Idiotype expression and the inheritance of mouse antibody clones.

The inheritance of idiotypes was investigated using idiotypic antisera against two monoclonal antibodies to streptococcal carbohydrates derived from A/J mice. Each of the two idiotypes was characterized by a special frequency of expression. One of the idiotypes was expressed in more than 80% of A/J mice, the other in less than 20%. The idiotypes of both antibodies were strain specific and were transmitted to (A/J x BALB/cJ)F(1) hybrid mice. Furthermore, both idiotypes remained associated with the A/J heavy chain C region allotype in (A/J x BALB/cJ)F(1) hybrid mice. The results suggest that idiotypes are specified by allelic V genes, and that the heavy chain idiotype locus is linked to the heavy chain allotype locus.

Relationships between relative binding affinity and electrophoretic behavior of rabbit antibodies to streptococcal carbohydrates.

After repeated intravenous injections with Group C streptococcal vaccine, most rabbit antisera were shown to contain one or more IgG antibody components, as revealed by microzone electrophoresis. A procedure for the fractionation of multiple IgG antibody components from such streptococcal antisera is described. Separation is achieved on the basis of differences in relative binding affinities of the antibody components to immunoabsorbent columns. The evidence suggests that the electrophoretic mobility, and thus the net charge of an antibody, bears a reciprocal relationship to its binding affinity for the streptococcal Group C antigens. Furthermore, the relative binding affinity affords another means to assess the functional homogeneity of streptococcal antibodies. A possible relationship between light chain variable-region subclasses and binding affinities of streptococcal antibodies is discussed.

The inheritance of individual antigenic specificities of rabbit antibodies to streptococcal carbohydrates.

The inheritance of individual antigenic specificities (IAS) of rabbit antibodies to the Group C streptococcal carbohydrate was demonstrated in a selectively bred rabbit family. The IAS of the antibodies from 3 proband rabbits were also observed in the Group C antibodies in as many as 7 out of 42 related rabbits, but in none of the Group C antibodies from 48 unrelated rabbits, Immunodiffusion analyses and quantitative radioprecipitin experiments revealed that this cross-specificity may be either partial or complete. Quantitative inhibition of the precipitin reaction between the proband antibody and its antiserum by preimmune IgG revealed 30-fold differences in the proportion of molecules with cross-specificity for the proband antibody. This proportion is higher in the preimmune IgG of the proband rabbit and of those relatives which produced cross-precipitating antibodies than it is in the IgG of rabbits which had the same group a allotype, but did not produce cross-precipitating antibodies. The proportion is much lower in the IgG of rabbits with a group a allotype different from that of the proband antibody. These data suggest that serologically detected individual antigenic specificities are inherited markers of immunoglobulins.

Human immune response to group A streptococcal carbohydrate (A-CHO). I. Quantitative and qualitative analysis of the A-CHO-specific B cell population responding in vitro to polyclonal and specific activation.

The immune response to the group-specific carbohydrate of group A streptococci (A-CHO) provides an informative in vitro model for the investigation of several aspects of human anticarbohydrate immune responses. A-CHO-specific B cells can be polyclonally activated by pokeweed mitogen (PWM), and, specifically, by in vitro immunization with streptococcal vaccine. High levels of A-CHO-specific antibodies, mainly directed to the immunodominant side chain N-acetyl-D-glucosamine (GlcNAc), occur in healthy adult individuals. Serum antibody levels are reflected in high frequencies of precursor B cells among peripheral blood lymphocytes. In one particular case, greater than 15% of all B cells activated by PWM for IgM production were found to produce IgM anti-A-CHO antibodies, as determined in limiting dilution experiments, as well as by analyzing Ig concentrations in bulk culture experiments. The case with the lowest proportion observed had 0.3% A-CHO-specific B cells among IgM-producing B cells. Preferential PWM activation of anti-A-CHO-producing B cells could be excluded. The comparison of the proportions of anti-A-CHO IgM produced in vivo, and of B cells producing antibodies of this specificity in peripheral blood, suggests a similar distribution of specific precursor B cells in the antibody-producing lymphoid tissue compartments and in peripheral blood. However, nearly all specific antibodies produced in vitro belong to the IgM isotype, whereas IgG anti-A-CHO in high amounts, mostly exceeding the specific IgM, was found only among anti-A-CHO antibodies produced in vivo. Low anti-A-CHO IgG production was seen in polyclonally activated as well as in antigen-activated cultures, whereas, in contrast, total IgG was produced in considerable amounts after polyclonal activation. This suggests a different distribution pattern, and/or diverse differentiation requirements for anti-A-CHO-producing B cells, compared with other B cell species.

