Relationships between liquid- and solid-phase antibody association characteristics: implications for the use of competitive ELISA techniques to map the spatial location of idiotopes

A liquid-phase assay system based on small-zone size-exclusion chromatography was used to examine the binding of a monoclonal anti-idiotopic antibody, F6, to its idiotope on the murine plasmacytoma IgA, TEPC-15. Chromatographic behavior revealed a strong association between T-15 and F6, which was previously characterized by solid-phase immunoassay as recognizing a nonbinding site epitope of the T-15. This chromatographic pattern suggests that the inability of the hapten phosphorylcholine to inhibit the anti-idiotope:idiotope relationship in solid-phase immunoassay might be equally explained by the low affinity of the hapten relative to the high affinity of the anti-idiotope antibody. Bivalent interactions between solid-phase IgA and liquid-phase IgG should enhance the binding of the anti-idiotope to an extent that would prevent the hapten from dissociating the complex. Under these solid-phase assay conditions, observation of hapten inhibition may, in some cases, indicate site specificity, but absence of inhibition cannot be interpreted. Computer simulations of solid-phase hapten inhibition scenarios were used to evaluate the qualitative nature of binding inhibition profiles expected under various conditions of liquid- and solid-phase reactant affinities and concentrations. The apparently unusual inhibition curves previously observed in the T-15:anti-T-15 studies in which the degree of binding inhibition by hapten appeared to be independent of anti-idiotope concentration may be predictable in cases of excess solid-phase epitope; the plateau inhibition value is a function of relative affinity constants and concentrations of solid-phase and inhibitor components. The results additionally suggest that the affinity of a liquid-phase antibody may modulate the effective concentration of solid-phase epitope.     

Regulation of idiotope expression. IV. Genetic linkage of two D region-dependent T15 idiotopes to the IgH allotype

The idiotopic (Id) repertoire of antibody response to phosphocholine was studied in mouse strains with different IgH allotypes. The T15 idiotype-bearing (T15+) serum antibody and antibody plaque-forming cells (PFC) were characterized with four monoclonal anti-Id that recognize distinct Id determinants on T15+ antibody encoded by VH-1 (of the S107 gene family), DH FL16.1, JH-1 and Vk22 germ-line genes. We have previously shown that expression of the Id designated AB1-2 and B36-82 depends on the third hypervariable loop (D region), whereas the other Id, MaId5-4 and B24-44, are influenced by VH structures outside of the D region. All four Id were expressed in the PC-response of all mouse strains tested, except the Ighj strains (C3H/HeJ, CBA/H-T6, PL/j), where the D region-dependent Id, AB1-2 and B36-82, were absent. The other Id, however, were normally expressed on individual PFC as well as the serum antibody of the Ighj strains. Expression of AB1-2 and B36-82 on 50% of PFC occurred in (BALB/c-Igha x C3H/HeJ-Ighj)F1 mice. The absence of Id correlated with a unique RFLP of the S107 gene family in Ighj strains. Finally, Id expression segregated with the appropriate RFLP pattern in individual (BALB/c x C3H/HeJ)F2 mice. These data demonstrate a selective genetic linkage of discrete T15 Id determinants, AB1-2 and B36-82 with the Igh allotype. By comparing these results with the available Ig sequences, we suggest that the Ighj allotype may be associated with an allelic form of the DH-FL16.1 segment which with VH-1, JH-1, and the Vk 22 code for the phosphocholine-specific antibody in the mouse.     

Regulation of idiotope expression. I. The effect of antigen dose on expression of certain T15 idiotopes during primary IgM response to S. pneumoniae R36a

Clonal heterogeneity among B cells reactive to the same epitope may be determined through differences in idiotypy. It appears that clones bearing distinct idiotopes may constitute functionally distinct subpopulations. Data suggest that idiotopically distinct clones of PC-reactive B cells may be regulated independently of one another. We have looked to see whether individual T15+ clones may also differ in their requirements for activation. Here we examine the effect of immunizing doses of antigen on expression of two T15 idiotopes, B36-82 and B39-38, during both in vivo and in vitro primary responses to Streptococcus pneumoniae R36a (Pn) in CB-20 mouse strain. The idiotopes were detected on the specific antibody plaque-forming cells (PFC) by using monoclonal anti-idiotopic antibodies. We find that distinct patterns of idiotope expression are generated by stimulation with different doses of antigen. Immunization with suboptimal and super-optimal doses of Pn produced responses dominated by PFC expressing both idiotopes, whereas PFC induced by optimal antigen concentrations were primarily B36-82+ and B39-38-. These data indicate that the varying of antigen concentration may induce the response of different B cells bearing distinct idiotypes.     

