Idiotypic analysis of polyclonal and monoclonal anti-p-azophenylarsonate antibodies of BALB/c mice expressing the major cross-reactive idiotype of the A/J strain

The idiotypic cascade allows the induction of silent idiotypes, and as such, the immune system can be reprogrammed towards predetermined goals. To understand the genetic origin of silent idiotypes, we have used a system in which detailed structural and genetic information is available. The major cross-reactive idiotype (CRIA) of A/J mice (positive strain) immunized with arsonate coupled to a carrier can be regularly induced in BALB/c mice (negative strain) by anti-idiotypic treatment with or without subsequent antigen immunization. By using a panel of monoclonal anti-idiotypic antibodies, we have found that the germline-encoded CRIA displays a mosaic of at least five idiotopes. Polyclonal and monoclonal anti-arsonate antibodies prepared from idiotypically manipulated BALB/c mice have been studied. Four germline idiotopes are shared between the CRIA of the A/J strain and the CRIA-like idiotype induced in BALB/c mice. Furthermore, CRIA-like antibodies can appear "spontaneously" in some BALB/c mice immunized with antigen only. The data suggest that anti-idiotypic treatment in BALB/c mice selects a preexisting subset of antibodies. From the serological analysis, it is predicted that CRIA molecules from A/J and CRIA-like molecules from BALB/c employ different VH subgroups but share some components of the hypervariable regions. These predictions are tested in a forthcoming paper that describes the amino acid sequences of BALB/c monoclonal antibodies displaying the major cross-reactive idiotype of the A/J strain.     

Characteristics of two idiotypic subfamilies of murine anti-p-azobenzenearsonate antibodies

A family of antibodies bearing a common or cross-reactive idiotype, termed CRIC, predominates in the response of most BALB/c mice to the p-azobenzenearsonate (Ar) hapten, but represents a minor component of the anti-Ar response of most A/J mice. Previous results have suggested that the VH region of CRIC is encoded by two different germ-line genes in both strains. We have determined extensive mRNA sequences for VH and VL, developed specific idiotypic reagents and measured affinities for two subfamilies of CRIC, designated CRIC1 and CRIC2. Both were found to be minor components of A/J anti-Ar antibodies, and CRIC1, but not CRIC2, is a major component of the BALB/c response. The two subfamilies utilize different VH germ-line genes but the same, or nearly identical V kappa genes. The VH nucleotide sequences of CRIC1 and CRIC2 exhibit approximately 90% homology. The D regions of both families are short (one or two amino acid residues) and some can be accounted for on the basis of known JH sequences alone. Affinity differences may account for the dominance of CRIA over CRIC1 and CRIC2 in A/J mice, but results obtained with allotype-congenic mice indicate that background (non-V region) genes are also important in controlling levels of expression of the CRIC1 idiotype. Our data suggest that the A/J germline VH gene that gives rise to the CRIC2 family of antibodies may be identical with a previously sequenced BALB/c germ-line VH gene. On the basis of these and earlier data it is suggested that extensive differences between inbred strains of mice in their complements of VH genes do not result from the accumulation of many mutations in these genes. An alternative possibility is that the differences arise from deletions and/or duplications of VH genes.     

The generation of major and minor idiotype-bearing families of anti-p-azophenylarsonate antibodies; stochastic utilization of VH gene segments

