Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup

Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.     

Inhibition of anti-IgM and growth factor-induced proliferation of guinea pig B cells by one of two distinct Fc gamma receptors on the cells

Guinea pig B cells were found to proliferate when co-stimulated with F(ab')2 of rabbit anti-guinea pig IgM and human 12-kDa B cell growth factor (BCGF), though the proliferation did not occur with the replacement of the F(ab')2 by its parent IgG antibody. In addition, the intact antibody inhibited the proliferation induced by F(ab')2 of anti-IgM and BCGF. Because both two distinct types of FcR for IgG on the B cells, one specific for IgG2 (Fc gamma 2R) and the other for both IgG2 and IgG1 (Fc gamma 1/gamma 2R), can bind rabbit IgG, we determined whether they participate in the inhibition of the B cell proliferation by intact anti-guinea pig IgM antibody. Blocking Fc gamma 1/gamma 2R by F(ab')2 of anti-Fc gamma 1/gamma 2R mAb significantly reversed the inhibitory effect of intact anti-IgM antibody. F(ab')2 of anti-Fc gamma 2R mAb, however, was not effective. Furthermore, guinea pig IgG1 and IgG2 anti-rabbit IgG antibodies suppressed similarly the B cell proliferation induced by F(ab')2 of rabbit anti-IgM and BCGF. These results show that between these two types of Fc gamma R on B cells, Fc gamma 1/gamma 2R alone is involved in the regulation of anti-IgM and BCGF-induced B cell proliferation, and inhibits the response when cross-linked to the surface IgM.     

A human monoclonal IgG1 with anti-idiotypic activity against anti-human thyroglobulin autoantibody

Sixty-one human myeloma proteins (HMP) from patients with multiple myeloma and Waldenstr?m macroglobulinemia were tested for anti-idiotypic (Id) activity against autoantibodies to double-stranded DNA, small nuclear ribonucleoproteins, and human thyroglobulin (HTg), by competitive radioimmunoassays and enzyme immunoassays. An IgG1, lambda HMP from patient BEN with anti-Id activity against antibodies to HTg is reported. IgG1 BEN was not directed toward human Fc fragments and its activity was not related to allotypic determinants. IgG1 BEN molecules recognized Id determinants (idiotopes) on F(ab')2 anti-HTg fragments, but not idiotopes of F(ab')2 fragments of antibodies of other specificities. This observation supports the general significance of Id network interactions in regulation and diversification of immune responses in man.     

Idiotypic interactions in type II mixed cryoglobulins

An analysis of interactions between immunoglobulin molecules within cryoglobulins has been carried out in 18 patients with type II mixed cryoglobulinemia. In this series, there was a prevalence of VH I and VK I variable regions subgroups in the monoclonal IgM component. Our analysis of the IgG component indicated a particular selection of IgG molecules during cryoprecipitation. There was a prevalence of IgG3 and of the VH I subgroup and the isoelectrofocusing pattern revealed a very restricted spectrotype in two thirds of these IgG. These results which suggested a restricted reactivity between cryo-IgM and IgG fractions were confirmed by the analysis of the interaction between each IgG and each IgM from the cryoprecipitates. All IgM reacted with intact IgG or Fc fragments but another reaction was observed between cryo-IgM and Fab fragments from a limited number of cryo-IgG, with a pattern suggestive of idiotypic specificity. Results of the absorption of each cryo-IgM on Fc or on Fab fragments from the corresponding cryo-IgG also suggested the existence of a reaction between IgM and IgG Fab in addition to that involving IgM Fab and IgG Fc. The coexistence of the 2 reactions should confer a higher stability to the IgM-IgG complex. Therefore, it is possible that the proliferation of one clone of IgM-RF producing B cells would be followed in certain cases by a relatively restricted anti-idiotypic IgG response. The IgM-RF would preferentially react with these anti-idiotypic IgG.     

On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.     