Structural characterization of idiotopes by using antibody variants generated by site-directed mutagenesis

Four anti-idiotopic mAB, 107, MB, AI, and AD8, react with mouse hybridoma protein 36-65 specific for the hapten p-azophenylarsonate. The four antiidiotypic antibodies do not react with hybridoma protein 36-71, a somatically mutated variant of 36-65 whose H and L chain V region sequence differs at 19 amino acid positions. To determine which regions of 36-65 are important for the interaction with each of the four anti-idiotypic antibodies, variants of 36-65 containing one or more of the 36-71 substitutions were generated by oligonucleotide-directed mutagenesis of the rearranged 36-65 H chain V region gene, followed by expression of mutant proteins containing either the 36-65 or the 36-71 L chain in transfected hybridoma cells. Idiotypic characterization of the mutant proteins showed that reactivity correlates with the 36-65 H chain, but some contributions from the 36-65 L chain come into play. In the 36-65 H chain V region, idiotopes were mapped to the first and third complementarity-determining regions for anti-idiotypic antibodies 107, MB, and AI, and to all three complementarity-determining regions for anti-idiotypic antibody AD8. The binding of all four anti-idiotypic antibodies to hybridoma protein 36-65 was hapten inhibitable. However, a comparison between the effect of individual 36-71 substitutions on idiotope expression and their effect on Ag-binding affinity suggests that none of the four anti-idiotypic antibodies bodies mimics the structure of Ag.     

Homologous monoclonal antibodies induce idiotope-specific suppression in neonates through maternal influence and in adults exposed during fetal and neonatal life

The fine specificity of idiotype suppression induced early in ontogeny was investigated in the murine A/J anti-azophenylarsonate (Ar) response. Suppression was induced with two hapten-inhibitable, homologous monoclonal anti-idiotopic antibodies, AI and MB, that recognize partially overlapping sets of Ar-immune antibodies. Suppression was found to be idiotope-specific when adult mice were exposed to anti-idiotope as neonates; suppression was also idiotope specific when adult mice were exposed to anti-idiotope during fetal (through maternal inoculation) and neonatal life. Of particular interest, anti-idiotope, administered maternally, induced suppression in offspring first immunized with Ar as neonates, and this suppression was idiotope specific too. Thus, AI and MB induce idiotope-specific suppression in mice exposed to anti-idiotope early in ontogeny. These results parallel previous findings in adult mice and suggest that the mechanism of suppression in very young mice is the same as that in adults.     

Regulation of an idiotype+ B cell lymphoma. Effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion

The B cell surface Ig molecule plays an important regulatory role in delivering inductive/tolerogenic signals to the cell. In this paper, the effect of Ag and anti-idiotopic antibodies on the in vitro proliferation and Ig secretion of a B cell tumor was studied. The tumor (BCL1), which had been transfected with the TEPC-15 VH and VL Ig genes, expresses surface Ig and secretes antibody that binds the hapten phosphorylcholine. We found that Ag (C polysaccharide and phosphorylcholine carrier Ag) and two different anti-idiotopic antibodies, in the absence of T cells, all inhibited the proliferation of the T15+ transfectant cell line. The anti-idiotopic antibodies, but not Ag, also inhibited the secretion of T15 Ig by this cell line, suggesting different functional roles for Ag vs anti-Id in the regulation of B cell inactivation. The inhibition of secretion and proliferation appears to be cell cycle phase related. In addition, mouse rIL-4 could override the inhibition of proliferation induced in these studies. These phenomena, demonstrating that binding of surface Ig can result in the transduction of negative growth signals to a B cell tumor, can be viewed as a manifestation of immunologic tolerance. These findings collectively demonstrate that Ag and anti-Id mediate different signals to B cells via interaction with the surface Ig. Because of the monoclonal nature of the T15 transfectant and the anti-idiotypic antibodies, this system can be used to investigate the underlying molecular reactions involved in the B cell response and induction of tolerance.     