An average of 50% of anti-p-azophenylarsonate (Ars) antibodies bear a cross-reactive idiotype, IdCR, and an average of 15% bear a relatively minor idiotype, Id, in A/J mice. To begin to investigate the processes that influence the expressed levels of these idiotype-bearing antibodies in serum, we have examined the frequency among preimmune B cells of cells that utilize the heavy chain variable region gene segment (VH) needed for IdCR and that which is needed for Id anti-Ars antibody expression. Our results indicate these VH gene segments are functionally rearranged at frequencies one would expect for random usage. The frequency of VH gene segment utilization is similar to, if not higher than, that of VHCR, arguing that the predominance of IdCR-over Id-bearing antibodies is not due to preferential usage of the VHCR gene segment. In addition to the analysis of Ars-immune sera pooled from several mice, we have examined 20 individual A/J mice to determine whether the relative serum levels of IdCR- and Id-bearing antibodies are strictly regulated relative to each other. Among individuals, we find that IdCR and Id antibody levels fluctuate over a 28-fold and a 120-fold range, respectively. The ratio of IdCR to Id antibody levels was found not to be strictly regulated, varying over a 300-fold range. Linear regression analysis of IdCR relative to Id concentrations shows a correlation coefficient of only 0.093. Indeed, rare mice can be found that generate greater levels of Id-bearing antibodies than those bearing IdCR. These results are indicative of a stochastic process involved during the generation of these IdCR-and Id-bearing antibody families. Models accounting for the generation of this highly variable serologic response derived from a preimmune repertoire in which VH gene segments are equivalently utilized are discussed.     

The association of various D elements with a single-immunoglobulin VH gene segment: influence on the expression of a major cross-reactive idiotype

A large fraction of the anti-p-azophenylarsonate antibodies of strain A/J mice share a major cross-reactive idiotype (IdCR). Structural analysis of monoclonal antibodies expressing this idiotype (IdCR+) indicates that a particular combination of variable region gene segments (Vk, Jk, VH, D, and JH) encodes the variable regions of the light and heavy chains of these IdCR+ antibodies. With the use of serologic methods, hybridoma cell lines have been isolated that produce monoclonal antibodies lacking IdCR determinants (IdCR-), but that are derived from most of the same combination of variable region gene segments that encode IdCR+ monoclonal antibodies. Structural analysis of these IdCR- monoclonal antibodies demonstrates that they are very homologous to each other and to IdCR+ monoclonal antibodies with respect to VH and VL sequences, but are markedly different from IdCR+ monoclonal antibodies in their utilization of D region segments. Comparisons of antigen avidity of these IdCR+ and IdCR- antibodies indicates that conservation of D region structure is not crucial for effective antigen binding. These results indicate the importance of the D region in idiotypy in the IdCR system and demonstrate the variation permitted in D region structure while maintaining antigen recognition.     

Complete amino acid sequences of the heavy and light chain variable regions from two A/J mouse antigen nonbinding monoclonal antibodies bearing the predominant p-azophenyl arsonate idiotype

The immune response to p-azophenyl arsonate (Ars) in A/J mice is dominated by a cross-reactive idiotype (CRI or IdCR). IdCR+ hybridoma proteins 1F6 and 3D10 produced in a single mouse by immunization with a monoclonal anti-IdCR antibody did not bind Ars [Wysocki, L., & Sato, V. (1981) Eur. J. Immunol. 11, 832-839]. The preservation of idiotype coupled with lack of antigen binding in the same molecules provoked an examination of their primary structures in order to localize sites involved in binding to antigen and to anti-idiotypes. The VH sequence of antibody 3D10 was determined by Edman degradation of intact chains and fragments generated by CNBr, hydroxylamine, and o-iodosobenzoic acid cleavage, by trypsin and V8 protease digestion, and by sequence analysis of mRNA. The 1F6 VH sequence was reported previously [Smith, J. A., & Margolies, M. N. (1984) Biochemistry 23, 4726-4732]. The VL sequences of 1F6 and 3D10 were determined by Edman degradation of intact chains and peptides generated by cleavage with o-iodosobenzoic acid and digestion with trypsin and chymotrypsin. Both 1F6 and 3D10 are encoded by the same VH, VK, D, and JK gene segments as are IdCR+ Ars-binding antibodies. However, 1F6 and 3D10 employ the JH4 gene segment rather than JH2. Antibodies 1F6 and 3D10 share several somatic mutations, suggesting a common clonal origin, but manifest individual mutations as well. By comparison with Ars-binding IdCR+ molecules, the substitutions in 1F6 and 3D10 likely responsible for the lack of Ars binding are localized to the heavy chain D-JH junction and/or to a substitution in light chain CDR 3.     

