Robert W. Rosenstein

The location and expression of idiotypic determinants in the immunoglobulin variable region--II. Chain location of variable region determinants.

Abstract Both myeloma protein 460 and myeloma protein 315 each exhibit two sets of idiotypic determinants. A ‘private’ determinant set which distinguishes protein 460 and protein 315 from each other, and a common or ‘public’ set of idiotypic determinants which are shared by proteins 315 and 460 and which distinguish these two proteins from many other mouse IgA myeloma proteins. One other mouse myeloma protein which shares the ‘public’ idiotype is myeloma protein S.23 which has heavy chain amino acid sequences which resemble in part those of protein 460. Heavy-light chain recombinant molecules of these three proteins were made. The amino acids constituting both the public and private idiotypic markers were located on the heavy chains. The public idiotypic is expressed in recombinatant molecules no matter which light chains were associated with the heavy chains. Antigenic expression of private idiotypes, however, depended on the type of light chain which was present. Modulation of antigenic expression of structural idiotypic determinants by associated chains must be considered when idiotypic determinants are used as genetic markers.

The distance between the contact sites for DNP and menadione ligands in the combining region of myeloma proteins binding both haptens--II. Estimation of distance using haptenic probes with variable length spacers.

Abstract Proteins 315 and 460 are IgA 2 mouse myeloma immunoglobulins which bind the haptens 2,4-Dnp and Menadione, and their derivatives competitively in the combining region. We have studied the binding of these proteins to a series of Dnp and Menadione based solid phase immunoabsorbents with different length spacer groups to which the Dnp and Menadione groups are attached, in order to determine how deep in the combining region each hapten binds. Both protein 315 and protein 460 share similar combining region dimensions with respect to the haptens used. Menadione binds in the combining regions of both proteins at a depth of 22 A, while Dnp binds at a depth of 8.8 A in protein 460 and 11 A in protein 315. The difference between these values, 11–13 A, measures the separations between the contact sites for these two haptens. In the preceding paper (pp. 929–937) calculations from energy transfer data gave an average minimum distance of 11.4 A between the contact sites of Dnp and Men in the protein 460 combining regions. Thus, there is agreement between these distances obtained by two independent experimental methods.

The location and expression of idiotypic determinants in the immunoglobulin variable region—III: Expression of the protein 315 and 460 idiotypic determinants in mouse anti-Dnp antibodies

Abstract Sera directed against the public and private idiotypic determinants of protein 460 and protein 315 were used to detect the presence of these idiotypic markers in serum immunoglobulins of various inbred mouse strains. Private protein 315 determinants were not detected in any of the mouse strains tested. Presence of protein 460 private idiotype was correlated with the presence of the Ig-1a allotype, but showed no obvious correlation with any H-2 haplotype. Analysis of allotype congenic strains demonstrated that the expression of the 460 private idiotype is controlled by an allotype linked gene. This is probably a VH structural gene coding for regions which include the heavy chain binding site of 460-like anti-Dnp antibodies. Isoelectric focusing of BALB/cN mouse serum showed that the protein 460 private idiotype was present in a pI 4.4 and a pI 6.8 fraction. The pI 4.4 fraction contained IgA showing both the private and public protein 460 idiotypic markers. The pI 6.8 fraction contained IgG1 and here, only the private protein 460 idiotype was detected.

The distance between the contact sites for DNP and menadione ligands in the combining region of myeloma proteins binding both haptens—I: Estimation of distance by fluorescent energy transfer in protein 460

Abstract Protein 460 is a mouse γA 2 myeloma immunoglobulin which binds competitively two haptens, ϵ-2,4 dinitrophenyl lysine and Menadione thioglycollate at the immunoglobulin combining region. In close relation to this combining region, protein 460 contains a heavy chain cysteine residue which has been substituted with 2 fluorescent donor probes. We have measured energy transfer between the donor probe and each of the 2 haptens bound to protein 460 using the technique of nanosecond fluorimetry. The distances calculated from the energy transfer data indicate that there is a substantial spatial separation between the contact site of each of the 2 haptens. Thus, the binding region of this immunoglobulin has distinct sub-sites at which 2 structurally diverse haptens are bound.

Analysis of detergent-extracted membrane immunoglobulins of T and B lymphocytes by ultracentrifugation and column chromatography.

