Anti-hypervariable region antibody induced by a defined peptide: an approach for studying the structural correlates of idiotypes.

Abstract

The structural correlates of idiotypes have been sought in several antibody systems. However, the precise molecular basis of idiotypes are exceedingly difficult to define.Antibodies of predetermined specificity can be induced by immunization with synthetic peptides. In the present experiments, a synthetic peptide corresponding to a hypervariable region on a monoclonal human IgM rheumatoid factor (Sie) has been used to induce specific anti-hypervariable region antibodies. The antibodies bound to the intact Ig molecule and to its isolated heavy chains but not to other IgM paraproteins nor to pooled human IgG. The binding of the antibody to the intact IgM was inhibited specifically by the free peptide, and the antibody activity was removed by a peptide-coupled affrmity column. These results clearly demonstrate that specific antiidiotypic antibody of predefined specificity can be induced by a hypervariable region peptide. Antibodies of this class may provide a new tool for defining the structural correlates of idiotypes.The study of idiotypes (1) has greatly advanced our understanding of immunoglobulin genetics and antibody structure (2, 3). In addition, idiotypes have been postulated to play an essential role in the generation and regulation of diverse immune responses (4-6).The structural correlates of idiotypes have been sought in several well-defined antibody systems (7)(8)(9)(10)(11)(12). Because of the common occurrence of monoclonal human IgM paraproteins with anti-IgG activity (rheumatoid factors), the idiotypes and the primary amino acid sequences of these proteins were compared in detail (9, 10, 13). The heavy and light chains from two monoclonal IgM rheumatoid factors (IgM-RF) with crossreactive idiotypes (e.g., Lay and Pom) had identical amino acid sequences in three of six complementarity-determining regions but differed substantially in the framework regions (9, 10). These results suggested that the hypervariable regions of the heavy and light chains were the structural correlates of idiotypic determinants. Similar comparisons of amino acid sequences and idiotypes among mouse monoclonal antibodies with antigen binding activity have yielded much the same conclusions (8,11,12). Noticeably, in the murine anti-Dextran system, one private idiotype and one public idiotype were assigned, respectively, to the third and the second hypervariable region of the heavy chain (11). However, in most other cases, it has proven to be extremely difficult to associate a particular idiotypic determinant with a specific amino acid sequence (7 MATERIALS AND METHODSPreparation of Synthetic Peptide. Peptides were prepared by the solid-phase method (17-20), using a Beckman model 990B peptide synthesizer. Briefly, 1.00 g (ca. 0.5 milliequivalent) of N-t-butoxycarbonylglutamic acid (BocGlu) resin was used along with the following side-chain protecting groups: O-bromobenzyloxycarbonyl for tyrosine and lysine; O-benzyl for glutamic acid and aspartic acid; S-methoxybenzyl for cysteine. Protected amino acids were recrystalli...

General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids.

“…Over the past 5 years there has been an escalating need for synthetic peptides in a wide variety of applications, including the development of synthetic peptide vaccines (1)(2)(3)(4)(5), the detailed study of antigen-antibody interactions (6)(7)(8)(9)(10)(11), the preparation of optimal analogs of biologically active peptides (12)(13)(14), the optimization of peptide antigens of clinical diagnostic utility (15)(16)(17), the mapping of protein products of brain-specific genes (18,19), and the study of protein conformational parameters (20). In the majority of these studies, the limiting factor has been the availability and cost of the desired peptides.…”

Characterization of an epibody. An antiidiotype that reacts with both the idiotype of rheumatoid factors (RF) and the antigen recognized by RF.

“…Since IgM-RF Glo reacted with rabbit IgG, an immunoblot assay was used to demonstrate the de facto antiidiotype activity of the anti-PGL1 antiserum (7). IgM-RF GIo and the control, pooled human IgG (Cohn fraction II), were Immune 2 ----449…”

“…This was done exactly as described previously (7,8). Briefly, antigens (2 #g/ ml) in 0.1 M borate, 0.2 M NaC1, pH 8.2 (BBS) were used to coat microtiter plates (3590; Costar, Data Packaging, Cambridge, MA) at 100 #l/well.…”

“…The reactivity of the anti-PGL1 antibody with immunogiobulin molecules was analyzed by the immunoblot method (11,12) with some modifications. Briefly, ~ 10 #g of individual monoclonal IgM-RF proteins (5,7,13) or pooled human IgG, in 20 #i sample buffer supplemented with 0.01% 2-mercaptoethanol, was loaded onto each slot of a 10% polyacrylamide slab gel containing 0.1% sodium dodecyl sulfate (14). After electrophoresis (2.5 h at 30 mA), the proteins in the gel were transferred electrophoreticaily to nitrocellulose paper (2 h at 70 V).…”

“…heavy chain of the IgM-RF Sie) has been described previously (7). It is composed of GluTrp-Lys-Gly-Gln-VaI-Asn-Vai-Asn-Pro-Phe-Asp-Tyr-Gly-Gly-Cys.…”