Rotational dynamics of immunoglobulin G antibodies anchored in protein A soluble complexes

The rotational dynamics of rabbit IgG anti-dansyl antibodies anchored in staphylococcal protein A (SpA) soluble complexes were studied by both steady-state and nanosecond fluorescence spectroscopy. To aid in the interpretation of the anisotropy data, the results of recently reported hydrodynamic and electron microscopic studies of IgG-SpA complexes were used to calculate global tumbling times of the various complexes and to estimate the steric hindrance of the antibody Fab segments. The anisotropy decays, fitted to the sum of two exponentials, indicated that the Fab arms of antibodies bound to SpA by their Fc regions exhibit considerable flexibility. For the different IgG-SpA mixtures examined, changes in the IgG preexponential anisotropy weighting factors, fS and fL, and the short rotational correlation time, phi S, were relatively small. On the other hand, the long rotational correlation time, phi L, increased systematically when the percentage of larger IgG-SpA complexes in a mixture was increased. The greatest restriction of Fab flexibility was observed for antibodies anchored in the exceptionally compact IgG4-SpA2 complexes. Available electron microscopic data suggest that increases in phi L correlate with increased steric hindrance of the antibody segments. Both native and hinge-disulfide-cleaved IgG experienced similar percentage increases in phi L when bound in SpA complexes. In agreement with our earlier interpretation, the results of this study provide rather striking evidence that phi L mainly represents flexible motions of the Fab segments and not global tumbling: the phi L-values of IgG bound in the various SpA complexes ranged from 101 to 162 nsec, whereas the calculated global tumbling times of the different complexes ranged from about 300 to 3000 nsec.     

Spatial structure of immunoglobulin molecules

Summary Immunoglobulin molecules of the class G (antibody molecules) consist of two heavy chains (50,000 dalton molecular weight) and two light chains (25,000 dalton). The overall shape is a Y with the arms formed by the light chains and the N-terminal half of the heavy chains in tight association. The stem is formed by the C-terminal halfs of the heavy chains.The heavy and the light chains fold into globular domains of molecular weights of 12,000 dalton. There are four domains of the heavy chain and two of the light chain. All these domains show a similar fold, consisting of two -sheets but display considerable differences in detail.The N-terminal variable domains of heavy and light chains and specifically the hypervariable polypeptide segments of the domains, located at the tips of the Y, constitute the antigen and hapten binding site. The nature of the amino acid residues of the hypervariable loops determines the shape and the specificity of the antibody.All domains pair tightly laterally, except the C_H2 domains of the heavy chain. This domain has carbohydrate bound which prevents lateral association.Longitudinal interaction between the domains is loose and allows flexibility in the arrangement. Flexibility is probably of significance for antibody function.Arm (Fab) and stem (Fc) parts are linked by the hinge peptide which contains a segment with a unique conformation of two parallel poly-proline helices.Antigen binding triggers effector functions of antibodies. Antigen binding is at the tips of the Y-shaped antibody, but effector functions are displayed by the stem part. It is an open question whether conformational changes of the antibody molecule play a significant role in the trigger mechanism.     

Cleavage of protein A-binding IgA1 with IgA1 protease from Streptococcus sanguis

Protein A-binding fractions of two IgA1 myeloma proteins failed to produce Fc fragments on digestion with IgA1 protease from Streptococcus sanguis. A polymeric protein A-binding IgA1 fraction yielded a protein A-non-binding monomer, which was further cleaved into Fab fragments but it did not yield Fc fragments. The protein A-binding fraction of a monomeric IgA1 yielded an IgA molecule lacking one Fab fragment. Subsequently, the remaining part of its cleaved alpha chain was degraded. Further digestion yielded Fab but not Fc fragments. Similarly, F(abc)2 and Fabc fragments, which lack the CH3 domain (8), yielded Fab fragments but not CH2 domains. Thus, the enzyme in addition to cleaving IgA in the hinge region, under certain conditions, also degrades its Fc fragments.     

