Rapid and sensitive procedure for assigning idiotypic determinants to heavy or light chains: Application to idiotopes associated with the major cross-reactive idiotype of A/J anti-phenylarsonate antibodies

A rapid and sensitive procedure is described for assigning idiotypic determinants to heavy or light polypeptide chains. Heavy and light chains are resolved by electrophoresis in the presence of sodium dodecyl sulfate. The electrophoretically resolved polypeptides are then transferred to nitrocellulose filters. Filters containing bound heavy and light chains are incubated with ¹²⁵I-labelled anti-idiotypic antibody, and idiotype-anti-idiotype reactivity visualized by autoradiography. This procedure is illustrated with three monoclonal anti-idiotopic antibodies which recognize determinants associated with the major cross-reactive idiotype family of A/J anti-phenylarsonate antibodies. All three anti-idiotopic antibodies are shown to react with electrophoretically resolved idiotype heavy chain, but not with idiotype light chain.     

The genetic basis of antibody production: the dominant anti-arsonate idiotype response of the strain A mouse

Immunization of the A strain of mice with the hapten p-azophenylarsonate (Ars) results in an immune response in which approximately 50% of the anti-Ars antibodies share cross-reactive idiotypic determinants (IdCR). A gene or genes linked to the heavy chain constant region locus is required for the production of this idiotype. The expressed VH gene from a hybridoma cell line which expresses the IdCR has been cloned. DNA hybridization studies utilizing the VH gene have revealed that there are many related genes in both idiotype-producing and idiotype-nonproducing strains of mice. However, under stringent hybridization conditions, only a single band of 6.4 kb is present in Eco R1-digested A strain DNA. Strains of mice which are phenotypically idiotype-negative either lack this band completely or possess a much weaker one at this position. Utilizing DNA from Igh recombinant strains of mice, it has been shown that the VH locus controlling idiotype expression contains the structural gene information for the idiotype-positive heavy chains. It has also been shown that DNA at this locus appears to be sufficient for the production of the cross-reactive idiotype. Utilizing a DNA probe derived from regions flanking the structural gene has confirmed the relatedness of V genes in a variety of mouse strains and revealed a significant degree of polymorphism at the Igh locus.     

Structural studies on induced antibodies with defined idiotypic specificities--VIII. NH2-terminal amino acid sequence analysis of the heavy and light chain variable regions of monoclonal anti-para-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype.

Monoclonal antibodies with specificity for the hapten p-azophenyl arsonate (Ar)^* were generated by somatic cell hybridization of A/J spleen cells and the non-secreting cell line, Sp2/0-Ag14, derived from a Balb/c myeloma. Of four hybridoma products examined, only one bore the previously described A/J anti-Ar cross-reactive idiotype (CRI). The heavy and light polypeptide chains of this CRI positive molecule along with one of of the CRI negative molecules were subjected to amino-terminal sequence analysis and compared with the induced CRI positive antibodies produced upon immunization of A/J mice. The results confirm the extremely restricted expression of V_H and V_L frameworks in the A/J anti-Ar population. Unexpectedly, when the hybridoma proteins were compared to the induced population of antibodies, from one to three amino acid substitutions were detected within the first framework segment (1–30) of each chain of both hybridoma products. The complete covalent structure of two monoclonal antibodies known to differ serologically only with respect to idiotype should prove useful in defining the exact structural basis of an idiotypic determinant.     

Characterization of variable-region genes and shared crossreactive idiotypes of antibodies specific for antigens of various influenza viruses

Several syngeneic monoclonal anti-idiotypic antibodies were obtained against PY206, a monoclonal antibody specific for X-31 (H3N2) influenza virus hemagglutinin. This idiotype was found in the sera of BALB/c mice immunized with various influenza viruses. Adsorption experiments indicated that the PY206 Id was borne by antibodies specific for viral hemagglutinin (HA) and/or neuraminidase (NA). This idiotype was identified on other monoclonal antibodies specific for various influenza HAs (H3 and H1). Study of the variable-region (V) genes of these monoclonal antibodies showed that its expression is independent of variable kappa (VK)21 light-chains and that the heavy-chains of the strongly idiotype-positive hybridomas derive from either the variable heavy (VH) J558 or VH 7183 family. Finally, Western blot analysis demonstrated that PY206 idiotypic determinants are located exclusively on the heavy chain.     

