Isoelectric focus analysis of rat anti-phosphocholine antibodies.

Anti-phosphocholine (PC) antibodies in sera from four strains of rats were examined before and afterimmunization with either Streptococcus pneumoniae R36A, which contains PC as a cell wall component, or with PC-coupled keyhole limpet hemocyanin (PC-KLH). PC-specific protein was purified from pooled immune sera and shown by a combination of isoelectric focus (IEF) in acrylamide and crossed immunoelectrophoresis, as well as by molecular weight determination in NaDodSO4-acrylamide, to be immunoglobulin. An additional, small molecular weight, nonimmunoglobulin protein (pI = 7.1-7.3) was present in sera from normal and germ-free rats which had the ability to bind the C-carbohydrate of S. pneumoniae R36A, but without specificity for PC. The IEF profile of normal and immune sera showed marked sharing of bands of anti-PC antibody between individual rats as well as between strains. In addition, other anti-PC antibodies which focused between pH 8.5 and 9.5 were less regularly shared. The uniformity of IEF profile of the bulk of anti-PC antibodies in rats is most consistent with their being the products of germ line genes.     

Functional, structural, and antigenic similarities of guinea pig anti-phosphorylcholine antibodies.

The humoral immune response to PC was measured in guinea pigs. PC-vaccine stimulated IgM and IgG2, but little IgG1, anti-PC -antibodies. No memory was induced and immunization in CFA produced tolerance. PC-KLH, on the other hand, stimulated IgM, IgG2, and IgG1 anti-PC antibodies with carrier-specific memory. Hapten inhibition of plaque formation showed uniform binding patterns with minor, but significant, differences between antiPC-vaccine and anti-PC-KLH antibodies. The antibodies were characterized by IEF and idiotypic analyses. Early after immunization with PC-vaccine, guinea pigs had restricted IEF patterns which in inbred, but not outbred animals were indistinguishable between individuals. These patterns remained restricted but more individualized with time after immunization. Anti-PC-KLH antibodies showed more heterogeneity and individuality. However, these structurally heterogeneous antibodies reacted equivalently with rabbit anti-idiotype antisera and therefore must share common structural features, regardless of isotype or the genetic background of the guinea pig.     

Subpopulations of antibodies to phosphocholine in human serum

We investigated the heterogeneity of anti-phosphocholine (PC) antibodies present in human serum taken from individuals before and after immunization with a multivalent pneumococcal vaccine. The fine specificity of IgM, IgG, and IgA anti-PC antibodies was determined in an ELISA by using phosphocholine or p-nitrophenyl phosphocholine (NPPC) to inhibit binding of antibody to PC-histone. We identified two populations specific for PC that differed in their binding properties. One population is inhibited by NPPC much better than by PC and is most evident in IgG antibodies. The second population has similar avidity for PC and NPPC and is consistently associated with the IgM and IgA isotypes as well as with IgG. The IgG antibodies in both populations were predominantly of the IgG2 subclass. Both populations were found in serum samples taken before immunization with pneumococcal vaccine, suggesting that they had been stimulated through prior environmental contacts with PC-containing antigens. Previously, we found populations with similar fine specificity patterns in the murine response to PC. The two murine antibody populations have been shown to derive from different immunoglobulin variable region genes. The presence of comparable antibody populations in the human suggests the possibility that these two fine specificity families have been conserved in evolution.     

Subclass distribution of human antibodies to Haemophilus influenzae type b capsular polysaccharide

The polysaccharide (PS) capsule of Haemophilus influenzae type b (Hib) is a "simple" antigen, polyribosylribitolphosphate. Although similar carbohydrate antigens have been reported to elicit IgG antibodies relatively restricted to the IgG2 subclass in man, we report here that Hib PS elicits substantial quantities of both IgG1 and IgG2 serum antibodies in most individuals. Because the determination of IgG subclass distribution can be technically difficult, we used four different approaches to establish our finding. First, we used an IgG subclass-specific, antigen-specific "sandwich assay." Second, we measured IgG subclasses of purified antibodies to Hib PS. Third, we showed that significant amounts of IgG anti-PS can be absorbed with a monoclonal anti-IgG1 affinity column. Fourth, we showed that IgG1 and IgG2 fractions of immune sera have clonally restricted anti-Hib PS antibodies that are easily distinguishable by their isoelectric points. The data indicate that both IgG1 and IgG2 contribute substantially to the IgG antibody response of most adults to immunization with Hib PS.     