Conformational changes in the antibody constant domains upon hapten-binding

Bacterial proteins A and G (SpA and SpG) are immunoglobulin receptors that can be used as probes for monitoring change in the conformation of heavy chain constant (C(H)) domains. Interaction of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody (Ab) with SpA and SpG were measured by isothermal titration calorimetry and surface plasmon resonance in order to address the question of whether hapten-binding induces a conformational change in the C(H) domain. The interactions of IgG2a or its enzymatic fragments with SpA were measured in the presence or absence of the hapten. Although binding of Fab and F(ab')2 fragments were not observed to free SpA, they did bind to immobilized SpA. In addition, the association constant (K(a)) for interaction of IgG2a with immobilized SpA was approximately 20-fold higher than that with free SpA. This was explained in terms of high avidity resulting from multivalent interaction between IgG2a and immobilized SpA on the chip. Interestingly, the hapten-binding weakened the interaction between the F(ab')2 fragment and SpA. Furthermore, approximately half of the IgG2a was incapable of binding to immobilized SpA in the presence of hapten. These results were explained using a model which assumed the formation of two kinds of SpA/IgG complexes; one through sites on F(ab')2 arms and the other through sites on the Fc region. The former type dissociated as a result of hapten-binding, as did the F(ab')2 fragment and suggested that a conformational change had occurred around the Fab arms, while the latter type did not dissociate because of the higher avidity of the Fc region. However, using a mutant SpA with a lower K(a) value for the interaction with IgG2a, it was shown that hapten-binding induced long range conformational changes in the Fc region of IgG2a. Similar evidence of conformational change upon hapten-binding was also obtained using SpG as a probe.     

Construction and characterization of affibody-Fc chimeras produced in Escherichia coli

Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human IgG. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture. Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of "artificial antibodies" was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such "artificial antibodies" should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.     

Effect of hapten binding on antibody-water interaction. A concept of fluctuating cavities

Solutions of pig anti-Dnp antibodies, Fab and Fc fragments, human IgG, human serum albumin and cytochrome c were examined by infrared spectroscopy with the aim to investigate the influence of these proteins on the surrounding water. Anti-Dnp antibodies, human IgG and oxidized cytochrome c induced a shift of the maximum of the deformation—liberation band of water towards lower wave numbers. Anti-Dnp antibodies complexed with hapten, free and haptenated Fab fragments, reduced cytochrome c and serum albumin induced a shift of the maximum to higher wave numbers, while Fc fragment did not induce any significant shift. Perturbating agents such as 0.5 M sodium chloride and 20% saccharose caused a shift of the maximum of water in protein solutions to higher wave numbers.The observed phenomena are interpreted in terms of the ability of cavities and clefts in proteins such as immunoglobulins to fluctuate between two states, more or less accessible to water. This ability is thought to result in a generation of anisotropic fluctuations in the surrounding water medium, associated with lowering of the number and/or strength of hydrogen bonds between water molecules. Proteins lacking cavities or clefts affect the water medium in an opposite way; the hydrogen bond network of water becomes more firm and consolidated. Binding of hapten to the intact antibody apparently alters the domain oscillations and the geometry of the antibody molecule. Consequently in contrast to free antibody, the antibody—hapten complex consolidates the hydrogen bond network of surrounding water.     

Pyridoxal-5'-phosphate-dependent catalytic antibodies

Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5'-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten N(alpha)-(5'-phosphopyridoxyl)-L-lysine, a stable analog of the covalent coenzyme-substrate adducts were screened by a competition ELISA for binding of the PLP-amino acid Schiff base adduct. The Schiff base with its C4'-N alpha double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent alpha,beta-elimination reaction of beta-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k(cat)'=0.42 min(-1) with D-alanine at 25 degrees C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme-amino acid adduct. (2) The bond at C alpha to be broken lies together with the C alpha-N bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The alpha-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme-substrate adduct assumes a cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10(4) times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9 k(cat (Ab x PLP))'/k(cat (PLP))'=5 x 10(3)). The analogies of antibody 15A9 with PLP-dependent enzymes suggest that the selection criteria in the screening protocol were similar to those that have been operative in the molecular evolution of enzyme-assisted pyridoxal catalysis.     