Idiotype-specific T helper cells are required to induce idiotype-positive B memory cells to secrete antibody.

We have tested the proposition that induction of certain sets of B cell clones to produce antibody requires a signal from T helper cells that recognize idiotypic determinants expressed on Ig receptors of the relevant B cell clones. The approach is based on the analysis of T cell populations required to induce B cells to secrete anti-arsonate antibodies that are marked by a cross-reactive idiotype (CRId). crid+ anti-azophenyl arsonate (Ar) antibodies are produced in A/J strain mice after immunization with Ar keyhole limpet hemocyanin and represent 20--70% of the total anti-Ar antibody response. These studies indicate that antibody secretion by idiotype+ B memory cells requires two signals: one provided by "carrier"-specific Ly-1 cells, and a second delivered by idiotype-specific Ly-1 cells. Both signals are required for optimal induction of idiotype+ B memory clones.     

Structural studies on induced antibodies with defined idiotypic specificities—VIII.: NH2-terminal amino acid sequence analysis of the heavy and light chain variable regions of monoclonal anti-p-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype

Monoclonal antibodies with specificity for the hapten p-azophenyl arsonate (Ar)^* were generated by somatic cell hybridization of A/J spleen cells and the non-secreting cell line, Sp2/0-Ag14, derived from a Balb/c myeloma. Of four hybridoma products examined, only one bore the previously described A/J anti-Ar cross-reactive idiotype (CRI). The heavy and light polypeptide chains of this CRI positive molecule along with one of of the CRI negative molecules were subjected to amino-terminal sequence analysis and compared with the induced CRI positive antibodies produced upon immunization of A/J mice. The results confirm the extremely restricted expression of V_H and V_L frameworks in the A/J anti-Ar population. Unexpectedly, when the hybridoma proteins were compared to the induced population of antibodies, from one to three amino acid substitutions were detected within the first framework segment (1–30) of each chain of both hybridoma products. The complete covalent structure of two monoclonal antibodies known to differ serologically only with respect to idiotype should prove useful in defining the exact structural basis of an idiotypic determinant.     

Cross-reactivity of the NPa and NPb idiotypic responses of BALB/c and C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl (NP)

A comparative antigenic analysis was carried out to determine whether cross-reactivity exists between the major idiotypic responses to (4-hydroxy-3-nitrophenyl)acetyl (NP) in BALB/c and C57BL/6 mice. Extensive cross-reactivity exists between the NP^a (BALB/c) and NP^b (C57BL/6) allotype-linked idiotypic responses to NP. The cross-reactive determinants of the NP^b idiotype are confined to one particular group of NP^b-positive monoclonal antibodies. The extent of cross-reactivity between this group of C57BL/6 antibodies and idiotype-positive monoclonal antibodies of BALB/c is so great that they cannot be readily distinguished as NP^b- or NP^a-positive antibodies with polyclonal anti-idiotypic reagents. That this cross-reactivity is not unique to monoclonal antibodies was confirmed by the demonstration of these cross-reactive determinants in the immune sera of individual BALB/c and C57BL/6 mice. Additionally, evidence was obtained from these experiments and from earlier ones from this laboratory which suggests that the BALB/c idiotypic response to NP-protein conjugate is more homogeneous than the C57BL/6 idiotypic responses.     

Idiotype-Anti-Idiotype Interactions of VHIX-Coded Anti-Progesterone and Anti-Arsonate Antibodies

The reactivity and specificity of potyctonal and monoclonal anti-idiotypic antibodies raised against monoclonal anti-progesterone and anti-arsonate antibodies have been studied by solid phase radioimmunoassay (RIA) with immobilized idiotype and by passive haemagglutination with idiotype-coupled red cells. The sensitivity of the two methods was comparable, though some cross-reactions were only detected by RIA. Passive haemagglutination was found to be especially suitable in screening for monoclonal anti-idiotypes in hybridoma supernatants and ascites. and had advantages over RIA in detection of syngeneic anti-idiotypes. Demonstration of binding site-associated idiotopes was possible by haemagglutination inhibition. RIA and haemagglutination were used to investigate the idiotypic relationships between BALB/c antiprogesterone and anti-arsonate monoclonal antibodies which share heavy chains encoded by V_HIX variable region genes.     