Regulation of thymocyte development through CD3. II. Expression of T cell receptor beta CD3 epsilon and maturation to the CD4+8+ stage are highly correlated in individual thymocytes.

Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.

Proliferation kinetics associated with T cell receptor-beta chain selection of fetal murine thymocytes.

After productive rearrangement of a TCR beta chain gene, CD4-8- double negative (DN) thymocytes express TCR beta polypeptide chains on the cell surface together with pre-T alpha and the CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select TCR beta + DN thymocytes for further maturation to the CD4+8+ double positive stage, whereas DN cells that fail to generate a productive TCR beta gene rearrangement do not continue in development. This process is termed TCR beta chain selection. Although it is likely that differences between proliferation dynamics of TCR beta + and TCR beta-cells may play a role, the exact mechanisms of TCR beta chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCR beta + and TCR beta-thymocytes during fetal development, i.e., when TCR beta chain selection takes place for the first time. We analyzed in situ accumulation of TCR beta + thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCR beta + and TCR beta-populations by flow cytometry. About 600 TCR beta + cells/thymic lobe are generated by independent induction events between days of gestation (dg) 13.5, and 15.5. As of dg 14.5, most TCR beta + cells have entered S/G2 phase of cell cycle, followed by seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCR beta + cells has slowed down to less than 1/d. About three quarters of TCR beta-cells divide at a slow rate of 1/d on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d. From dg 16.5 onwards, TCR beta-cells, but not TCR beta + cells, contain a significant proportion of apoptotic cells. The results suggest that failure to become selected results in shutdown of proliferation and eventual programmed cell death of fetal TCR beta-cells. Positive selection of fetal TCR beta + cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.

Asynchronous coreceptor downregulation after positive thymic selection: prolonged maintenance of the double positive state in CD8 lineage differentiation due to sustained biosynthesis of the CD4 coreceptor.

In several experimental systems analyzing the generation of single positive (SP) thymocytes from double positive (DP) thymocytes, CD4 SP cells have been shown to appear before CD8 SP cells. This apparent temporal asymmetry in the maturation of CD4 SP and CD8 SP thymocytes could either be due to divergent molecular differentiation programs of the two T cell lineages, or merely to slower degradation kinetics of the CD4 protein. To study this question in unmanipulated in vivo differentiation, we developed a four-color flow cytometry protocol which identifies a recently activated TCRintCD69pos thymocyte population containing DP cells and early CD4 SP cells but no CD8 SP cells. We show that these TCRintCD69pos thymocytes represent a transitory stage in the mainstream alphabeta T cell lineage. The precursors of the CD8 SP cells are contained in this population as incompletely selected DP cells. Moreover, we show that expression of both coreceptors in the TCRintCD69pos population depends on transcriptional and translational activity, thus excluding differences in turnover rates of the CD4 and CD8 proteins as the cause of the asynchrony in differentiation of the CD4 and CD8 lineages.

Quantitative studies on T cell diversity. IV. Mathematical analysis of multiple limiting populations of effector and suppressor T cells.