Interaction of C3 and C3b with immunoglobulin G

Human C3b as well as native C3 were found to bind to solid phase human and rabbit IgG. Haemolytically active C3 had significantly higher binding capacity to IgG than the C3b fragment. Inhibition experiments proved that C3 and C3b have common binding sites on the Fab and Fc part of the IgG molecule but the character of these binding sites was different. As a functional consequence of C3-IgG interaction, C3 binding was found to inhibit the specific precipitation of an IgG antibody preparation.     

Human IgG anti-F(ab'')2 antibodies possess rheumatoid factor activity.

Individual preparations of affinity purified anti-F(ab')2 antibodies and anti-Fc antibodies isolated from the sera of patients with rheumatoid arthritis (RA), were examined for reactivity with the Fab and Fc fragments of human IgG. Western blot assays demonstrated specific interaction of affinity-purified anti-Fab antibodies with both Fab and Fc molecules. Approximately one-half of the anti-Fab antibody preparations studied contained IgG antibodies reactive with Fab and Fc fragments in ELISA, suggesting the existence of naturally occurring epibody-like autoantibodies in these patients. Thirteen of 14 affinity-purified anti-Fc antibody preparations contained IgG cross-reactive with Fab molecules in ELISA. Double-adsorption assays on affinity columns demonstrated that a minimum of 14%, and possibly as much as 50%, of the IgG anti-Fab antibodies reacted with the Fc of IgG. Conversely, a minimum of 12%, and possibly as much as 70%, of the IgG anti-Fc antibodies reacted with IgG Fab molecules. Anti-Fab antibodies isolated from non-RA individuals also exhibited anti-Fc reactivity in ELISA, demonstrating the presence of these dual-reactive antibodies in other autoimmune and normal individuals. These studies establish the presence of naturally occurring IgG autoantibodies reactive with both the Fab and Fc fragments of human IgG. Their existence emphasizes the potential of anti-immunoglobulin antibodies to recognize a multiplicity of antigens, possibly including other members of the immunoglobulin supergene family.     

Immunochemical studies on the binding properties of Brucella abortus lipopolysaccharides to bovine precipitating antibodies

The binding properties of Brucella abortus smooth lipopolysaccharides (LPS) to the precipitating antibodies in the serum of a B. abortus S2308 infected cow were studied by quantitative precipitin and precipitin-inhibition assays. Crude B. abortus lipopolysaccharides (cLPS) and chemical modification products of strains 1119.3, 19 and 2308 (virulent) were used for this study. In the quantitative precipitin assay, 13 micrograms of cLPS precipitated about 4.0-5.2 micrograms nitrogen of precipitating antibodies in 10 microliter of serum. After alkaline treatment (0.125 N NaOH at 56 degrees C for 1 hr), the alkali-treated lipopolysaccharides (aLPS) from S19 and S1119.3 maintained the maximum precipitability, but the precipitation profile was changed from a sigmoidal to a bell-shaped curve, and aLPS from S2308 was reduced to about 40% of its reactivity. These results indicate that some of the antigenic determinants blocked by other molecules were reshielded by alkaline treatment, and possibly that structural differences exist between virulent (S2308) and nonvirulent (S19 and S1119.3) strains. Among the monosaccharides and glycosides tested for inhibition of precipitation, methyl alpha-D-mannose was most active and about 3.3 times as active as D-mannopyranose (DMan) and only 1/200 as active as acid hapten or the carbohydrate moiety of cLPS prepared from S1119.3 (expressed as a monosaccharide of mol. wt 200) implying that the carbohydrate determinants of this antigen are probably related to a DMan alpha 1----linked compound. The precipitability of the antibodies in this serum was greatly reduced after absorption with cLPS and the precipitin reaction was completely inhibited by the carbohydrate moiety of LPS (O-antigen) S1119.3 demonstrating that the common antigenic determinants (hapten) of B. abortus LPS reacting with these precipitating antibodies are in the region of the carbohydrate moiety of the LPS.     