The Fc portion of intact IgG blocks stimulation of human PBMC by anti-T3

The means by which normal human serum inhibits the activation of PBMC by OKT3 has been investigated. The Fc portion of intact IgG is shown to be the major inhibitor in human serum of this OKT3-induced stimulation. Furthermore, inhibition by IgG subclasses correlated with their ability to bind to the monocyte Fc receptor, i.e., IgG1 and IgG3 produced greater inhibition than IgG2 and IgG4, and this inhibition was competitive. In contrast, hypogammaglobulinemic serum, IgA, IgM, and F(ab')2 of IgG were not inhibitory relative to intact IgG or Fc of IgG. Because of the similarities between T3 and the idiotype-defined T cell receptor for antigen, these investigations suggest that IgG might modulate the interactions between the T cell recognition complex and anti-idiotype antibody, thus regulating the idiotype network.     

Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Synthesis of bifunctional antibodies for immunoassays

The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.     

Requirements for the opsonic activity of human IgG directed to type 6 group A streptococci: net basic charge and intact Fc region

By two independent techniques for separating human opsonic IgG for group A type 6 streptococci into fast- and slow-migrating fractions, it was found that the opsonic activity was localized within the basic charge population. This charge dependence was found to be a characteristic of the IgG isolated from three individuals. When the fast- and slow-migrating IgG fractions were tested for their ability to bind to purified M6 protein, antibodies in both opsonic and nonopsonic populations exhibited binding activity, with the majority being located within the opsonic IgG in two of the three individuals; the third displayed greater binding in the nonopsonic population. The functional difference observed in the antibody populations to this M antigen may be a reflection of the net charge within the area of the antibody binding site, which suggests that the opsonic antibodies need to bind to acidic residues along the outer surface of the fibrillar M protein molecule. F(ab')2 fragments prepared from both human and rabbit type 6 opsonic IgG were still able to bind to the M6 molecule but were unable to mediate opsonization of type 6 streptococci. However, the F(ab')2 fragments had the capacity to enhance or amplify the opsonic activity of low concentrations of opsonic IgG molecules. The results suggest that the M protein molecule may function as an active inhibitor of phagocytosis and that F(ab')2 fragments from opsonic IgG have the capacity to neutralize the "active" determinants on the molecule, thus allowing lower concentrations of IgG with functional Fc receptors to mediate phagocytosis.     

F(ab')2 reagents are not required if goat, rather than rabbit, antibodies are used to detect human surface immunoglobulin.

The present report provides evidence that whole goat anti-human immunoglobulin, unlike similar reagents produced in the rabbit, binds both to the same number of and the same individual cells as the F(ab')2 fragments of rabbit or goat anti-human immunoglobulin. These results suggest that goat IgG has a lower affinity for the Fc receptors of human lymphocytes and monocytes than rabbit IgG. Because of this property, whole goat antibodies against human immunoglobulin can be used as simple, convenient relatively inexpensive reagents for the routine detection of immunoglobulin on cell surfaces by immunofluorescence microscopy. The preparation of F(ab')2 fragments of anti-immunoglobulin, which are necessary when rabbit antibodies are used, does not appear to -e required if goat antibodies can be empolyed. This observation has multiple practival applications in cellular immunology.     

Human anti-IgG1 hinge autoantibodies reconstitute the effector functions of proteolytically inactivated IgGs

A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab')(2) possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab')(2)s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab')(2), as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab')(2). Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab')(2) in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.     

Monoclonal rheumatoid factor-secreting cells in a patient with mixed cryoglobulinemia. Homogeneity and stability of the idiotypic production and in vitro idiotypic suppression

The potential therapeutic value of anti-idiotypic antibodies during B cell proliferations largely depends on the stability of the target Ig idiotopes. We investigated this stability in a clinical condition of so called nonmalignant monoclonal B cell proliferation, mixed cryoglobulinemia. The idiotypic profile of a single IgM kappa monoclonal auto-antibody with anti-IgG activity (rheumatoid factor (RF] which originated from a patient suffering from a nonmalignant mixed cryoglobulinemia was followed over a period of 3 yr. As judged from the reactivity of a panel of five different mouse monoclonal anti-idiotypic antibodies mapping the RF variable regions, there was no idiotypic change in the serum IgM RF. At a cellular level, in vitro stimulation of the patient's PBL gives rise to IgM kappa auto-antibodies that were shown to bear the same idiotypic determinants as the serum IgM kappa. We then investigated the effects of the anti-idiotypic antibodies on the in vitro IgM kappa production. When stimulated with PWM and in the presence of anti-idiotypic antibodies (10 micrograms/ml), the patient's PBL produced less IgM RF (18 to 62% inhibition). The same inhibition of IgM RF production was observed after EBV infection of the patient's PBL (from 19 to 90% inhibition). In both cases, the remaining IgM RF production was idiotypically indistinguishable from the serum IgM RF. The implications of the idiotypic stability and of the results of in vitro idiotypic manipulation could be important in view of both the understanding of nonmalignant cryoglobulinemia and of the possible therapeutic use of anti-idiotypic antibodies in diseases.     