Heterogeneity and specificity of human anti-insulin antibodies determined by isoelectric focusing

Anti-insulin antibodies are present in the majority of insulin treated diabetics, and in some cases these antibodies have been found to be highly specific for limited epitopes on the molecule. To determine how the human response differs from that seen in inbred animals, we have examined the heterogeneity and specificity of human anti-insulin antibodies by isoelectric focusing (IEF). In addition, we have used human insulin to examine the extent of autoreactivity in the serum of subjects treated with animal insulins. The majority of diabetic sera exhibited complex IEF spectra that were composed of discrete bands and unresolved smears. Autoradiography using 125I-beef, pork, and human insulin revealed some affinity differences; however, the predominant antibodies were capable of binding all insulins, including human. These specificity studies were extended by comparing competitive inhibition with excess cold insulins, and sera with highly specific binding of the A chain loop of beef insulin were identified. The spectra by IEF of these highly specific sera were found to be variable. Our results indicate that the majority of insulin-treated diabetics develop a heterogeneous antibody response that is more complex than the response of inbred animals and includes reactivity with autologous insulin. Although infrequent, individuals having antibodies directed at limited regions of the molecule can be identified and will provide valuable tools for dissecting this complex response.     

Isotype concentrations of human antibodies to group A meningococcal polysaccharide

Antibodies to meningococcal group A polysaccharide (MenA) in the sera of 34 vaccinated adults were quantitated by an isotype-resolving solid-phase RIA (IgA, IgM, IgG1, IgG2, IgG3, and IgG4). All individuals had antibodies before vaccination. The geometric mean concentration was 2.9 micrograms/ml. Two weeks after vaccination the mean antibody concentration had trebled. Average proportions of the three isotypes were then as follows: IgA 15%, IgM 48%, IgG 37%. No differences were found between individuals who had been immunized with the polysaccharide 7 to 8 yr earlier and "primary responders." The subclass composition of IgG antibodies was determined in the 24 postvaccination samples with a definite IgG response (greater than 2-fold increase). IgG1 was the predominant subclass in antibodies of some sera and IgG2 in others, but the average proportions of both subclasses were nearly the same. IgG3 and IgG4 were only found in occasional sera, but when present, each subclass accounted for up to 6%. Although the ratio of kappa and lambda chains could not be determined, there was evidence to suggest that it was higher in anti-MenA antibodies than in antibodies to protein antigens.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type B polysaccharide. Demonstration of three types of V regions and their association with H and L chain isotypes

The antibody response to Haemophilus influenzae type b polysaccharide (Hib-PS) is pauciclonal but can vary between different individuals. To estimate the size of this antibody repertoire we examined the constant and V regions of human IgG anti-Hib antibodies from 14 individuals at the clonal level using various serologic and IEF methods. Examination of H chains showed that 11 of 14 individuals produced both IgG1 and IgG2 antibodies, two individuals produced only IgG2 and one individual produced only IgG1 antibody. All 14 individuals produced kappa-containing antibody clones and three persons also produced significant lambda-containing antibody clones. V region heterogeneity was examined by comparing cross-reactivity of anti-Hib-PS antibodies to the capsular polysaccharide of Escherichia coli K100 carbohydrate (K100 CHO). These studies showed that clones of IgG anti-Hib-PS antibodies cross-reactive with K100 CHO were present in 5 of 14 (36%) individuals and also revealed at least three types of V regions among these antibodies. The first type has no cross-reaction with K100 CHO and was found in 13 of the 14 individuals. The second type, found in three of 14 individuals, cross-reacts with K100 CHO and uses a lambda L chain V region. The third type, found in 2 of 14 individuals, cross-reacts with K100 CHO and uses a kappa L chain V region. Although the lambda type V region was found only in association with IgG2, the other two V region types associate with both IgG1 and IgG2. Thus, five IgG antibody clones are serologically discernable. An individual generally responds to Hib-PS by expressing several clones selected from these discernable antibody clones. Indeed, we can observe six individual response patterns among these 14 individuals and conclude that considerable variability in individual responses to Hib-PS can be achieved with very few V regions.     