Effect of posttranslational modifications on the thermal stability of a recombinant monoclonal antibody

The effect of oligosaccharides and C-terminal lysine residues on the thermal stability of a recombinant IgG(1) monoclonal antibody was investigated using differential scanning calorimetry (DSC). The C-terminal lysine did not appear to affect the thermal stability of this IgG(1) molecule. However, oligosaccharides, which are buried between the two CH(2) domains, provided significant stabilizing energy. Characterization of the Fab and Fc after papain digestion suggested that the stabilizing effect of oligosaccharides on this molecule was through stabilizing CH(2) domains. Oligosaccharides had little effect on the thermal stability of Fab region and CH(3) domains. It was also interesting to note that both intact IgG(1) antibody and its Fab, but not the Fc regions, appeared to form precipitate after thermal unfolding under the experimental conditions.     

Binding of human IgA1 and IgA1 fragments to jacalin

Interaction of jacalin, an N-terminal galactose specific lectin, with human IgA1 and IgA1 fragments was investigated. IgA1 and all galactose containing fragments bound to jacalin-Sepharose, including Fab fragments containing only the galNac linked to serine-224 and Fc fragments containing four gal-galNac sequences. These data indicate that both the galNac and gal-galNac sequences can interact with jacalin. Jacalin precipitated IgA1 and the fragments F(abc)2, F(ab')2 and Fc in agar gel and from solutions. It also precipitated Fab' fragments in agar gel. Jacalin did not precipitate Fab fragments significantly. This suggests that, except for the single binding site on the Fab fragments containing the galNac linked to serine-224, jacalin itself also has a limited number of sites to interact with N-terminal galactose residues. ELISA studies revealed that intact IgA1 had a lower jacalin binding capacity than F(abc)2 fragments which lack CH3 domains, than F(ab')2 which lack the CH2 and CH3 domains, and than Fc fragments containing four gal-galNac sequences. This led to the conclusion that part of the galNac or gal-galNac sequences in intact IgA1 molecules are inaccessible to interaction with jacalin. Cleaving the C-terminal domains off may have induced a reorientation of the hinge region structure, including the orientation of the carbohydrate units.     

Differential binding characteristics of protein G and protein A for Fc fragments of papain-digested mouse IgG

It has been previously reported that staphylococcal protein A (SPA) bound only to the Fc region of mouse immunoglobulin G (IgG) and streptococcal protein G (SPG) bound to both Fab and Fc regions of mouse IgG and the binding sites for SPG and SPA on Fc were overlapped. In this study the binding characteristics of SPG and SPA for papain-digested mouse IgG were analysed. Papain digestion of mouse IgG purified from CAy-IFNg99C hybridoma (secreting IgG1 monoclonal antibody specific for human interferon gamma)-induced ascites resulted in Fab and two major Fc fragments referred to as the high molecular weight (HMW) and the low molecular weight (LMW) Fc fragments. SPG bound to Fab and the LMW Fc fragments of the papain-digested IgG. However SPG did not bind to the HMW Fc fragment. SPA showed practically no reactivity with the Fab and the LMW Fc fragments of the papain-digested mouse IgG but only to the HMW Fc fragment. SPG and SPA binding assays showed that papain digestion discriminated the SPG and SPA binding sites in the Fc fragment of mouse IgG. These results demonstrated a clear evidence for the presence of two independent SPG and SPA binding sites in the Fc fragment of mouse IgG.     

Immunological studies of the glycoprotein allergen Ag-54 (Cla h II) in Cladosporium herbarum with special attention to the carbohydrate and protein moieties

The glycoprotein allergen Ag-54 (Cla h II) isolated from Cladosporium herbarum was split into its carbohydrate and protein moieties by using alkaline-borohydride treatment and deglycosylation, respectively. The native Ag-54, the deglycosylated protein and the protein-free carbohydrate moieties were tested for IgE-binding activity by radioallergosorbent test inhibition using selected sera from individuals with C. herbarum allergy. The deglycosylated material had a stronger allergenic activity than the native Ag-54 with all the sera tested. The Ag-54 carbohydrate moieties were not found to possess any IgE-binding activity. The galactoglucomannan part of the carbohydrate moiety was isolated and it was demonstrated to precipitate IgG rabbit antibody. Removal of the galactofuranose units by mild acid hydrolysis did not alter the IgG-binding capacity of the polysaccharide, indicating that galactofuranose was not immunodominant.     

