Modulation of the murine immune response to streptococcal group A carbohydrate by immunization with monoclonal anti-idiotope

Using monoclonal anti-idiotopes with previously defined specificities for the variable (V) domain of HGAC 39, a monoclonal antibody against streptococcal group A carbohydrate (GAC), we have studied the effect of anti-idiotope on an anticarbohydrate immune response. Anti-IdI-3a and anti-IdI-3b are anti-idiotopes which recognize binding site-associated determinants, whereas anti-IdX recognizes a framework-associated determinant on the HGAC 39 V kappa domain. Each of three anti-idiotopes elicited a specific idiotope response, as measured by inhibition radioimmunoassay, in A/J and C57BL/6J mice. A single immunization with conjugated anti-IdI-3a elicited an idiotope(+), GAC-binding(+) response in C57BL/6J and (BALB/c X CBA/N)F1 male mice, but not in A/J or (CBA/N X BALB/c)F1 male, X-linked immunodeficient mice. When C57BL/6J mice immunized initially with anti-idiotope were further treated with group A vaccine, those receiving anti-IdX had the greatest increase in anti-GAC activity. Stimulation of an anticarbohydrate response with anti-idiotope may therefore be enhanced by selecting anti-idiotopes against both binding site- and framework-associated determinants.     

Serologic and topographic characterization of idiotopes on murine monoclonal anti-streptococcal group A carbohydrate antibodies

We have employed five spectrotypically distinct monoclonal anti-variable region antibodies in the definition and characterization of a set of idiotopes expressed on murine monoclonal antibodies specific for streptococcal group A carbohydrate (GAC). By evaluating which of a panel of monoclonal anti-GAC antibodies were bound by the various anti-idiotopes, we observed four distinct reactivity profiles for the five anti-idiotopes ranging from highly restricted (binding of the homologous anti-GAC monoclonal antibody only) to broadly cross-reactive (binding of 18 of the 38 IgG3 anti-GAC antibodies). With N-acetyl-D-glucosamine and soluble GAC used as haptens, this spectrum of reactivity profiles was paralleled by a gradient of susceptibility to hapten inhibition of anti-idiotope binding to idiotope. The degree of cross-reactivity exhibited by a given anti-idiotope was found to be inversely related to its susceptibility to hapten inhibition. The topographic relationships among the idiotopes, defined by the results of competitive binding assays, were suggestive of a linear idiotope map spanning the variable region from the antigen-binding site to the vicinity of the constant region. Additional data from competitive inhibition assays with isolated and recombined H and L chains from a prototype monoclonal anti-GAC antibody (HGAC 39), and from isoelectric focusing of whole or reduced and alkylated HGAC 39, suggested that one of the idiotopes was located, at least primarily, on the VL domain.     

Analysis of idiotope variability as a function of distance from the binding site for anti-streptococcal group A carbohydrate antibodies

We have extended our analysis of rat monoclonal anti-idiotopes (anti-Id) specific for previously mapped binding site-associated (distal) and less-or nonbinding site-associated (proximal) idiotopes on a murine monoclonal anti-streptococcal group A carbohydrate (GAC) antibody. By utilizing other monoclonal anti-GAC antibodies and anti-idiotypic antibodies as radiolabeled probands in both competitive and direct radioimmunoassays, we have detected previously unsuspected reactivities of some of the anti-Id. Although the anti-Id recognizing the most proximal idiotopes manifest relatively narrow ranges of binding strengths for anti-GAC antibodies, the anti-Id recognizing the most distal idiotopes display broader, more continuous distributions of binding strengths. These results suggest that mimicry of antigen structure by anti-Id might best be understood from a quantitative perspective, and that idiotopes intimately associated with binding sites display a broader range of variants than those not associated with binding sites. In addition, for one monoclonal anti-Id recognizing a distal determinant, changing the radiolabeled proband in inhibition radioimmunoassays results in dramatic changes in relative inhibitory efficacies for certain anti-GAC antibody inhibitors. This observation suggests the possibility that this anti-Id represents an example of a multispecific (polyfunctional) anti-idiotypic antibody.     