Limiting dilution (LD) analyses of polyclonally activated T cells yielded results suggesting the existence of multiple paired populations of effector and suppressor precursors for a number of different T cell functions and specificities analyzed. These populations occur at graded frequencies and suppression occurs within a pair but not between pairs. In this paper, we establish the mathematical basis for the interpretation of these multi-component limiting dilution results. First, we derive equations for a number of mathematical models and identify one model that both makes biological sense and can be used to reproduce experimental data. Second, within this model, we identify parameters such as the frequency of suppressive cells and the number of suppressive cells required for suppression. The results suggest that within each paired population, suppressor precursors are 20 times more frequent that effector precursors. Furthermore, a similar but variable excess of suppressor cells is required for suppression to become effective. Together with the high frequency (1/50-1/500) of most effector T cell precursors previously reported, the results suggest that up to 40% of the T cells can become involved in suppression of an antigen-specific effector T cell population. These studies may provide exact estimates for predictions to be tested in experiments on immune regulation.

Murine macrophages secrete interferon gamma upon combined stimulation with interleukin (IL)-12 and IL-18: A novel pathway of autocrine macrophage activation.

Interferon (IFN)-gamma, a key immunoregulatory cytokine, has been thought to be produced solely by activated T cells and natural killer cells. In this study, we show that murine bone marrow- derived macrophages (BMMPhi) secrete large amounts of IFN-gamma upon appropriate stimulation. Although interleukin (IL)-12 and IL-18 alone induce low levels of IFN-gamma mRNA transcripts, the combined stimulation of BMMPhi with both cytokines leads to the efficient production of IFN-gamma protein. The macrophage-derived IFN-gamma is biologically active as shown by induction of inducible nitric oxide synthase as well as upregulation of CD40 in macrophages. Our findings uncover a novel pathway of autocrine macrophage activation by demonstrating that the macrophage is not only a key cell type responding to IFN-gamma but also a potent IFN-gamma-producing cell.

Regulation of thymocyte development through CD3. I. Timepoint of ligation of CD3 epsilon determines clonal deletion or induction of developmental program.

Several recent observations suggest that successful rearrangement of the T cell receptor (TCR) beta locus induces several important events in thymocyte maturation. Allelic exclusion is achieved by interruption of further rearrangement of the beta locus, and CD4-8- interleukin (IL)-2R+ cells enter the CD4+8+IL-2R- stage. The actual molecular events regulating this important control point are unknown, but may be related to the expression of the TCR-beta locus in immature CD4-8- thymocytes. It is not clear whether maturation is induced by intracellular appearance of TCR-beta chain or by signal transduction through an immature TCR complex on the thymocyte membrane, possibly involving TCR-beta chain homodimers and CD3. Here we show that early addition of anti-CD3 mAb to fetal thymic organ cultures induces all known events associated with the acquisition of the CD4+8+ stage. Expression of CD4 and CD8 is accelerated, IL-2R alpha is downregulated, and the cells fail to produce TCR-beta, possibly based on premature cessation of beta gene rearrangement. Upon stimulation with anti-CD3 antibodies, we see calcium mobilization in 15% of all CD4-8- thymocytes with no detectable surface TCR expression. These results suggest that functional CD3 is expressed on immature thymocytes at very low concentrations before the appearance of a complete TCR-beta chain. Ligation of CD3 at this stage may mimic the maturation signal normally generated by the immature TCR-beta homodimer-CD3 complex. The results are consistent with the notion that acquisition of the CD4+8+ stage involves signal transduction through an immature TCR complex. Later in thymocyte development, ligation of CD3 results in deletion of CD4+8+ cells. Thus, signal transduction through CD3 may result in entirely different cellular responses, depending on the stage of thymocyte differentiation. These results suggest an involvement of CD3 as a link in signal transduction for at least two different decision points in the development of a thymocyte.

Rabbit antibodies to streptococcal carbohydrates. Influence of primary and secondary immunization and of possible genetic factors on the antibody response.