Subclass restriction of murine anti-carbohydrate antibodies.

Examination of the subclass distribution of murine antibodies directed against groups A and C streptococcal carbohydrate, alpha-(1 leads to 3) dextran and phosphocholine yields the surprising observation that these carbohydrate antigens stimulate IgG responses largely restricted to the rare IgG3 subclass. This subclass restriction is particularly impressive in light of the low circulating levels of IgG3 in nonimmune mouse serum and the failure of a variety of other antigens including proteins and aromatic haptens to stimulate IgG3 antibody production. Attempts to alter the subclass restriction of antibodies with carbohydrate specificity by immunization with carbohydrate-coupled protein have been unsuccessful and indicate that immunoregulation of subclass expression probably occurs at the level of the antibody forming (B) cell. It is therefore conceivable that VH regions of murine immunoglobulins may be restricted to particular IgG subclasses. A similar type of subclass restriction has been reported in human and rat anti-carbohydrate antibodies. This recruitment of a minor immunoglobulin isotype by carbohydrate antigens in several species further supports the concept of immunoregulation at the level of subclass, and suggests that these and other mammals may share a structurally similar isotype with perhaps a common evolutionary origin.     

A restricted human antitetanus clonotype shares idiotypic cross-reactivity with tetanus antibodies from most human donors and rabbits: reactivity with antibodies of widely differing electrophoretic mobility

Antitetanus antibodies from each of 20 hyperimmunized human donors were isolated on a tetanus immunoadsorbent, eluted with acidic buffer, and examined by isoelectric focusing (IEF). There was electrophoretic restriction, as determined by IEF, in the IgG of only 20% of the purified antibodies studied. The remaining 80% showed a more diffuse polyclonal spectrotype. Several IEF bands from the most electrophoretically restricted sample were isolated and used to immunize rabbits. Virtually every IgG-IEF band in the antitetanus antibodies of the original donor shared idiotypic cross-reactivity as detected by one or more of the three rabbit anti-Id reagents, even though major qualitative differences in binding from one rabbit anti-Id reagent to another were noted. Antitetanus antibodies of each of the 20 donors were separated by IEF and transferred to a nitrocellulose membrane. By using a sensitive and specific ELISA detection method, cross-reactivity was detected with the rabbit anti-Id reagents in 1 to 50% of the antitetanus antibodies of individual donors. This cross-reactivity was greater than 10% in 15 of the 19 antisera studied. In addition, these cross-reactive antibodies had very different electrophoretic mobility. Binding of the rabbit anti-Id reagents to the tetanus antibodies was almost completely blocked by pretreatment with soluble tetanus toxoid antigen. This idiotypic cross-reactivity with antibodies of different electrophoretic mobility from the same and unrelated donors suggests sharing among these antibodies of one or more of the germ-line DNA-encoded hypervariable regions present in the antibody-combining site.     

Further characterization of cooperative interactions of monoclonal antibodies

We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays.     

Alteration in H chain V region affects complement activation by chimeric antibodies

Chimeric antibodies to the synthetic polypeptide (Tyr, Glu)-Ala-Lys ((T,G)-A-L) were used to examine C activation by human IgG1. Two IgG1 antibodies, which contained mouse L chains and H chains with mouse V domains and human C domains, differed only in their VH domain. Ag binding and C activation by these antibodies were analyzed by ELISA. When limiting amounts of Ag were used in the assays, the antibodies required different quantities of Ag for optimal binding, suggesting that the antibodies bind to different epitopes on the (T,G)-A-L molecule. However, when competitive inhibition assays were performed with an optimal concentration of Ag, there were no differences in relative binding affinities for (T,G)-A-L or dissociation characteristics of the antibodies. C activation was examined at optimal Ag concentration to ensure equivalent binding of two IgG1 antibodies to Ag. After combination with immobilized Ag, these two antibodies bearing different V regions exhibited marked differences in the binding of C components C1q and C3d. When present in equal amounts in the assay, antibody 10B activated C and bound more C1q and C3d than antibody B11. These results indicate that V region differences can affect C activation by IgG.     