Immunological properties of a single-chain fragment of the anti-idiotypic antibody ACA125

Vaccination with anti-idiotypic antibodies has been described as a promising concept for treatment of several malignant diseases. The murine monoclonal anti-idiotypic antibody ACA125 imitates a specific epitope of the tumor-associated antigen CA125 expressed by 80% of ovarian carcinomas. In the first clinical trial it could be shown that mAb ACA125 is able to elicit anti-anti-idiotypic antibodies (Ab3) with anti-CA125 specificity in patients with advanced ovarian cancer. In order to improve the capabilities of anti-idiotype vaccines we generated a genetically engineered single-chain fragment (scFv) ACA125 composed of heavy- and light-chain variable regions connected by a flexible linker. The antigenicity of scFv ACA125 was demonstrated by immunizing rats i.p. with scFv or complete mAb in complete/incomplete Freund's adjuvants (CFA/IFA) or precipitated by aluminium hydroxide. Negative control groups included applications of irrelevant mouse IgG or adjuvants alone. Anti-anti-idiotypic antibodies (Ab3) directed against the mAb ACA125 as well as specific anti-CA125 antibodies (Ab1′) could be detected in all animals treated with scFv in CFA/IFA. Nevertheless, antibody titers were lower than when the complete mAb ACA125 was used. Suprisingly, an increase of specificity could not be observed in scFv-immunized animals, which had been expected because of the lack of heavy- and light-chain constant regions that could raise rather unspecific anti-isotypic and anti-allotypic rat anti-(mouse Ig) antibodies (RAMA). In contrast, the RAMA responses detected in these rats were even stronger than those following immunization with complete mAb ACA125. In conclusion, the anti-idiotypic scFv ACA125 alone cannot improve the immunogenic features of the corresponding mAb, but provides a useful tool for the further development of genetic vaccines. Received: 20 January 2000 / Accepted: 24 April 2000     

Polyclonal anti-idiotypic opioid receptor antibodies generated by the monoclonal beta-endorphin antibody 3-E7

Anti-idiotypic antibodies were raised in rabbits against the monoclonal beta-endorphin antibody 3-E7. These antibodies inhibit beta-endorphin binding to the 3-E7 antibody, binding of 3H-diprenorphine to solubilized opioid receptors and the binding of 125I-beta-endorphin to rat brain membranes. Exposure of NG-108CC15 hybrid cells to anti-idiotypic antibodies produces an opioid-like inhibition of PGE1-stimulated cAMP accumulation. These data suggest that the antibodies raised by the anti-idiotypic route both bind to and activate opioid receptors.     

Idiotope structure and genetic diversity in anti-streptococcal group A carbohydrate antibodies

Three cross-reactive idiotopes(Id), termed IdX, IdI-1, and Id5, that are present on free L chains from murine anti-group A streptococcal carbohydrate antibodies have been mapped; these Id distinguish between products of three homologous V kappa genes. For each determinant, sequence analysis of anti-streptococcal group A carbohydrate antibody V domains yielded small numbers of amino acids invariably associated with Id expression. Flow micro-fluorimetry was used to isolate three IdI-1- spontaneous mutants of the IdI-1+ hybridoma GAC 39; all had single amino acid changes in the L chain at position 60 and 77, all retained other Id, and all bound group A carbohydrate. Computer modeling was used to examine spatial relationships between Id. A number of the conserved Id5 and IdX residues cluster in the L chain framework region 1 around the first back loop connecting strands of the beta pleated sheets, and overlap at residue 15 (Id5, proline; IdX, leucine). This overlap accords with the mutually exclusive expression of Id5 and IdX. The IdI-1 loss variants have mutations of residues 60 or 77 on adjacent back loops, approximately 7.5 and 14 A from residue 15. Competitive inhibition of anti-IdX and anti-IdI-1 binding to antibodies expressing both Id can be attributed to steric hindrance. The framework back loops may be favored sites for cross-reactive Id expressed by products of a single V region gene. IdI-3a, an individual Id not associated with use of a particular gene segment, has been localized in part to residue 31 (hypervariable region 1) of the H chain.     

一种抗AIV共有独特型抗体具有AIV血凝素分子的内影象

用纯化的鸡抗禽流感病毒(AIV)IgG作免疫原,通过单克隆抗体技术制备出1株分泌针对鸡抗AIV和兔抗AIV共有独特型抗体的杂交瘤细胞。竞争抑制试验、特异性检验和诱导产生血凝抑制抗体的功能实验证明,此抗体具有AIV血凝素分子的内影象。     

Identification of müllerian inhibiting substance specific binding in human cell lines.