Single-step purification of pepsin-derived monoclonal antibody fragments from crude murine ascitic fluids by ceramic hydroxyapatite high-performance liquid chromatography

Ceramic hydroxyapatite (CHT) high-performance liquid chromatography (HPLC) is used to purify a variety of classes of monoclonal antibodies (mAbs) from crude murine ascites fluids. We report here that this method is also applicable for simple and efficient purification of many mAb fragments that are generated by pepsin treatment of crude ascites. F(ab')(2) fragments were quantitatively generated from IgG(1) mAbs in ascitic fluids by incubation with pepsin for 6 h at pH 3.9-4.1. Under the same conditions, pepsin also cleaved unwanted ascites components, such as albumin and transferrin to very low molecular weight polypeptides. The F(ab')(2) fragments, but not the low molecular weight products, selectively bound to and were eluted from the CHT column using a linear gradient of phosphate ion concentration over 15 min. The recovery of the F(ab')(2) fragments by CHT-HPLC was >90%. This method also allowed single-step purification of mAb fragments from distinct IgG subclasses (IgG(2a) and IgG(2b)) and IgM directly from crude digested ascitic samples. This CHT-HPLC method combined with direct pepsinolysis of murine ascites is a useful strategy for rapid purification and characterization of many types of mAb fragments.     

Comparative studies on monotypic IgMlambda and IgGkappa from an individual patient. I. Evidence for shared VH idiotypic determinants.

A comparative idiotypic antigenic analysis of an IgMlambda and an IgGkappa paraprotein obtained from sera of an individual patient, Br, revealed the presence of very similar idiotypic determinants associated with the VH regions of the Br mu- and gamma-chains. In addition, the IgGkappa protein expressed light (L) chain-associated idiotypic determinants which were not evident on the IgMlambda protein or its isolated L chains. Extensive tests of specificity revealed that the shared VH idiotypic determinants were not present in large weight amounts of heterologous nonspecific IgM and IgG, nor on Ig molecules contained in a large number of normal and myeloma sera.     

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Immunization strategies to block the herpes simplex virus type 1 immunoglobulin G Fc receptor

Herpes simplex virus type 1 (HSV-1) glycoprotein gE functions as an immunoglobulin G (IgG) Fc receptor (FcgammaR) that promotes immune evasion. When an IgG antibody binds by the F(ab')(2) domain to an HSV antigen, the Fc domain of some of the same antibody molecules binds to the FcgammaR, which blocks Fc-mediated functions. gE is a type 1 membrane glycoprotein with a large ectodomain that is expressed on the virion envelope and infected-cell surface. Our goal was to determine if immunizing with gE protein fragments could produce antibodies that bind by the F(ab')(2) domain to gE and block the FcgammaR, as measured by competitively inhibiting nonimmune human IgG binding to the FcgammaR. Three gE peptides were constructed in baculovirus spanning almost the entire ectodomain and used to immunize mice and rabbits. Two fragments were highly effective at producing antibodies that bind by the F(ab')(2) domain and block the FcgammaR. The most potent of these two antibodies was far more effective at blocking the FcgammaR than antibodies that are only capable of binding by the Fc domains to the FcgammaR, including anti-gC, anti-gD, and nonimmune IgG. These results suggest that immunizing with gE fragments has potential for preventing immune evasion by blocking activities mediated by the HSV-1 FcgammaR.     

Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies.

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.