In a search for possible genetic factors which may influence the immune response to the streptococcal carbohydrates, over 100 rabbits have been immunized with streptococcal vaccines, and representative examples of high and low response pairs mated. The concentration of precipitins to the group-specific carbohydrates has been measured in the antisera following primary intravenous immunization with heat-killed streptococcal vaccines, Group A, Group A-variant, and Group C. For the majority of rabbits, the concentration of precipitins varied between 1 and 10 mg/ml of antiserum; while in the minority, it was between 11 and 32 mg/ml. The offspring of rabbits with high antibody levels had a significantly higher concentration of antibody than was seen in the offspring of rabbits of low response parents. Such data suggest that the magnitude of the immune response to these carbohydrate antigens is under some form of genetic control. Not uncommonly in rabbits with hyper-gamma-globulinemia following primary immunization, the group-specific precipitins are the predominant component of the gamma-globulin. An unusual feature of such components is that they are electrophoretically monodisperse, and possess individual antigenic specificity. In this respect they resemble the myeloma proteins. When a response of this sort is not seen after primary immunization, it may occur after secondary immunization. Therefore, prior exposure to the same or closely related antigen may also have an influence on the occurrence of high concentrations of such uniform antibodies.

Quantitative studies on T cell diversity. III. Limiting dilution analysis of precursor cells for T helper cells reactive to xenogeneic erythrocytes.

Splenic T cells exposed to concanavalin A (Con A), and subsequently to factors produced by rat spleen cells in response to Con A (Con A sup), acquire the ability to function as helper T (TH) cells in response to xenogeneic erythrocytes (RBC). Help is measured as the reconstitution of the plaque-forming cell response of a spleen cell population depleted of T cells by treatment with anti-Thy-1 serum and complement. We propose that precursor TH cells differentiate during the in vitro treatment into mature TH cells. As differentiation occurs under limiting dilution conditions, an estimation of the precursor frequency should in principle be possible. However, a single-hit Poisson distribution does not fit our data. Instead, we observe, dependent on the T cell concentration, three separate "peaks" of response. In many experiments, using sheep, horse, and chicken RBC as antigens, we reproducibly find these "peaks" at 40-190, 600-3,000, and 20,000-100,000 T cells, placed into limiting dilution cultures, respectively. By various experiments we can show that the helper activity is not due to passively transferred rat factors, but to the titrated cells themselves. The active cell is a T cell that appears to function in an antigen-specific way and to require direct cell contact to do so. It thus resembles the classical helper T cell. As we find precursor TH cells already at very low concentrations of T cells, we titrated the range between 0 and 100 T cells/well carefully. The bent shape of the titration curves does not always allow a statistically satisfying regression analysis, and we therefore cannot estimate precise precursor frequencies from every experiment. However, a common sense argument can be made that these frequencies must be on the order of 1/10-1/100 T cells. We propose that the limiting dilution curves obtained in this system most likely reflect fundamentally important cellular interactions that regulate immunological effector functions. We favor a concept of independently interacting sets of helper and suppressor T cells of various frequencies, but other models are possible.

Antigen receptors on major histocompatibility complex-restricted T lymphocytes. I. Preparation and characterization of syngeneic antisera against trinitrophenyl-activated T cell blasts and demonstration of their specificity for idiotypes on cytotoxic T lymphocytes.

This paper describes the specificity of AKR anti-(a) [AKR anti-trinitrophenylated AKR (AKR-TNP)] [AKRa (AKRaAKR TNP)] antisera raised in syngeneic AKR mice against AKRaAKR-TNP cell populations enriched for H-2-restricted aTNP cytotoxic lymphocytes (CTL) by blast-cell isolation. The activity of the antisera resided in the Ig fraction. All antisera were shown to reproducibly react with AKRaAKR-TNP-CTL-containing cell populations in indirect immunofluorescence and all removed the major fraction of CTL in complement-dependent lysis causing a considerable depression of cell-mediated lympholysis. The antisera were nonreactive with alloreactive AKRaC57BL/6 CTL and other H-2-restricted AKR CTL against fluorescein-isothiocyanate-conjugated AKR-target cells. It could be excluded that the antisera contained contaminating antibodies against TNP, TNP-neoantigenic determinants (NAD), or processed CTL-receptor-bound TNP-NAD, thus demonstrating specificity for determinants on T cell receptors of AKRaAKR-TNP CTL. These receptors were produced by the CTL themselves. These observations are interpreted to suggest that AKRa (AKRaAKR-TNP) antisera contain anti-idiotypic antibodies directed against specificity-associated determinants (idiotypes) on T cell receptors of H-2-restricted AKRaAKR-TNP CTL. The antisera provide a new tool to study the genetic control of idiotype expression on H-2-restricted CTL, the biochemistry of T cell receptors, and the regulation of the generation of H-2 restricted CTL on the idiotype level.