The receptor for Müllerian Inhibiting Substance (MIS), a gonadal glycoprotein hormone, has not been previously identified. Plasma membranes from MIS-sensitive human tumor cell lines (HTB-111, endometrial carcinoma; and A-431, vulvar squamous carcinoma) were detergent extracted and incubated with 125I-labeled MIS anti-idiotypic antibody, or radioiodinated human recombinant MIS (125I rhMIS), with and without unlabeled competitors. 125I anti-idiotypic MIS antibody bound to HTB-111 membrane extracts was displaceable by unlabeled anti-idiotypic antibody, but not by anti-isotypic antibody prior to cross-linking. Specific binding of the anti-idiotypic MIS antibody to endometrial carcinoma cells was verified using fluorescence activated cell analysis and fluoresceinated antibody. Furthermore, unlabeled anti-idiotypic MIS antibody competed for 125I rhMIS binding to A-431 vulvar carcinoma membranes. The labeled anti-idiotypic MIS antibody binding complex could be separated from 32P labeled EGF receptor in the A-431 membranes, indicating that EGF, a natural inhibitor of MIS activity, and MIS itself bind to different receptors. These studies demonstrate a specific, displaceable binder for MIS in the plasmalemmae of two human tumor lines. Purification of this cell surface receptor protein will be greatly aided by using the MIS anti-idiotypic antibody.     

Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

We previously described the production of four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG2a, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes [125I]iodocyanopindolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and R9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9 [k-1(+R9) = 0.025 min-1 vs. k-1(-R9) = 2.04 min-1], consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Monoclonal antibodies to streptococcal group A carbohydrate. II. The VK1GAC light chain is preferentially associated with serum IgG3

A kappa-light chain variable region (V kappa) dominantly employed in the serum antibody response of A/J mice to streptococcal group A carbohydrate (GAC) has been termed VK1GAC. Examination of in vitro recombinants between the isolated heavy and light chains of VK1GAC+ and VK1GAC-anti-GAC hybridomas and non-GAC-binding myeloma proteins indicated that two antisera (anti-Id5 and anti-Id20) recognized the VK1GAC light chain when it was free in solution or paired with several heterologous heavy chains. Screening of a panel of A/J anti-GAC monoclonal antibodies with these antisera showed almost complete concordance between Id5 and Id20 expression and the presence of VK1GAC light chain as detected by its unique isoelectric focusing spectrotype. These antisera were used to examine serum expression of the VK1GAC light chain in normal and hyperimmune serum of A/J mice. Normal A/J serum contained from 20 to 100 micrograms Id5/ml serum, whereas only 1 to 10 micrograms Id20/ml serum was detected. The levels of both VK1GAC idiotypes increased dramatically 10- to 20-fold after hyperimmunization of mice with group A vaccine. When serum IgG from normal and immune mice was fractionated into the IgG subclasses (IgG1, IgG2a, and IgG3), it was found that the VK1GAC light chain does not pair randomly with heavy chains of the IgG subclasses, but rather is associated preferentially with heavy chains of the IgG3 subclass whether or not it is associated with antibodies to GAC. These results suggest that the heavy chain pairing exhibited by this VK product may not be random.     

The idiotypic characterization of the immune response to a defined epitope of a protein antigen and the specific in vivo suppression of the immune response to this epitope by anti-idiotypic antibodies

C10, a monoclonal antibody of C3H.SW (CSW) origin, binds a decapeptide epitope of the tobacco mosaic virus protein (TMVP) representing residues 103-112 of the protein. In vivo administration of syngeneic anti-idiotypic antibodies to C10 (anti-C10) prior to immunization with TMVP suppressed the expression of antibodies to this decapeptide determinant in CSW mice without a significant reduction of the total anti-TMVP titer. The suppression could not be overcome with repeated challenges by antigen even 6 months after administration of anti-C10. Analysis of anti-C10 showed that it contains antibodies to at least two idiotopes found on C10. One of these idiotopes, C10-Idm, is found on a very small fraction of CSW anti-TMVP antibodies capable of binding the decapeptide epitope. The other idiotope, C10-IdX, is found on most of the anti-TMVP antibodies which bind the decapeptide determinant. With synthetic analogues of the decapeptide determinant, a correlation was established between the presence of the C10-IdX and the fine specificity of the decapeptide-binding antibodies. The studies reported herein demonstrate that anti-idiotypic antibodies are potent modulators of the immune response and that the C10-IdX is important in the determination of the fine specificity of antibodies to this decapeptide epitope of TMVP.     