Analysis of glycoforms present in two mouse IgG2a monoclonal antibody preparations.

Lectins have been used for the determination of the oligosaccharide structures expressed by two monoclonal IgG antibodies, MN12 and RIV6. Dot blot experiments revealed the presence of terminal Fuc alpha (1-->6)GlcNAc, Gal beta (1-->3)GalNAc, Gal beta (1-->4)GlcNAc, Man alpha (1-->6, 1-->3)Man, NeuAc alpha (2-->6)Gal and NeuAc alpha (2-->6)GalNAc on both monoclonal antibodies. MN12 was shown to contain a carbohydrate moiety within the Fc region only. RIV6 contained carbohydrate moieties within both the Fc and Fab regions. Additional O-glycosidic linked carbohydrate chains were detected within the Fc region of both monoclonal antibodies. High mannose structures were also detected on both Mabs.     

Comparison of the Binding of Radiolabelled Human IgG and Fc Fragments to Murine Spleen Cells

The binding properties of an IgG1 human myeloma protein, normal IgG, and the Fc and Fab fragments of each were compared in cultures of murine spleen cells. Both 125I-labelled IgG and Fc fragments bound to splenic lymphocytes, whereas Fab fragments did not bind significantly at the highest concentrations tested. On a molar basis, more Fc bound than intact IgG. According to Scatchard plot analysis, the affinity constand of IgG1 was 1.5 x 10(6) +/- 1 x 10(5) L/M and that of the Fc fragments was 7.8 x 10(5) +/- 2.6 x 10(5) L/M. Approximately 25,000 binding sites/cell were calculated for IgG1 and 102,000 for Fc. Deaggregation of the Fc preparation did not change these values, suggesting that the difference in binding of IgG and Fc did not result from Fc aggregation. Unlabelled IgG inhibited about 25% of the labelled Fc binding, whereas unlabelled Fc inhibited approximately 80% of the labelled Fc binding. IgG antigen-antibody complexes, however, inhibited 75% of the Fc binding. In the reciprocal experiment both intact IgG and Fc inhibited the binding of labelled IgG by 100%. The major cell population that bound IgG and Fc fragments in the spleen cell preparation were the B lymphocytes. Removal of macrophages did not significantly affect the binding of labelled Fc fragments. In addition, T-cell-enriched populations bound an insignificant quantity of Fc fragments.     

Presence of IgG4 on the membrane of human basophils. Histamine release is induced by monoclonal antibodies directed against the Fab but not the Fc region of the IgG4 molecule

We have studied the possible role of human IgG4 as an anaphylactic antibody. For that purpose, we have determined the induction of histamine release (HR) from human basophils by anti-IgE and anti-IgG4 monoclonal antibodies (MoAbs) recognizing different epitopes located at the Fc and Fab regions of the IgG4 molecule. The results show that anti-IgG4 (Fab) MoAb was able to induce HR in 93% of donors tested, with no differences between atopics and non-atopics. That HR is calcium dependent and is accompanied by the synthesis and release of leukotriene C4. In contrast, no HR could be induced by anti-IgG4(Fc) MoAbs in any individual, even in the presence of D2O or after a second challenge with a polyclonal goat anti-mouse IgG antibody. The results obtained suggest the presence of IgG4 on the basophil membrane and that the epitope recognized by the anti-IgG4 (Fc) MoAbs is probably hidden in cell-bound IgG4. This was demonstrated by immunofluorescence techniques: IgG4 bound to the basophil membrane could be detected with anti-IgG4(Fab) but not with anti-IgG4(Fc) MoAbs. In addition, we found that nine donors were unresponsive to an anti-IgE stimulus, while they released histamine efficiently after challenge with anti-IgG4(Fab), suggesting the existence of different receptors for both immunoglobulins.     