Biological activities of rabbit antibodies against synthetic human thyrotropin receptor peptides representing thyrotropin binding regions.

Recently, we have shown that the thyrotropin (TSH) binding regions of human thyrotropin receptor (TSHR) reside in two areas within residues 12-44 and 308-344. Serial antisera were raised against four overlapping synthetic peptides representing these two regions of TSHR (peptides 12-30, 24-44, 308-328, and 324-344) and were investigated for their ability to stimulate or block the cultured porcine thyroid cells. In addition, serum concentrations of triiodothyronine (T3) and thyroxine (T4) in serial sera obtained from each rabbit were examined. It was shown that residues of 12-30 and 324-344 of TSHR, respectively, are the site (at least a part of the site) where stimulating (TSAb) and blocking type (TSBAb) immunoglobulins are directed.     

The structure of an antigenic determinant in a protein

The immunogenic and antigenic determinants of a synthetic peptide and the corresponding antigenic determinants in the parent protein have been elucidated. Four determinants have been defined by reactivity of a large panel of antipeptide monoclonal antibodies with short, overlapping peptides (7-28 amino acids), the immunizing peptide (36 amino acids), and the intact parent protein (the influenza virus hemagglutinin, HA). The majority of the antipeptide antibodies that also react strongly with the intact protein recognize one specific nine amino acid sequence. This immunodominant peptide determinant is located in the subunit interface in the HA trimeric structure. The relative inaccessibility of this site implies that antibody binding to the protein is to a more unfolded HA conformation. This antigenic determinant differs from those previously described for the hemagglutinin and clearly demonstrates the ability of synthetic peptides to generate antibodies that interact with regions of the protein not immunogenic or generally accessible when the protein is the immunogen.     

Idiotypic determinants on human anti-insulin antibodies are cyclically expressed

Insulin-treated diabetics develop a heterogeneous antibody response to the protein hormone. To understand the repertoire of this diverse immune response, we developed monoclonal antibodies (Mab) that recognize idiotypic determinants on anti-insulin antibodies. Two Mab that recognize distinct idiotopes on anti-insulin antibodies were studied in detail. These idiotopes were present on IgG, but not IgM, anti-insulin antibodies from the proband and from four of 11 insulin-treated diabetics. The determinants recognized were distinct from conventional allotype (Gm) markers and were found in both Type I and Type II diabetics. The partial inhibition of anti-idiotope binding by beef insulin suggests that idiotopes related to the binding site for beef insulin may be recognized. When the presence of idiotope-positive molecules was determined sequentially during insulin therapy, in one patient, idiotope expression was found to be cyclical. In contrast, the total amount of anti-insulin antibody was constant, whereas idiotope expression varied. These data suggest that common V region determinants are employed in the anti-insulin response, and their variable expression may reflect dynamic regulation during chronic insulin therapy.     

Binding of phosphorylcholine by non-immunoglobulin molecules on mouse B cells

Phosphorylcholine (PC), a molecule found in the cell wall of most serotypes of pneumococcus, has been used extensively as a probe for the study of network interactions during immune responses. The frequency of B lymphocytes capable of interacting with PC has not been directly examined. We used immunofluorescence to study the binding of PC and monoclonal anti-TEPC15 anti-idiotopic antibodies to murine lymphocytes. In addition to identifying PC-specific Ig molecules, PC was bound by a non-Ig molecule on the surface of a relatively large subset of B cells; this non-Ig marker shared an idiotypic determinant with the PC-binding myeloma protein HOPC8 (H8). PC-bearing R36a pneumococci bind to a similar subset of lymphocytes. This binding is inhibited specifically by PC coupled to bovine serum albumin and also by a monoclonal anti-H8 antibody. We suggest that bacterial interaction with B cells through non-Ig molecules capable of binding a dominant antigen like PC may possess functional significance, possibly during the events that lead to antibody induction by these microorganisms.     