Recognition of human polymorphonuclear leukocyte receptors for leukotriene B4 by rabbit anti-idiotypic antibodies to a mouse monoclonal antileukotriene B4

Rabbit anti-idiotypic IgG antibodies to the combining site of a mouse monoclonal IgG2b antibody to leukotriene B4 (LTB4) cross-reacted with human polymorphonuclear (PMN) leukocyte receptors for LTB4. Anti-idiotypic IgG and Fab both inhibited the binding of [3H]LTB4, but not [3H]N-formylmethionyl-leucylphenylalanine (fMLP), to PMN leukocytes with similar concentration-effect relationships, whereas neither nonimmune rabbit IgG nor Fab had any inhibitory activity. At a concentration of anti-idiotypic IgG that inhibited by 50% the binding of [3H] LTB4 to PMN leukocytes, the antibodies preferentially recognized high affinity receptors. Anti-idiotypic IgG and Fab inhibited PMN leukocyte chemotactic responses to LTB4, but not fMLP, with concentration-effect relationships resembling those characteristic of the inhibition of binding of [3H] LTB4, without altering the LTB4-induced release of beta-glucuronidase. Chemotaxis and increases in the cytoplasmic concentration of calcium equal in magnitude to those elicited by optimal concentrations of LTB4 were attained at respective concentrations of anti-idiotypic IgG equal to and 1/25 the level required for inhibition of binding of [3H]LTB4 by approximately 50%. Thus, the anti-idiotypic antibodies bound to PMN leukocyte receptors for LTB4 with a specificity, preference for high affinity sites, and capacity to alter PMN leukocyte functions that were similar to LTB4.     

The valence for ligand of the human mononuclear phagocyte 72 kD high-affinity IgG Fc receptor is one

The valence for ligand of the 72 kD high-affinity IgG FcR present on human mononuclear phagocytes was evaluated. Lysates of U937 cells whose high-affinity FcR had been saturated with equivalent quantities of 125I-IgG1 kappa and unlabeled IgG1 lambda or with 125I-IgG1 lambda and unlabeled IgG1 kappa were incubated with Sepharose-anti-kappa. Eighty-nine percent of the applied 125I-IgG1 kappa was bound, whereas 0.35% of the applied 125I-IgG1 lambda bound (mean of two experiments), indicating that if the receptors are occupied with ligand, the receptors bind only one ligand molecule at a time. Two experiments were performed to show that the receptors were ligand-occupied. First, a monoclonal antibody directed against the 72 kD FcR (FcRmab32) was added to lysates of U937 cells saturated with equal quantities of 125I-IgG1 lambda and IgG1 kappa. This anti-FcR antibody caused a dose-dependent sevenfold increase in the amount of 125I-IgG1 lambda bound to the anti-kappa immunoadsorbent (presumably by cross-linking receptors bearing 125I-IgG1 lambda with receptors bearing IgG1 kappa), whereas monoclonal antibodies (MMA and IV3) directed against two other determinants on U937 caused no such increase. In the second experiment, Sepharose-FcRmab32 adsorbed 60% of the 125I-IgG1 kappa and 46% of the 125I-IgG1 lambda applied in a U937 lysate (bearing high-affinity FcR), whereas only 3% of 125I-IgG1 kappa and 6% of 125I-IgG1 lambda applied in a K562 lysate (bearing no high-affinity FcR) were adsorbed. We interpret these data to indicate that in detergent solution the valency of the high-affinity FcR on U937 cells is one.     

Attempts to obtain anti(D2 dopamine receptor) antibodies via the anti-idiotypic route.

Five stable hybridomas have been obtained that secrete monoclonal antibodies against the D2-dopamine receptor-selective drug spiperone. Each monoclonal antibody has been characterized in terms of its ability to bind a range of dopamine-receptor-selective ligands. One monoclonal antibody has been purified by Protein A affinity chromatography and used to immunize mice. Anti-idiotypic antisera and one hybridoma secreting an anti-idiotypic monoclonal antibody were obtained and shown to inhibit [3H]spiperone binding to the anti-spiperone antibody used for immunization. Neither the antisera nor the anti-idiotypic monoclonal antibody, however, inhibited binding of [3H]spiperone to D2-dopamine receptors.     