An example of idiotypic mimicry

We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibody repertoires. The transgenic system was chosen as to allow for normal recombination of endogenous Ig genes, secretion of TGIg from early development on, and distinguishing the TGIg from endogenous Ig by several serological markers on the C and V regions of the molecules. The transgenic construct encodes a complete anti-(4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NP) antibody molecule carrying a well-defined idiotype, bearing a λ1 light chain and a chimeric heavy chain encoded by a human α2 C region devoid of its membrane exon, and the murine B1.8 VDJ-region. Endogenous antibody repertoires were analyzed in mitogen-driven limiting dilution cultures, in single-cell assays for naturally activated Ig-secreting cells, and in hybridomas derived by direct fusion of spleen cells from unmanipulated animals. The results show that a very high frequency of splenic resting B cells and plasma cells in transgenic animals produce IgM with B1.8-cross-reactive idiotypes. This was confirmed by hybridoma analysis which also established that the levels of transgene expression and of idiotype-positive IgM production by the same cell are not correlated. The affinities of idiotype-positive endogenous Ig varied, but were generally several orders of magnitude lower than the transgene-encoded idiotype. V regions from idiotype-cross-reactive IgM heavy chains showed marked diversity in sequences that were all different from the transgenic B1.8. These results are compatible with idiotypic mimicry resulting from intercellular selection based on degenerate, whole V region reactivities.     

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: Localization and characterization of idiotypic determinants

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (K_a values ranging from 5 × 10⁶ to 10¹⁰ M⁻) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (γ, κ) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15–20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained γl heavy (H) chains and 4-6-10 contained both γl and γ2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which denned binding mechanism diversity. The location of determinants recognized by antiidiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotye by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (˜25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the V_H- and V_L-chain interaction must exist for the expression of these idiotypes.     

Idiotypic heterogeneity of VKIII autoantibodies to red blood cell antigens.

VKIII light (L) chains are commonly expressed by human autoantibodies with diverse binding specificities, including red blood cell antigens. To better understand the physiologic and pathologic expression of these L chain variable region genes, we have created a panel of murine monoclonal anti-idiotypic antibodies by immunization with a human lymphoblastoid B cell line that secretes an IgM VKIII autoantibody specific for the I red blood cell carbohydrate determinant. The binding specificities of these nine murine monoclonal antibodies, termed IV.1-IV.9, were evaluated against a large panel of monoclonal Ig proteins and compared to two previously well-characterized monoclonal anti-idiotypes, 6B6.6 and 17.109; these two anti-idiotypes have been shown to primarily identify VKIII rheumatoid factors derived from the kv328 (VKIIIa) and kv325 (VKIIIb) genes, respectively. In contrast, our anti-idiotypic antibodies identified (public) cross-reactive idiotypes present on many VKIII proteins that included both anti-erythrocyte and rheumatoid factor autoantibodies. Certain anti-idiotypic antibodies (IV.2 and IV.6) were restricted to VKIIIa L chains but differed from the 6B6.6 anti-idiotype by binding to a larger subset of VKIIIa proteins representing the products of at least two VKIIIa genes. One antibody of our panel (IV.5) recognized a private idiotope expressed only by the immunizing antibody. Using the panel of anti-idiotypic antibodies to evaluate erythrocyte autoantibodies with different serologic specificities, we found striking heterogeneity of L chain idiotype expression, even among known VKIII anti-i/I autoantibodies. These findings differ from the recently described structural and idiotypic conservation associated with the H chain of anti-i/I autoantibodies. From correlations of idiotypic reactivity with L chains of known sequence, it is postulated that the observed heterogeneity of L chain idiotype expression is due to differences in the genetic origin and/or somatic diversification of L chain variable region genes. Furthermore, subtle variability of L chain structure may contribute in part to the differences in fine binding specificity among anti-I and anti-i autoantibodies.     