Idiotype vaccines against human T cell acute lymphoblastic leukemia. I. Generation and characterization of biologically active monoclonal anti-idiotopes

A murine monoclonal anti-tumor antibody termed SN2 (Ab1), isotype IgG1-kappa, that defines a unique human T cell leukemia-associated cell-surface glycoprotein, gp37 (m.w. 37,000), was used to generate monoclonal anti-idiotype antibodies (Ab2) in syngeneic BALB/c mice. The Ab2 were screened on the basis of their binding to the F(ab')2 fragments of SN2 and not to the F(ab')2 of pooled normal BALB/c mice sera IgG1 or to an unrelated BALB/c monoclonal antibody of the same isotype. Fifteen Ab2, obtained from two fusions, were specific for the SN2 idiotope and not against isotype or allotype determinants. To find out whether these Ab2 are directed against the paratope of SN2, the binding of radiolabeled SN2 to leukemic MOLT-4 and JM cells which contain gp37 as a surface constituent was studied in the presence of these anti-idiotopes. Clone 4EA2 inhibited the binding 100% at a concentration of 50 ng and 4DC6 inhibited 90% at a concentration of 250 ng. A third clone 4DD6 gave about 50% inhibition. Similar was the inhibition of SN2 binding to insolubilized MOLT-4 antigen or cell membrane preparation. The binding of SN2 (Ab1) to 4EA2 and 4DC6 was also inhibited by semipurified preparation of gp37 antigen. These results demonstrate that at least two of the anti-idiotope antibodies are binding either at or near the binding site idiotope of SN2. Next, the purified Ab2 was used to immunize syngeneic mice to induce antibody binding to MOLT-4 cells or gp37. Sera from mice immunized with 4EA2 and 4DC6 coupled to keyhole limpet hemocyanin contained antibodies which bind to semipurified gp37 antigen and MOLT-4 cells. Immune sera inhibited the binding of iodinated Ab2 and Ab1 indicating that an anti-anti-idiotopic antibody (Ab3) in mice shares idiotopes with Ab1 (SN2). Also, the binding of iodinated Ab2 to Ab1 was inhibited by rabbit antisera specific for gp37. Collectively, these data suggest that anti-idiotype antibodies 4EA2 and 4DC6 may be useful in the generation of idiotype vaccines against human T cell leukemia.     

Restricted antigenicity of thyroxyls in human thyroglobulin.

Interactions between two human iodothyronine-binding autoantisera and three preparations of human thyroglobulin (Tg) were not proportional to the latter's thyroxyl residue content. Probably only one of the several thyroxyl-containing sites in Tg reacted with the immunoglobulins from both antisera. In the case of one of the antisera, which was thyroxine (T4)-specific, the thyroxyl residue was the immunodominant feature of the antigenic site. The other antiserum, which had a specificity for 3,5,3'-tri-iodothyronine (T3), recognized different determinants around the same thyroxyl residue, but this residue was not itself an important element of the binding site. Thus, despite the specificity for T3 free in solution, the presence of T4 in the complete antigenic site was tolerated, since other structures supplied the bulk of the binding energy. 'Specificity' of this antiserum for T3 in solution is therefore coincidental and need not be ascribed to the presence of T3 in the original immunogen. Some results obtained in these studies may be interpreted as supporting the possibility that a modified Tg was the immunogen for the generation of these naturally occurring human antisera.     

Paratope determination of the antithrombotic antibody 82D6A3 based on the crystal structure of its complex with the von Willebrand factor A3-domain