Abstract Lactoperoxidase-catalyzed radioiodination was used to incorporate 125I or 131I into membrane proteins of murine thymocytes, spleen cells or spleen cells from mice depleted of T cells by adult thymectomy, X-irradiation and reconstitution with T cell-depleted bone-marrow cells (ATXBM mice). Radiolabelled cells were lysed with 0.05% Triton X-100 and the lysates were fractionated by ultracentrifugation through sucrose gradients and/or chromatography in Sephadex or Sepharose gels. Membrane immunoglobulins in the fractionated lysate were detected with rabbit antiserum to murine Ig L-chains. Membrane Ig associated with thymocytes sedimented more slowly than serum IgG in sucrose gradients but had a molecular size of 180,000 or 360,000d based on column chromatography. Eighty % of M-Ig obtained from B cells of ATXBM mice sedimented in the 8–10s region of sucrose gradients and the sedimentation rate of normal spleen cell M-Ig was split into two populations sedimenting slower or faster than IgG. The results provide further support to the proposal that T cell M-Ig and B cell M-Ig are structurally distinct.

The location and expression of idiotypic determinants in the immunoglobulin variable region—I: Characterization of antibodies directed against the variable region of mouse myeloma immunoglobulins 315 and 460☆

Abstract The preparation and characterization of rabbit antibodies directed against structures associated with thehapten binding site of proteins 315 and 460 is described. That these antibodies are site-directed was shown by: (a) specific elution of anti-idiotypes by hapten; (b) the inability of these rabbit antibodies to bind to affinity labeled proteins 315 and 460; and (c) the inability of anti-constant region serum to block the association of the rabbit antibody with proteins 315 and 460. Rabbits producing anti-315 and anti-460 binding site specific antisera, which were bled at one month show strong shared reactions between the two myeloma proteins 315 and 460. Later bleeds (4 months) are essentially monospecific, with little or no shared reaction. We conclude that anti-315 and anti-460 purified by hapten elution from myeloma-Sepharose columns are anti-idiotypic-antibody populations.

Characterization of idiotopes on MOPC 315 IgA using monoclonal antiidiotypic antibodies

The isologous antiidiotypic response in BALB/c mice to immunization with the DNP-binding IgA myeloma protein, MOPC 315, alters the expression of the anti-DNP antibody repertoire and confers immunity against MOPC 315 myeloma tumors. In order to characterize the idiotopes on MOPC 315 IgA which elicit this response we have isolated four monoclonal antiidiotypic antibodies (AIA) D10 (IgG2a), A2(IgG1), G3 (IgG2b) and F1 (IgG2a), produced by splenocytes of BALB/c mice immunized with MOPC 315 IgA in three independent fusion experiments. These AIA react with MOPC 315 IgA, reassociated H315 L315 and FV31¿5 but not with free H315, L315, VH315 or VL315. In addition the AIA do not react with the closely related DNP-binding IgA myeloma protein, MOPC 460, suggesting that they are directed against private idiotopes on MOPC 315 IgA. These idiotopes can be divided into two groups. Group I, defined by D10, A2 and G3 consists of two overlapping idiotopes, one of which is related to the hapten-binding site. The two idiotopes are formed by an interaction of amino acids in H315 and L315. Group II defined by F1 consists of one idiotope which is related to the hapten-binding site. This idiotope is comprised of an amino acid sequence on H315 which requires an interaction with either L315 or L460 for expression. A2 and G3 react identically with the same idiotope but were derived from two independent fusion experiments. This indicates an identity of AIA clonotypes among individual mice and suggests that the isologous AIA response to MOPC 315 IgA is restricted.

Antibody Combining Regions

An immune serum usually consists of a very heterogeneous population of immunoglobulins (Ig’s) the appearance of which in the serum has been induced by antigen. The antigen-ligating function of the Ig molecule (see Figure 1) is confined to the combining regions, which are two symmetrical areas at the solvent-exposed ends of the Fab arms of the Y-shaped Ig molecules. The combining region is situated in the variable (V-region) domain, a compact region consisting of the N-terminal half of the light (L) chain and the N-terminal quarter of the heavy (H) chain that is linked by sulfhydryl bonds. Between the areas of this domain occupied by the L- and H-chain V regions is a cleft exposed to the solvent. Antigens have been shown to bind in, or close to, this cleft (Amzel et al., 1974). An induced antibody population is said to be specific because it usually binds most strongly to the immunizing antigen and with lesser binding energies to certain compounds that resemble the immunogen in structure. Heteroclitic antibodies may be induced that bind more strongly to some determinant other than the immunogen (Makela, 1965). In general, antibody populations show a high degree of specificity, in that they are able to discriminate among chemical compounds differing by as little as a single functional group, between stereoisomers, or between two proteins differing by as little as a single amino acid residue (Reichlin, 1974).