Complete heavy and light chain variable region sequence of anti-arsonate monoclonal antibodies from BALB/c and A/J mice sharing the 36-60 idiotype are highly homologous

Structural and serologic studies on murine A/J monoclonal anti-arsonate antibodies resulted in the identification of a second idiotype family (Id36-60) in addition to the predominant idiotype family (IdCR). Id36-60, unlike IdCR, is a dominant idiotype in the BALB/c strain but is a "minor" idiotype in the A/J strain. The complete heavy and light chain variable region (VH and VL) amino acid sequences of a representative Id36-60 hybridoma protein from both the A/J and BALB/c strains have been determined. There are only four amino acid sequence differences between the VH of antibody 36-60 (A/J) and antibody 1210.7 (BALB/c). Two of these differences arise from single nucleotide changes in which the A/J and BALB/c Id36-60 VH germline gene sequences differ. The two other differences are the result of somatic mutation in hybridoma protein 36-60. In addition, Id36-60 heavy chains employ the same D and JH3 segments in both strains. The entire Vk2 VL of 36-60 and 1210.7 differ by only two amino acids, suggesting that like the heavy chains, they are derived from highly homologous VL genes. The same Jk segment is used in both antibodies. A comparison of the amino acid sequence data from Id36-60-bearing hybridomas suggests that a heavy chain amino acid difference accounts for the diminished arsonate binding by the 1210.7 hybridoma protein. Because the 1210.7 heavy chain is the unmutated product of the BALB/c VH gene, somatic mutation in VH may be required to enhance Ars affinity in this system.     

Allogeneic manipulation of the GAT idiotypic cascade. Immunization of C57BL/6 mice by BALB/c anti-idiotypes stimulates similar strain-specific V genes as the original antigen

Antibodies specific for the immunizing Ag (Ab1) (Id+ Ag+) and Ab3 (Id+ Ag+ or Id+ Ag-) of the (Glu60 Tyr10 Ala30) (GAT) idiotypic cascade express similar pGAT public determinants in BALB/c and C57BL/6 strains. These determinants have been shown to be dependent upon both VH and Vkappa encoded segments. The VH of the BALB/c Ab1 (germ-line gene H10) and that of the C57BL/6 Ab1 (germ-line gene V186-2) are only 75% homologous, whereas VK are much more conserved. C57BL/6 mice were immunized with BALB/c Ab2 (anti-idiotypic) antibodies and monoclonal Ab3 were derived after fusion of immunized spleen cells with the nonsecreting hybridoma cell line Sp/2.0-Ag. From 13 cell lines, five clones (four Id+ Ag- and one Id+ Ag+) were isolated and the mRNA V regions sequenced. Immunization with BALB/c anti-idiotypes elicits expression of the same or closely related C57BL/6 VH and Vkappa genes as when C57BL/6 mice were immunized with GAT, although functional VH BALB/c equivalents have been isolated in the B6 strain. Our results suggest that manipulation of the repertoire via antigenic or idiotypic stimulation both lead to the expression of different genes in different strains. They further confirm that the immune system is largely degenerate, for both idiotype expression and Ag recognition.     

Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.     

Preliminary crystallographic studies of the Fab fragment of an anti-azophenylarsonate antibody

Single crystals of the Fab fragment of a murine A/J anti-azophenylarsonate monoclonal antibody have been prepared by the vapor diffusion method. Antibody 3A7 uses the same combination of variable region gene segments (VK, JK, VH, JH) as do anti-azophenylarsonate antibodies bearing a predominant cross-reactive idiotype, but utilizes a different D gene segment. The crystals grow in the presence of beta-octylglucoside as tetragonal bipyramids in the space group of either P4(1)2(1)2 or P4(3)3(1)2 and with unit cell dimensions of a = b = 77.9 A, and c = 146.7 A. They diffract X-rays to better than 2.7 A resolution. Data up to 2.7 A resolution have been collected.     