Genetics of the idotype of BALB/c myeloma S117: multiple chromosomal loci for Vh genes encoding specificity for group A streptococcal carbohydrate.

A small proportion of the antibodies to Group A streptococcal carbohydrate (A-CHO) elicited in BALB/c mice by immunization with Group A streptococci, has idiotypic determinants in common with the BALB/c myeloma protein S117 which has specificity for N-acetyl-glucosamine, the major antigenic determinant of A-CHO. The expression of these idiotypic determinants is under the control of a gene which is linked to the Ig-1a+ allotype locus in strain BALB/c and in other strains carrying the same Ig-1 haplotype. This gene (S117+) segregates in breeding experiments as if it were an allele to the gene A5A+ which controls the expression of the A5A idiotype in association with antibodies to A-CHO in strain A/J and which is linked to the Ig-1e allotype locus. Another possible allele, linked to the Ig-1c allotype locus, controls the expression of both S117 and A5A cross-reactive determinants (S117cr, A5Acr). The distribution of these idiotypic determinants in various lines that carry recombinant Ig-1 haplotypes suggests that the A5A and S117 loci are nonallelic and map at different positions in the Ig-1 region. The data suggest complex pseudollelic relationships between different Ig-1 haplotypes that allow the expression of the same genes in allelic and in nonallelic fashion.

Absolute frequencies of lipopolysaccharide-reactive B cells producing A5A idiotype in unprimed, streptococcal A carbohydrate-primed, anti-A5A idiotype-sensitized and anti-A5A idiotype-suppressed A/J mice.

The absolute frequencies of B cells-producing A5A idiotype have been determined in vitro by limiting dilution analysis in a culture system in which every LPS-reactive B cell grows into a clone of IgM-secreting cells. Spleen cells from normal A/J mice contain 1 A5A-idiotype-producing B-cell precursor in 2.5 X 10(3) LPS-reactive B cells. Approximately a 10-20-fold increase in frequencies of precursor cells from antigen priming with Strep A-CHO (1 in 2.8 X 10(2)) or from sensitization with IgG1 anti-A5A idiotype (1 in 1.3 X 10(2)). Injection of IgG2 anti-A5A idiotype which has been shown to suppress A5A idiotype in vivo results in only a marginal and maybe insignificant decrease in precursor frequencies (1 in 6.7 X 10(3)). On the other hand, priming does not result in a detectable qualitative difference in the specific precursor cells, since each clone of B cells secretes 30 ng of A5A-bearing Ig within 8 days of culture, regardless of being unprimed or primed. Nearly half of all A5A idiotype-producing clones, both from unprimed as well as from primed mice, show antigen specificity in binding A-CHO. Priming by antigen, therefore, also results in a 10-fold increase in the frequency of idiotype positive B cells without antigen specificity. This result is a prediction of the network hypothesis.

Influence of genetic factors on the magnitude and the heterogeneity of the immune response in the rabbit.

Selective breeding of rabbits immunized with Group C and Group A streptococcal vaccines was employed to reveal genetic influences on the magnitude and on the restriction in heterogeneity of the immune response to the group-specific carbohydrates. After two generations of selective breeding, complete segregation was achieved between a high-response population (>18 mg precipitins/ml serum, average 33 mg/ml) and a low-response population (<13 mg precipitins/ml serum, average 7.5 mg/ml) to Group C carbohydrate. This suggests that a limited number of genes controls the magnitude of the immune response to this antigen. Selective breeding of rabbits which were representative of heterogeneous, restricted, and monoclonal responses revealed that the degree of antibody heterogeneity in the parental rabbits is reflected in the offspring. More than 95% of the offspring derived from rabbits which had a heterogeneous immune response developed heterogeneous antibodies. 33% of the offspring derived from rabbits which had restricted and monoclonal immune responses developed monoclonal antibodies. This suggests that the degree of heterogeneity of the antibody response to the streptococcal carbohydrates is under genetic control. The degree of heterogeneity and the magnitude of the immune response appear to be independent variables.