Analysis of a common inheritable idiotype in IgA-deficient sera using monoclonal antibodies

In these studies we describe the production of three mAb raised to an idiotype on an IgG anticasein antibody isolated from the serum of one IgA-deficient blood donor. These are IgM kappa and block the binding of casein Ag to anticasein antibody. Sera of unrelated IgA-deficient donors were tested for the presence of the idiotype; 15 of 56 IgA-deficient sera (25%) contain the anticasein idiotype, whereas 1 of 45 normal sera was positive. Anticasein antibodies as a whole were predominantly of the IgG1 and IgG3 subclass; idiotype-positive anticaseins are predominantly of the IgG1 subclass. For IgA-deficient donors, the relative amount of idiotype-positive anticasein antibody was correlated with the level of anticasein present in the serum. Studies were done to investigate the potential inheritance of the idiotype in families; in three of four families the idiotype was inherited in an apparent autosomal dominant pattern. Our data show that a common cross-reactive idiotype can be detected in the sera of IgA-deficient individuals and their family members. This suggests that V region markers may be conserved in this humoral immunodeficiency disease.     

IgA isotype-restricted idiotypes associated with T15 Id+ PC antibodies

Idiotypes are believed to be due to the structural conformation of the variable region of immunoglobulins (Ig). We have found an idiotype (C3-24) that requires both variable and constant regions of the heavy chain to be expressed. C3-24 Id is associated with both the T15 variable region from anti-phosphorylcholine (PC) antibodies and the constant region for the alpha-heavy chain. High titer anti-PC serum from a variety of inbred strains of different Ig haplotypes failed to express C3-24 Id. However, when IgA but not IgG or IgM fractions were isolated from a pool of anti-PC serum from BALB/c mice, more than 70% of the molecules expressed C3-24 Id. The high frequency of the expression of C3-24 Id in IgA anti-PC hybridoma proteins from mice of different Ig haplotypes and in the IgA fraction of normal anti-PC antibodies from BALB/c and presumably other strains of mice suggests that idiotypic determinants produced by the three-dimensional product of VH and CH regions may not be unusual.     

Patterns of mutations and selection in antibodies to the phosphocholine-specific determinant in Proteus morganii

The contribution of somatic mutation to the generation of an antibody response was investigated by using the phosphocholine (PC) determinant in the bacterium Proteus morganii as the model Ag. The response to this determinant is restricted to a single VH/VL pair and apparently is derived from only one or two precursors per mouse. In this study we examined hybridoma antibodies from nine individual mice which produced representatives of 12 different clones. We found that all antibodies reactive with the PC Ag of P. morganii contained somatic mutations; the number ranged from 2 to 20. Two clusters of mutations were observed, one in complementarity-determining residue (CDR) 2 and the other in CDR 3 of VH. Examination of a three-dimensional model of M603, an antibody with the same V region composition as the anti-PC antibodies under study, showed that these clusters occupied an area of the binding site which presumably interacts with carrier elements of the PC epitope in P. morganii. A high incidence of recurring mutations were found in both clusters, and one of these was invariant, leading to an Asn for Asp substitution at 95. Ag binding studies with these antibodies and an additional one, which was unmutated except for the invariant substitution at 95, showed that: 1) antibodies having only the 95Asn mutation failed to bind the PC Ag of P. morganii, 2) addition of a second recurring mutation, at 52a (CDR 2), was sufficient to create strong binding to the P. morganii Ag, and 3) accumulation of mutations was directly correlated with increased binding activity for Ag. These results show that somatic mutations play a critical, if not essential, role in generating specificity for this PC Ag, and that Ag, and most likely a carrier element of the epitope, is a primary force in the continued selection and expansion of Ag-reactive B cells.     