The antithrombotic monoclonal antibody 82D6A3 is directed against amino acids Arg-963, Pro-981, Asp-1009, Arg-1016, Ser-1020, Met-1022, and His-1023 of the von Willebrand factor A3-domain (Vanhoorelbeke, K., Depraetere, H., Romijn, R. A., Huizinga, E., De Maeyer, M., and Deckmyn, H. (2003) J. Biol. Chem. 278, 37815-37821). By this, it potently inhibits the interaction of von Willebrand factor to collagens, which is a prerequisite for blood platelet adhesion to the injured vessel wall at sites of high shear. To fully understand the mode of action of 82D6A3 at the molecular level, we resolved its crystal structure in complex with the A3-domain and fine mapped its paratope by construction and characterization of 13 mutants. The paratope predominantly consists of two short sequences in the heavy chain CDR1 (Asn-31 and Tyr-32) and CDR3 (Asp-99, Pro-101, Tyr-102 and Tyr-103), forming one patch on the surface of the antibody. Trp-50 of the heavy and His-49 of the light chain, both situated adjacent to the patch, play ancillary roles in antigen binding. The crystal structure furthermore confirms the epitope location, which largely overlaps with the collagen binding site deduced from mutagenesis of the A3-domain (Romijn, R. A., Westein, E., Bouma, B., Schiphorst, M. E., Sixma, J. J., Lenting, P. J., and Huizinga, E. G. (2003) J. Biol. Chem. 278, 15035-15039). We herewith further consolidate the location of the collagen binding site and reveal that the potent action of the antibody is due to direct competition for the same interaction site. This information allows the design of a paratope-mimicking peptide with antithrombotic properties.     

The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes

Two murine mAb have been generated with a reactivity toward a 30,000 m.w. DNA binding protein found on the cell surface of human leukocytes; mAb 12A has an IgG1/k isotype, and mAb 24T has an IgG2b/k isotype. Both react with the DNA binding domain or adjacent region of the putative DNA receptor and inhibit the binding of [3H]DNA to PBMC at concentrations as low as 100 ng/ml. Stoichiometric studies indicate that both mAb react with monocytes and T cells with a kDa of 10(-7) M; about 0.5 x 10(6) molecules bind per cell at saturation. Flow cytometry indicated that 67% of lymphocytes and 98% of monocytes bore the DNA receptor. Dual labeling studies showed that 90% of B cells and 50% of T cells express the receptor; 50% of CD4+ T cells are receptor positive. Immunomatrices constructed with both mAb 12A and 24T allowed the receptor to be purified to a high degree of purity. A single protein of Mr 30,000 was readily observed after SDS-PAGE and silver staining of the gel; after electropheretic transfer of nitrocellulose this protein was shown to be a DNA binding molecule by use of a probe of biotin labeled DNA. These experiments provide further evidence to support the existence of a specific DNA receptor on human leukocytes; the availability of mAb to the receptor should be useful in its further characterization.     

Regulation of idiotope expression. III. H-2 influences the magnitude and the idiotypy of a T-independent antibody response in mice of certain genetic backgrounds

Antibody response to the phosphocholine (PC) epitope on Streptococcus pneumoniae R36a (Pn), a T-independent Ag type 2, was studied in H-2 congenic mouse strains. The PC-specific antibody plaque-forming cells (PFC) were enumerated in the spleen at various intervals after the primary Pn injection, and the proportion of PFC that produced antibody expressing the AB1-2 idiotope (Id) was determined by using the corresponding monoclonal anti-Id. AB1-2 is a cross-reactive Id, detectable on germline-encoded PC antibody of the T15 family, and on most, but not all, somatic variants of that antibody. The specific PFC responses in BALB/c (H-2d) and BALB.B (H-2b) strains were of comparable magnitude and most, if not all, PFC were ABl-1 Id-positive (AB1-2+). This was not the case in the responses of the B10D2 (H-2d) vs C57BL/10 (H-2b) strains and the D1.C (H-2d) vs D1.LP (H-2b) strains (on DBA/1 background). In each of these pairs, the H-2d mice were high responders, and the response was dominated by AB1-2 Id (greater than or equal to 80% AB1-2+ PFC at the peak, on day 5). The H-2b mice were low responders, and only a minor proportion of PFC (less than or equal to 30%) were AB1-2+; an increase of AB1-2+ was seen later in the response (d.10). The results of PFC assays were confirmed by measuring the PC-binding antibody and AB1-2 Id in the sera of D1.C and D1.LP mice immunized repeatedly with Pn. Moreover, D1.LP mice that had very low levels of AB1-2 Id had higher serum levels of antibody expressing two other T15 Id, B36-82, and B24-44. The B36-82 and B24-44 Id have been previously found on somatic variants of PC antibody expressed independently of the Ab1-2 Id. The concentrations of these two Id in D1.LP mice after repeated immunization approached those in D1.C. These results indicate that 1) the H-2 allelism may have a significant effect on TID antibody response in mice of a certain genetic background, but not in the BALB/c; and 2) the idiotypic repertoire of the response may be influenced by H-2 at the level of clonal variants of PC-reactive cells.     