The effect of light chain gene expression on the inheritance of an idiotype associated with primary anti-(4-hydroxy-3-nitrophenyl)acetyl(NP) antibodies.

Primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies from C57BL/6 mice were idiotypically defined by a rabbit anti-idiotypic antiserum. The idiotypic marker detected by this antiserum (the NP^b idiotype) requires for its expression a specific heavy (H) chain-lambda (Λ) light (L) chain combination as shown in chain reassociation experiments. The idiotypic binding reaction is inhibitable by hapten. BALB/c and CBA mice also produce large amounts of Λ-bearing anti-NP antibodies, but these do not express the NP^b idiotype as defined by the rabbit antiserum. Since the NP^b idiotype could be reconstituted by reassociating C57 BL/6 anti-NP antibody H chains with either HOPC 1, or BALB/c or CBA anti-NP antibody Λ chains, the absence of the NP^b idiotype in BALB/c and CBA antibodies implies that these animals express different sets of H chain variable (V) regions in the anti-NP response. SJL mice, in contrast to other mouse strains with the Ig-1^b allotype, express only traces of NP^b idiotype in their anti-NP antibodies, the L chains of which are almost exclusively of the χ type. Knowing that SJL mice possess a regulatory gene responsible for the low level of Λ (Λ¹⁰) chains in their immunoglobulins, we have analyzed the progeny of (SJL × BALB/C)F₁, (SJL × BALB/C)F₁ × BALB/C and (SJL × BALB/C)F₁ × SJL for the expression of Λ-bearing anti-NP antibodies and NP^b idiotype. In accord with previous work (SJL × BALB/c)F₁ mice produced substantial amounts of anti-NP antibodies with the NP^b idiotype. Backcross of these mice to BALB/c revealed linkage of the NP^b idiotype to the Ig-1^b allotype, in agreement with previous data. Backcross of the F₁ animals to SJL demonstrated control of the NP^b idiotype by the gene regulating Λ chain expression in serum immunoglobulin. Chain reassociation experiments showed that in SJL mice, the regulation against Λ chain expression prevents the selection in the anti-NP response of those H chains that together with Λ chains constitute the NP^b idiotype. Apparently, these H chains are unable to contribute to a binding site with specificity for the NP hapten when combined with χ chains.     

Assessment of a functional role of auto-anti-idiotypes in idiotype dominance

We have used a well-defined idiotypic system, the cross-reactive idiotype of A strain (CRI_A) (Ab1) idiotype generated in A/J mice injected with arsonate coupled to keyhole limpet hemocyanin (ARS-KLH), to determine the frequency of precursors for auto-anti-idiotypic antibodies (auto-Ab2) in naive and immunized A/J mice by limiting dilution analysis after polyclonal activation by lipopolysaccharide. In naive animals, the precursor frequencies of auto-Ab2 B cells were below the limit of sensitivity of the technique in the majority of A/J mice, and could be detected in only 20% of the animals. Upon immunization with ARS-KLH, a large increase in auto-Ab2 precursor frequency was observed. This shift in frequency was not found when A/J mice were injected with KLH alone, or when BALB/c mice, which do not express the CRI_A idiotype, were injected with ARS-KLH. To study the functional role of the auto-Ab2 B cells, we injected neonatal A/J mice with polyclonal rabbit Ab3 antibodies directed against a recurrent idiotype of auto-Ab2. Thereafter, these mice were injected with ARS-KLH. Although the anti-arsonate response level was normal, the CRI_A Ab1 expression was reduced tenfold. Thus, the suppression of auto-Ab2 affects Ab1 dominance. We further show that the presence of maternal Ab1 can strongly modify the immune response of the offspring by inducing higher levels of the idiotype after immunization. Furthermore, IgM anti-arsonate antibodies were detected before immunization with antigen. From these data, we conclude that the affinity of antigen alone cannot explain the dominance of CRI_A. Network selection is important in the shaping of the available repertoire.     