Complete amino acid sequence of the heavy-chain variable region from an A/J mouse antigen-nonbinding monoclonal antibody bearing the predominant arsonate idiotype

The 1F6 hybridoma protein, exhibiting the predominant cross-reactive idiotype (CRI) associated with the immune response to p-azophenylarsonate in A/J mice but failing to bind the hapten arsonate, was elicited following immunization with rat anti-CRI [Wysocki, L.J., & Sato, V. (1981) Eur. J. Immunol. 11, 832-839]. The dissociation of idiotype and antigen binding in this hybridoma provides an opportunity to determine structural features involved in antigen binding and idiotypic sites. The complete heavy-chain variable region (VH) amino acid sequence was obtained by automated Edman degradation of the intact chain and fragments due to CNBr cleavage, trypsin digestion, mild acid hydrolysis, and carboxypeptidase A digestion of a CNBr fragment. Comparison of the CRI+ arsonate-nonbinding 1F6 sequence with the CRI+ germ-line VH gene sequence reveals that the 1F6 heavy chain differs from the germ-line-encoded amino acid sequence at seven positions within VH [Siekevitz, M., Gefter, M. L., Brodeur, P., Riblet, R., & Marshak-Rothstein, A. (1982) Eur. J. Immunol. 12, 1023-1032]. The 1F6 VH appears to arise from the CRI+ germ-line VH by somatic mutation at at least seven amino acid residues, each of which could be due to a single nucleotide base change. The diversity (D) gene-encoded segment of 1F6 is similar to that of the CRI+ antigen-binding hybridoma 36-65 except for two amino acid substitutions. Further, the idiotype (CRI) is preserved despite use of a JH4 gene segment in 1F6 as compared to JH2 in all CRI+ arsonate-binding hybridomas examined to date.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new cross-reactive idiotype-defined family in the phthalate humoral immune response of mice. I. Linkage of VH-Xmp to IgCH allotype locus and mapping with respect to other known VH genes

A cross-reactive idiotype family was previously identified from a very large library of phthalate-specific hybridoma clones. The prototype of this idiotype family is the hybridoma, 2E9, secreting an IgM antibody with phthalate specificity. A portion of both primary and secondary anti-phthalate antibodies elicited in all BALB/c mice tested expresses the 2E9 cross-reactive idiotype. This idiotype has now been found in the anti-phthalate antibodies of several other inbred strains of mice (A/HeHa, DBA/2, and C3Hf/HeHa) tested but not in C57BL/6 mice. Anti-phthalate antibodies elicited from congenic mice BC.8, which express the same IgCH allotype as BALB/c mice but possess C57BL/6 genetic background, contain the 2E9 cross-reactive idiotype, whereas this idiotype is not expressed on the anti-phthalate antibodies derived from another congenic mouse CB.20, which expresses a C57BL/6 IgCH allotype and a genetic background of the BALB/c strain. These results indicate that the gene controlling the 2E9 idiotype is closely linked to the IgCH allotype locus. The 2E9 cross-reactive idiotype was also found in all of the F1 mice (BALB/c X C57BL/6) tested, and the level of expression of this idiotype in the F1 mice was quantitatively equivalent to the allotype/idiotype homozygous mice. The expression of the 2E9 idiotype in the phthalate repertoire has been followed in 12 different wild mouse populations. As expected, the 2E9 idiotype was observed in a large proportion of the wild mouse strains. Surprisingly, several examples of nonconcordance in the expression of idiotype and allotype were observed in these mice. One likely explanation for the linkage breakdown is a crossing over of the heavy chain constant and variable region gene complexes. In the SM/J inbred strain of mice, where such a crossover has occurred, nonconcordance between allotype and 2E9 idiotype expression was demonstrated. By using the recombinant inbred BXD strains of mice, the VH gene encoding the 2E9 idiotype has been mapped with respect to other known VH gene families. Relative to other VH genes the VH-Xmp is situated very close to the IgCH gene region.     