Identification of discontinuous antigenic determinants on proteins based on shape complementarities

Diverse procedures for identifying antigenic determinants on proteins have been developed, including experimental as well as computational approaches. However, most of these techniques focus on continuous epitopes, whereas fast and reliable identification and verification of discontinuous epitopes remains barely amenable. In this paper, we describe a computational workflow for the detection of discontinuous epitopes on proteins. The workflow uses a given protein 3D structure as input, and combines a per residue solvent accessibility constraint with epitope to paratope shape complementarity measures and binding energies for assigning antigenic determinants in the conformational context. We have developed the procedure on a given set of 26 antigen-antibody complexes with a known structure, and have further expanded the available paratope shapes by generating a virtual paratope library in order to improve the screening for candidate residues constituting discontinuous epitopes. Applying the workflow on the 26 given antigens with known discontinuous epitopes resulted in the correct identification of the spatial proximity of 12 antigen-antibody interaction sites. Combining solvent accessibility, shape complementarity and binding energies towards the identification of discontinuous epitopes clearly outperforms approaches solely considering accessibility and residue distance constraints.     

Detection of private and recurrent idiotopes on natural anti-tubulin antibodies by monoclonal anti-idiotopic antibodies

Two monoclonal anti-idiotopic antibodies (anti-Id) were raised in mice against a human monoclonal IgA,K displaying a monospecific anti-tubulin (anti-alpha- and anti-beta-tubulin) activity. One anti-Id (IgG,K) recognized a private idiotope, TID 3.2, present only on the IgA,K immunogen, close to or within the antigen-combining site. The other anti-Id (IgM,K) recognized a recurrent idiotope, TID 7.1, outside the paratope and present in normal human and BALB/c mouse serum, on 2 of 11 polyspecific human monoclonal immunoglobulins and on 6 of 11 murine natural monoclonal auto-antibodies exhibiting a widespread anticytoskeletal protein-binding activity. Both the idiotopes were absent on two induced anti-tubulin antibodies exhibiting a monospecific anti-alpha- and anti-beta-tubulin specificity. Utilizing competitive and additivity immunoassays, we could show that the polyspecific human and mouse anticytoskeletal antibodies tested, whether bearing the TID 7.1 Id or not, appeared to compete in variable degrees for epitopes on the tubulin molecule recognized by the monoclonal IgA,K but distinct from the epitopes recognized by the induced monospecific anti-tubulin antibodies. The high incidence of the recurrent TID 7.1 idiotope in man and mouse suggests an important physiologic and perhaps regulatory function of this idiotope. Furthermore our data suggest that a restricted family of germ-line genes, highly conserved during phylogeny, may encode for these idiotope-bearing Ig molecules.     

Shared idiotypy between phosphorylcholine-specific antibody and acetylcholinesterase detectable by a monoclonal antibody

Studies were undertaken to detect structural similarities between immunoglobulins and other proteins that bind to choline-containing ligands. Such proteins may share serologically detectable determinants that may not be predicted from the amino acid sequence alone. A monoclonal antibody was used that recognizes an idiotope near the phosphorylcholine binding site of the IgA myeloma TEPC15. This monoclonal anti-TEPC15 idiotopic antibody (anti-Id) also bound the enzyme acetylcholinesterase (AChE) as well as the nicotinic acetylcholine receptor from Torpedo californica. The anti-Id antibody also significantly decreased the AChE catalytic activity but did not affect the activity of an unrelated enzyme, horseradish peroxidase. These findings suggest that nonimmunoglobulin molecules share antigenic determinants with immunoglobulin that are associated with binding to structurally related ligands, and immune regulation may inadvertently affect the function of nonimmune systems.