The Major Rheumatoid Factor Cross-Reactive Idiotype in Rheumatic Disease

The major rheumatoid factor cross-reactive idiotype (RCRI), a tertiary structure formed by both light and heavy chains, is found on 60% of all monoclonal IgM kappa RFs. To determine if the RCRI is expressed in patients with rheumatic disease, we used polyclonal rabbit anti-idiotypic antibodies to detect RCRI in sera and in pokeweed mitogen cultures of blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). We detected increased expression of RCRI + plasma cells in PWM cultures, and in sera from these patients. We have determined that some 7S IgM molecules from RF+ RA patients are RCRI +, and can bind IgG in a sensitive RF ELISA. We have also observed that the CD5+ B cell subset, which is responsible for autoantibody production, generates RCRI+ antibodies. We review these data and discuss the relationship of the idiotypic network of interacting antibodies with rheumatic disease.     

Monoclonal antibodies raised against the idiotype of the murine B cell lymphoma, BCL1 act primarily with heavy chain determinants

Anti-idiotypic antibodies can be used as probes to distinguish neoplastic cells from their normal counterparts. In addition they have been used in the passive therapy of B cell tumors. In this report we describe a panel of 7 rat monoclonal antibodies raised against idiotypic determinants carried by the IgM molecule of the BCL1 lymphoma. The majority (6/7) of these antibodies recognize private idiotypic determinants that are carried on the isolated mu heavy chain of the molecule, and do not require the lambda chain for reactivity. This is unusual for antibodies raised against the idiotype of the whole immunoglobulin molecule, which normally require both chains for reactivity. The antibodies do not, however, bind peptides corresponding to the complementarity determining regions of the mu heavy chain of BCL1. The antibodies perform well in complement mediated cytotoxicity, and, in at least one case, are effective in the passive immunotherapy of BCL1 lymphoma.     

A private idiotype can become recurrent through genetic recombination and gene(s) unlinked to the Igh locus governs its expression

Any immune response is characterized by its idiotypic profile. Two different kinds of idiotype (Id) have been described. Private Id are restricted to a few individuals from a species while recurrent Id appear in a large majority of individuals from the same species immunized with the same antigen. We describe, in this report, an experimental model whereby a private Id can become recurrent through genetic recombination. The immune response of A mice against the hapten arsonate is characterized by a recurrent Id called cross-reactive idiotype A (CRIA). A strongly CRI, called CRIA-like, can be occasionally detected in some BALB/c mice (5% to 10%) immunized with arsonate. Molecular studies show that CRIA and CRIA-like antibodies have highly homologous D segments and identical light chains. By contrast, their VH segments are vastly dissimilar. We have examined the anti-arsonate response of inbred strains of mice whose Igh loci are recombinant between those of A/He and BALB/c. Interestingly, we have observed that the CRIA-like Id which is private in BALB/c becomes recurrent in the AXC-1 strain which harbors the VH genes from BALB/c, the DH and CH genes from A/He. Structural studies demonstrate that highly homologous, VH, VL and D segments are used in BALB/c and AXC-1 mice. The basis for this differential expression of highly similar genes could be linked to the DH locus. However, F1 mice stemming from the cross between AXC-1 and BALB/c do not express the Id. The backcross analysis shows that the non-expression of the Id in F1 mice depends on genes unlinked to the Igh locus.     

Analysis of a major rat idiotype associated with Anti-GAT antibodies

An anti-idiotypic antiserum was raised in a rabbit against a pool of purified F.344 rat anti-GAT antibodies. GAT-13, the idiotype defined by this serum, is present in all F.344 anti-GAT sera from primary and secondary anti-GAT responses. Anti-GAT sera of 13 inbred rat strains, with different RT1 haplotypes and with different heavy- and light-chain allotypes, all express idiotypic determinants cross-reacting with GAT-13. Thus, like in mice anti-GAT antibodies from rats express public idiotypic determinants. The anti-idiotypic serum also recognizes a highly conserved idiotypic specificity present on mouse and guinea-pig anti-GAT antibodies. The mouse, rat and guinea-pig express a similar highly conserved idiotypic specificity after immunization with GAT. All anti-GAT antibodies from the mouse and guinea-pig bear this idiotypic specificity. These results confirm the existence in the anti-GAT response of interspecies cross-reactive idiotypic determinants.     