Quantitative studies on T cell diversity. II. Determination of the frequencies and Lyt phenotypes of two types of precursor cells for alloreactive cytotoxic T cells in polyclonally and specifically activated splenic T cells.

Two different limiting dilution systems have been applied to compare precursor frequencies of alloreactive cytotoxic T cells (CTL-P) in the polyclonally and specifically activated lymphocyte populations and in selected Lyt T cell subsets. Both systems make use of T cell growth factor for T cell expansion but differ with respect to the activation step in that lymphocytes are either activated directly with allogenetic stimulator cells or are sensitized polyclonally with concanavalin A (Con A) in bulk culture before their expansion under limiting dilution conditions. In polyclonally activated C57BL/6 lymphocyte populations, two types of CTL-P specific for H-2d alloantigens could be identified: a frequent set with a frequency of 1/100-1/300, and a rare set with a frequency of 1/2,000-1/8,000. In contrast, only a single CTL-P set was found in specifically activated populations with a frequency similar to that of the frequent CTL-P found on Con A blasts. In Con A blasts, the frequent at higher cell concentrations by suppressor T cells, whereas rare CTL-P were insensitive to this suppressive mechanism. Whereas in specifically activated T cells, the predominant CTL-P phenotype was Lyt-123, the predominant Lyt phenotypes for the frequent and the rare CTL-P found in Con A blasts were Lyt-123 and Lyt-123, respectively, which suggests that they represent primary and secondary CTL-P, respectively. The results are discussed with respect to previous reports on the involvement of Lyt T cell subsets in the generation of cytotoxic responses and their regulation by T suppressor cells.

The emergence of antibodies with either identical or unrelated individual antigenic specificity during repeated immunizations with streptococcal vaccines.

Electrophoretically monodisperse antibody components in rabbit antisera to the carbohydrates of the Groups A and C streptococci have been examined for their individual antigenic specificity. In these antibody components which were isolated by preparative electrophoresis, individual antigenic specificity was confined to the specific antibody and was absent in the nonantibody gamma-globulin. Radioprecipitation experiments and the use of immune absorbent columns constructed from goat anti-antisera, which had been absorbed with fraction II, revealed that all the specific antibody in an electrophoretically monodisperse component was reactive with the homologous anti-antibody. Antibodies with either identical or distinct individual antigenic specificities may occur in the same rabbit with repeated immunizations. Antibodies with identical antigenic specificity had identical electrophoretic mobility, whereas antibodies with unrelated antigenic specificities had distinct electrophoretic mobilities. In the interval between immunizations, if antibody to the carbohydrate antigen was absent, there was no detectable antibody with individual antigenic specificity.

Induction of rabbit antibody with molecular uniformity after immunization with group C streptococci.

Antibodies with uniform properties may occur in rabbits after immunization with Group C streptococci. These precipitating antibodies possess specificity for the group-specific carbohydrate. Not uncommonly, their concentration is between 20 and 40 mg/ml of antiserum. Evidence for molecular uniformity in the case of one of these antibodies, described in detail here, includes: individual antigenic specificity; monodisperse distribution of the light chains by alkaline urea polyacrylamide disc electrophoresis; and a single amino acid in each of the first three N-terminal positions of the light chains. When the amino acid sequence of rabbit antibody b+ light chains (kappa type) are aligned against their human kappa counterparts, a definite homology is observed between the N-terminus of the human and the rabbit variable region.

Allotype exclusion in uniform rabbit antibody to streptococcal carbohydrate.

Rabbit antibodies to streptococcal polysaccharide are described which show selectivity of expression of the allotypic specificities on both the heavy (H) and light (L) chains. One of these antibodies binds weakly to Sephadex. A purification method based on this binding has yielded antibody completely lacking any group a allotypic marker on its H chains.