that whilst papain and pronase remove the whole IgD molecule, Fc delta but not Fab delta determinants survive trypsin. The implications of the results for structural differences between membrane and serum IgD and for models of insertion of SIgD into the plasma membrane are discussed.

Anti-idiotope antibodies produced by mice immunized with a human IgG paraprotein complexed with various mouse monoclonal antibodies (McAbs) have been measured. All animals receiving more than one injection of the paraprotein (free or complexed with a mouse McAb) produced antibodies to the idiotypes of the paraprotein. Complexing with a McAb, especially an anti-Fc-gamma McAb, enhanced the response. Antibodies to the idiotopes of mouse McAbs were more difficult to produce and their production was very dependent on the mode and schedule of the immunization. The best antisera were produced by mice receiving a course of injections of pre-formed complexes of the IgG paraprotein and McAbs. Four of five mice produced antibodies to the idiotopes of an anti-light chain McAb (C4) after a course of immunization (one primary plus four boosts) of an IgG-C4 complex. Two of the six mice receiving a similar course of injections of the paraprotein complexed with an anti-gamma McAb (A55) produced high titres of antibodies to A55 idiotypes. Responses were enhanced when complexes were prepared with a pool of McAbs. It is probable that the formation of large multi-cross-linked complexes containing the McAb under study is important in generating the response. Once a response is initiated, very high titres may be achieved.

Peripheral blood monocytes, but not other leucocytes, from healthy donors, were shown to enhance the DNA synthesis of three cell lines. The effect was very marked on an epithelial (Chang) and lymphoid (CLA-4) cell line derived from normal tissues, and less marked on a carcinoma-derived line (HT-29). The enhancement was demonstrable over a wide range of monocyte:cell line ratios, and some activity was present in supernates from monocytes cultured alone or with a cell line. Furthermore, monocytes from gastric carcinoma patients did not enhance Chang and CLA-4 cells to the same extent, relative to the healthy donors.