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.     

Molecular analysis of immunoglobulin heavy chain genes coding for idiotypic and anti-idiotypic antibodies involved in B-B cellular interaction.

We recently reported that a unique B cell clone (B19-1d), specific for a cross-reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh-restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19-1d-derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91% (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5'-noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5' upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B-B cellular interaction is discussed from a molecular point of view.     

Structural definition by antibody engineering of an idiotypic determinant

Using computer-aided techniques for predicting molecular structure, we constructed an atomic model of the variable domain of a murine anti-thyroglobulin antibody whose immunodominant idiotypic determinant (Id62) was mapped by site-directed mutagenesis and immunochemical analysis. We previously showed that under experimental conditions this idiotype activates anti-idiotypic B cells and T cells, and modulates the response to thyroglobulin in mice. Because idiotype interactions are considered of physiological importance for immune regulation, we studied this idiotype as a model to understand the relationship between function and structure. To determine the contribution of heavy- and light-chain variable domains to the idiotype structure, we constructed chimeric expression vectors and introduced them into the (non-secreting) P3X63Ag8.653 myeloma cell line. Mutants of the heavy-chain variable domain were obtained by site-directed mutagenesis and transfected into the murine (lambda 1) light-chain producer J558L cell line. The expressed proteins were purified from culture supernatants of transfected cells and characterized. We provide evidence that the third hypervariable loop (D region) of the heavy-chain variable domain is the structural correlate of the idiotypic determinant of this autoantibody and is independent from the nature of the associated light chain. Substitution of residues of the first and second complementarity-determining regions do not affect idiotype expression. The results described here are discussed in relation to our understanding, at a molecular level, of the interaction of idiotopes with B- and T-cell compartments.     

A common VH marker relating BALB/c alpha 1-3 dextran-binding and A/J P-azophenylarsonate-binding antibody families

Antibodies that define the idiotypic marker 104E-MPC11 were purified from rabbit anti-M104E sera. The expression of this marker was searched for in a panel of representative myeloma and hybridoma proteins differing in antigen-binding specificities and idiotype expression. By radioimmune competition assays, the marker was distinguished from the IdX Dex idiotype of BALB/c alpha 1-3 dextran-binding proteins (alpha 1-3 Dex-BP) in that it was expressed on both IdX Dex-positive and IdX Dex-negative proteins on the one hand, and on A/J p-azophenylarsonate-binding proteins (Ar-BP) on the other. Among the later, six of eight of the proteins also expressed the major cross-reactive idiotype (CRI). The marker was expressed weakly or not at all on A/J Ar-BP, which did not express the CRI, as well as on a number myeloma and hybridoma proteins of different antigen-binding specificities, and normal IgG. The marker was localized in VH, but its full expression required the light chain, and was influenced by quaternary structure.     

Complete variable region sequences of five homologous high affinity anti-digoxin antibodies

High affinity murine A/J anti-digoxin monoclonal antibodies exhibit diversity in binding specificity for structurally related cardiac glycosides. They utilize several VH and VL genes. Among this diverse set, however, five antibodies share V region amino-terminal sequences that are remarkably homologous. The five antibodies were divided into three subsets based on different fine specificity-binding patterns. Therefore, complete V region sequences were determined by Edman degradation and by nucleotide sequence analysis. The VH region homology among the five antibodies was 84 to 100% and the VL region homology was 89 to 99%. The sequence data are consistent with the use of single (or closely related) VH and VL genes encoding the five antibodies. Four antibodies, derived from the same fusion (40-40, 40-120, 40-140, and 40-160), use identical D, JH2, and JK5 gene segments and identical junctions suggesting that they are clonally related. The fifth antibody (35-20) uses different D and JH1 gene segments but the same JK5 gene segment. All five antibodies share a cross-reactive idiotype. The three antibodies that exhibit the greatest degree of homology (40-40, 40-120, and 40-140) also share indistinguishable antigen-binding patterns as well as private idiotopes not present on the other two antibodies. Antibody 40-160, which has the next most homologous sequence, shares idiotopes with the first set but binds preferentially to different sites on the hapten, whereas antibody 35-20 has the most divergent sequence. In general, the degree of sequence homology among the five antibodies correlates with their hierarchical order based on hapten and idiotype fine specificity patterns.     