Production and characterization of two monoclonal antibodies against human myeloperoxidase

Two monoclonal antibodies against human myeloperoxidase, designated 3-2H3 and 4-2C11, were produced and characterized. Both bound to the native enzyme, but neither bound to the denatured enzyme or to its two denatured subunits. 4-2C11 bound to the three types of leukocyte myeloperoxidase, I, II, and III, as well as to the four types of myeloid leukemia HL-60 cell myeloperoxidase, IA, IB, II, and III. 3-2H3 did not bind to enzyme IB, but bound to the other types of leukocyte and HL-60 cell enzymes. On incubation with myeloperoxidase III, 4-2C11 inhibited the enzyme activity, but 3-2H3 did not. Both antibodies belong to the IgG1 subclass.     

The subclass distribution of human IgG rheumatoid factor

The subclass distribution of IgG rheumatoid factor (RF) was determined by a sensitive ELISA assay in sera from patients with rheumatoid arthritis and from normal controls. In both instances, the most important subclasses were IgG1 and IgG4. The IgG4 RF was directed against the Fc region of IgG, and recognized human as well as rabbit IgG. Although human IgG4 myeloma proteins bound to rabbit IgG better than did myelomas of other IgG subclasses, the IgG4 RF activity in rheumatoid sera showed an additional specificity, because the fraction of IgG4 RF/total IgG4 for rheumatoid arthritis sera was far greater than for myelomas. This inference was supported by the observation that there was persistent, albeit diminished, IgG RF activity in pepsin-digested, RF-containing sera (but not myeloma proteins), indicating that a critical component of IgG4 RF activity was contained within the Fab region of the IgG4 molecule. The finding of large quantities of IgG4 RF was not due to a bias of the assay, because the preponderance of IgG4 did not extend to the subclass distribution of antibodies directed against other antigens. The demonstration of an important role for IgG4 as a RF is of special interest because of the relative inability of this subclass to fix complement or to bind to Fc receptors, and because of its potential role as a mediator of increased vascular permeability.     

Human antibodies to group A streptococcal carbohydrate. Ontogeny, subclass restriction, and clonal diversity

To investigate immuno-incompetence to polysaccharide Ag in young children, antibodies to the polysaccharide and protein Ag of Streptococcus pyogenes were studied. S. pyogenes was chosen because it commonly causes natural infections and has well-characterized polysaccharide and protein Ag. In children over the age of 2 yr it was found that the maturation of antibody responses to the polysaccharide Ag of S. pyogenes (A-CHO) appeared to occur in parallel with, or even earlier, than the responses to streptococcal protein Ag. When antibodies to group A carbohydrate (A-CHO) were studied in detail, qualitative differences between the antibodies of children and adults were demonstrated. Although anti-A-CHO antibodies of adults were strikingly restricted to the IgG2 subclass, those of children were found in both the IgG1 and IgG2 subclasses. In addition, the clonal diversity of IgG antibodies to A-CHO increased with age, and additional clonotypes were detectable in convalescent sera of some subjects of all ages after infection. Two cases with major additional clonotypes after group A streptococcal infection were studied in detail. In these two cases the additional clonotypes belonged to a different IgG subclass than the previously dominant clonotypes, and the expression of the additional major clonotypes occurred in both IgG1 and IgG2 subclasses.     

IgG antibodies to secretory component in normal human serum

While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.     

Human IgG2 antibodies display disulfide-mediated structural isoforms

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C(L)) and the heavy chain C(H)1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C(H)1-C(L)-hinge)(2) complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either kappa or lambda light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.     

Natural antibodies to IFN-gamma in man and their increase during viral infection.

Natural antibodies to IFN-gamma were found in healthy individuals ranging from newborn babies to adults and, at higher levels, in patients suffering from different viral infections. During a viral infection, the titer of anti-IFN-gamma antibodies was observed to be correlated with the stage of the disease. Antibodies specific to IFN-gamma were affinity purified both from sera taken from healthy individuals and sera from viral-infected patients, by using a rIFN-gamma-coupled CNBr-activated Sepharose 4B column. The antibodies were found to be of the IgG class, and maintained their ability to bind rIFN-gamma. They were then tested for neutralizing activity and none of the IgG preparations we analyzed impaired the antiviral activity of rIFN-gamma. This finding suggests that the antigenic determinants recognized by these antibodies on the IFN-gamma molecule are located outside the site, on the IFN-gamma molecule, responsible for its antiviral activity.