The antitumor monoclonal antibody MOv2 recognizes the Lewis A hapten

Monoclonal antibody MOv2, produced against ovarian carcinoma, was previously found to bind a carbohydrate epitope (CAMOv2) present on mucins, glycoproteins and a neutral glycolipid. In this paper, the structure of the carbohydrate epitope is determined by immunological reactivity with purified glycolipids and oligosaccharides. Using solid-phase radioimmunoassay and immunostaining of thin layer chromatograms, MOv2 binds strongly to Le(a)-active pentasaccharide ceramide. A smaller neutral glycolipid also weakly binds MOv2. Fifty percent inhibition of binding to Le(a)-active pentasaccharide ceramide is achieved with approximately 8 microM concentration of lacto-N-fucopentaose II (LNF II). Lacto-N-tetraose (LNT) also partially inhibits at about 10(3) times higher concentration suggesting that the faster migrating glycolipid antigen contains this carbohydrate sequence. Binding to Le(a)-active hapten is further confirmed by the specific inhibition of binding by authentic anti-Le(a) monoclonal antibodies but not by anti-Le(b) MOv2 antibody in a serum assay among healthy blood donors also supports these results. In conclusion, we have obtained direct evidence from several independent experiments that antibody MOv2 recognizes the Le(a)-active hapten.     

Properties of two monoclonal antibodies directed against the Fc and Fab' regions of rat IgE

Hybridoma antibodies directed against the Fc and Fab portions of rat IgE were produced by immunizing BALB/c mice with rat IgE and fusing the spleen cells with the nonsecreting plasmacytoma P3/X63Ag8.653. Two of the antibodies, designated as A2 and B5, were extensively characterized. Competitive binding experiments using rat IgE from the IR 162 and IR2 immunocytomas and rat IgG indicated that both A2 and B5 were epsilon-chain specific and not anti-idiotype. A2 also exhibited some cross-reactivity with mouse IgE. When IgE was treated with chymotrypsin so as to produce both F(ab')2 and Fab fragments, the enzyme-treated IgE retained reactivity with B5, but the reactivity to A2 was lost. Heat denaturation of IgE at 56 degrees C resulted in a progressive loss of reactivity of the IgE for both A2 and the Fc receptor on rat basophilic leukemia cells; the reactivity of B5 remained unchanged. A2 does not evidently interact with the same site on the Fc of IgE that is involved in binding to the rat basophilic leukemia cell Fc receptor; A2 exerted little influence on the binding of IgE to rat basophilic leukemia cells. Thus, the data indicate that the antigenic site for B5 is in the Fab region of the IgE molecule and that A2 reacts with the IgE Fc. Use of these antibodies to measure cell-bound IgE was also evaluated in dual label experiments, and potential problems in using divalent antibodies to quantitate cell surface antigens are discussed.     

Binding of IgG to B cell via HLA molecules

Binding of immunoglobulins to major histocompatibility complex (MHC) molecules was demonstrated by two different assays: the binding of IgG to B cells by flow cytometry, and purified MHC antigens with an Elisa assay. Fc fragment from immune-complex binds to the Fc receptor on B lymphocytes. Here, Fab was also shown to bind to B cells. This binding was inhibited by specific human allo anti-HLA Class I and II sera directed at the polymorphic sites. Thus, in addition to the Fc receptor, MHC can also serve as a binding site for IgG. In an Elisa assay using purified antigens, IgG was shown to bind to HLA Class I and II molecules. Other proteins such as transferrin, human serum albumin, gelatin, etc., did not bind to the MHC proteins. Immunoglobulins bound to MHC molecules by sites on the Fab fragment independent of the hypervariable region. This was demonstrated by the retention of antibody activity even after binding of antibody (anti-lactoferrin) to MHC. The relative avidity between Fab and HLA Class I and II was 4-8 x 10(5) M-1.