The expression and functional involvement of nuclease- specific idiotype on nuclease-primed helper T cells

The expression and functional significance of idiotypic determinants on antigen-specific helper T (Th) cell populations for responses to Staphylococcal nuclease (Nase) were evaluated in an in vitro antibody response system. Trinitrophenyl (TNP)- specific plaque-forming cell responses to TNP-conjugates of Nase (TNP-Nase) were shown to require the cooperation of Nase-primed Th cells as well as unprimed B and accessory cells. The expression on these antigen-primed Th cells of idiotypic determinants cross-reactive with those on anti-Nase antibodies was demonstrated by the specific elimination of Th cells for TNP-Nase by treatment with affinity-purified anti-idiotypic antibodies plus complement. The susceptibility of Nase-primed Th cells to elimination by such treatment was specific in that anti-idiotypic antibodies affected Th cells only from strains normally expressing the same (or a cross-reactive) idiotype on anti-Nase antibodies. A functional role of the idiotypes expressed on Nase-primed Th cells was suggested by the fact that anti-idiotypic antibody present throughout the period of culture, in the absence of complement, suppressed responses to TNP-Nase in an antigen- and strain-specific manner. It was further shown, by cell mixing experiments, that this inhibition appeared to occur at the level of the Th cells and was not dependent on the strain of origin of the B cells. Thus, antigen-specific Nase-primed Th cells express strain-specific idiotypic determinants cross-reactive with, or identical to, those of anti-Nase antibodies. These cell surface idiotypic determinants appear to be functionally involved in the activity of Th cells for the induction of antibody responses to TNP-Nase in vitro.     

Amino acid sequence of a light chain variable region of a human rheumatoid factor of the Wa idiotypic group, in part predicted by its reactivity with antipeptide antibodies

Antipeptide antisera were raised to the second and third complementarity-determining regions of the light chain derived from a human monoclonal IgM (Sie) which had antigammaglobulin activity and belonged to the Wa cross-reactive idiotypic group of human rheumatoid factors, two of whose members (Sie, Wo1) had been previously sequenced in our laboratory (Andrews and Capra, Biochemistry 20, 5816-5822, 1981). These antisera were found to react with the light chain of another human monoclonal IgM (Go1) that shared the Wa idiotype while antipeptide antisera made to the third CDR of the Sie heavy chain failed to react. The amino acid sequence of the variable region of the Go1 light chain was found to be highly homologous to the light chain of Sie from which the synthetic peptides were derived, particularly in the framework regions and the second and third CDR. This study illustrates that antipeptide antisera are valuable and specific probes for determining the relationship between molecules which exhibit similar antigen binding or idiotypic specificities and, furthermore, such antisera are able to predict amino acid sequences with surprising precision.     

Uniformity in the clonal repertoire for the immune response to phosphorylcholine in mice.

A comparison of the clonal nature of the immune response to phosphorylcholine (PC) was made in nine different inbred mouse strains. Quantitative idiotypic analysis showed that anti-PC antibodies from each strain were composed of antibodies bearing binding-site idiotypic determinants indistinguishable from two different BALB/c myeloma proteins, T15 and M511. Idiotypic determinants of two other PC-binding proteins, M167 and M603, were not detected. Isoelectric focusing of the light (L) chains verified the presence of antibodies similar to T15 and M511 in each strain and indicated the presence of two additional antibodies, one of which has an L chain which cofocuses with M603. Fractionation of anti-PC antibody with anti-idiotypic antibody showed that immunoglobulins bearing T15 and M511 idiotypic determinants are separate and contain L chains that are unifore and resemble those of T15 and M511, respectively. Thus, these mice which differ genetically at multiple loci including the heavy chain allotype complex locus each possess, at least in part, an equivalent set of clonotypes specific for PC. This indicates that the genes encoding these antibodies must be contained in the germ line.