Selective activation by thymus-dependent antigens of distinct B cell subpopulations expressing a major cross-reactive idiotype

We determined the B cell subpopulations that produce the major cross-reactive idiotype (CRIA) associated with the anti-phenylarsonate (ARS) antibody response of A/J mice. Specifically, we examined the B2 subpopulation found in normal mice which, in H-2b mice, bears the I-Ab-encoded determinant Ia.W39; the B1 subpopulation found in mice expressing the CBA/N X-linked immunodeficiency trait (xid); and the B1 subpopulation found in normal mice after the cytotoxic elimination of B2 cells with anti-Ia. W39 and complement. CRIA is expressed in each of these B cell subpopulations. Antigen plays a selective role in the stimulation of distinct B cell sets. ARS conjugates of keyhole limpet hemocyanin (KLH) can activate both the B1 and B2 subpopulations. In contrast, ARS conjugates of synthetic polypeptides under Ir gene control selectively activate the B2 subpopulation in strains that are genetic responders to the carrier. This leads to the establishment of CRIA dominance where CRIA+ anti-ARS antibody is 70 to 95% of the total anti-arsonate antibody response. This class of antigens fails to activate the B1 cells in either normal or xid mice. We compared the CRIA+ antibody produced by selectively activated B2 cells to that produced by the B1 subpopulation in xid mice. For these comparisons, we used competitive radioimmunoassays that employed polyspecific anti-CRIA antiserum or monoclonal anti-CRIA antibodies specific for distinct idiotopes on the heavy chain of CRIA+ antibody. B2 cells produce a CRIA+ anti-ARS antibody that is idiotopically uniform among individual mice, and that closely approximates the hybridoma protein 36-65 (the heavy chain of 36-65 represents the germ line-encoded sequence of the unique CRIA structural gene (25]. In contrast, the CRIA+ antibody produced by the B1 cell subset of xid mice is idiotopically diverse among individual mice, and differs markedly from the 36-65 hybridoma protein. The extent of diversification found in CRIA+ antibody depends on the B cell subpopulation that produces it.     

Characterization of monoclonal antibodies specific for sequential influenza A/PR/8/34 virus variants

A panel of monoclonal antibodies specific for a corresponding panel of sequentially selected variants of influenza A/PR/8/34 virus has been established. Although the monoclonal antibodies are paratypically distinct, idiotypic relatedness has been observed. Two cross-reactive idiotypes have been defined that are associated with the 7183 and S107 VH gene families, respectively. Three of the four monoclonal antibodies utilize the VK21 group of light chains, and three VH genes belong to the VH7183 family and one to the VH S107 family. Antibodies encoded by genes deriving from the VH7183 family share a cross-reactive idiotype, a marker of the VH region as well as distinct individual idiotopes. These antibodies are produced by different clones using related VH and VK genes.     

Idiotypic heterogeneity of the cross-reactive idiotype associated with the anti-p-azophenylarsonate antibodies of BALB/c mice

The cross-reactive idiotype (CRI) associated with the BALB/c antibody response against the p-azophenylarsonate (Ar) hapten was studied by analyzing hybridoma anti-Ar derived from BALB/c mice. The BALB/c CRI (CRIc) was previously thought to be a single idiotype as defined by rabbit anti-idiotype. CRIc has been resolved into at least two separate families of anti-Ar antibodies that are unrelated idiotypically to each other. Analysis of the 5AF6 family revealed considerable idiotypic heterogeneity, including a number of public idiotopes that appeared to be primarily associated with combinatorial idiotopes (requiring H + L chains) or L chain-specific idiotopes.