Monoclonal antibodies to streptococcal group A carbohydrate. I. A dominant idiotypic determinant is located on Vk

In an effort to better define the antibody repertoire to streptococcal group A carbohydrate (GAC), somatic cell hybrids were prepared from A/J mice immunized with streptococcal vaccine. Most antibodies were IgG3K and IgMK, while 2 of 26 antibodies were lambda type. Each of the IgG3 antibodies had a distinct isoelectric point consistent with previous estimates of clonal repertoires of approximately 200. IEF analysis of the L chains, however, showed that about half of the antibodies produce a common L chain, called VK1GAC, previously identified in A/J anti-GAC serum antibodies. Additional support for the structural similarity of these L chains was gained by developing an idiotype antiserum to VK1GAC. All proteins with the common L chain spectrotype react strongly with anti-VK1GAC. Thus, it appears that anti-GAC antibodies are composed of H chains bearing a few VH regions pairing with a few L chains.     

Production of antisera specific for idiotype(s) of murine anti-(T,G)-A--L antibodies.

Antibodies to the synthetic polypeptide (T,G)-A--L were raised in C57BL/10 and C3H.SW mice. For each strain, the anti-(T,G)-A--L antibodies from 10 animals were pooled, affinity purified on a (T,G)-A--L-Sepharose column, and used to immunize Lewis rats. The resulting rat antisera were adsorbed with insolubilized normal mouse globulin in order to remove anti-isotypic and anti-allotypic antibodies. The residual antibodies specifically inhibited the binding of (T,G)-A--L by anti-(T,G)-A--L as measured by a radioimmunoassay. The specificity of this inhibition was demonstrated as follows: 1) failure of the anti-(T,G)-A--L anti-idiotype to inhibit the binding of nuclease to anti-nuclease antibody of the same allotype; 2) failure of Lewis anti-[B10 anti-(T,G)-A--L] to inhibit C3H.SW anti-(T,G)-A--L and vice versa; 3) ability to absorb anti-C3H.SW anti-idiotypic activity on insolubilized C3H.SW anti-(T,G)-A--L but not on B10 anti-(T,G)-A--L. The same or cross-reactive idiotype(s) was present in the majority of individuals of each of these strains.     

Shared idiotope on monoclonal anti-Ia.7 antibodies reactive with determinants in a structural domain of the I-E molecules

In previous studies, heterologous anti-idiotypic (anti-Id) antisera against the C3H.SW 14-4-4S or the A.TH 41.A anti-Ia.7 monoclonal antibodies (mAb) were shown to identify an interstrain cross-reactive idiotypic specificity (IdX.Ia.7) expressed on monoclonal or conventional anti-Ia.7 alloantibodies. The objective of the present investigation was to characterize further this IdX at the idiotopic level. To this end, 11 hybridomas producing IgG1, IgG2a, or IgM anti-Id mAb were derived from a rat immunized with a mixture of 10 A.TH or A.BY anti-Ia.7 mAb. The specificity of the latter anti-Id mAb was determined by direct Id binding radioimmunoassay (RIA) with the use of a panel of 52 anti-Ia mAb derived from hybridomas produced in various inbred mouse strains. These rat anti-Id mAb recognized idiotopes expressed on i) all anti-Ia.7 mAb against determinants in the topographic domain I of the I-Ek molecule but not on 18 other anti-I-Ek mAb directed at epitopes in domains II or III; ii) three of 19 anti-I-Ak mAb; and iii) one A.TL-derived anti-I-As mAb. Competitive Id binding assays revealed that among the 14 IdX+ anti-Ia.7 mAb, one (81.B) was bound to a lesser extent by various rat anti-Id mAb, suggesting that heterogeneity probably exists in this antibody family. By contrast, two isologous (B10.S(7R)) anti-Id mAb to the IdX.Ia.7+ mAb 41.A displayed a specificity restricted to 41.A individual idiotopes (IdI). Rat anti-IdX.Ia.7 and mouse anti-41.A IdI mAb inhibited the binding of 125I-labeled mAb 41.A to CBA spleen cells. These two sets of mAb bound in a noncompetitive fashion to mAb 41.A-coated plates, indicating that their corresponding public or private idiotopes were spatially distinct. These data may have implications for in vivo manipulations of anti-Ia immune responses.     

Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.     

Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.