Subclasses of human IgG. I. Production of antisera to IgG subclasses]

Guinea pigs were used for preparing antisera to human IgG subclasses for anti-IgG1, and rabbits--for anti-IgG2, anti-IgG3, and anti-IgG4. Schemes of laboratory animals immunization with myeloma paraproteins of four IgG subclasses were determined. Methods of antisera absorption for bringing them up to strict monospecificity were worked out. Antisera specificity were determined by the precipitation test after Ouchterlony with standard myeloma proteins in the concentration of 1 mg/ml, and in the passive hemagglutination test with erythrocytic antigenic diagnostic agents. Precipitating antisera to four human IgG subclasses were obtained.     

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s⁰_20,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier R_G values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.     

Anti-cord factor (trehalose 6,6'dimycolate) IgG antibody in tuberculosis patients recognizes mycolic acid subclasses.

The detection of anti-cord factor (trehalose 6,6'-dimycolate) IgG antibody in active (smear-and/or culture-positive) and inactive (smear-and culture-negative) tuberculosis patients is a useful serodiagnostic tool that can be used for early clinical diagnosis of the disease. We estimated the titers of anticord factor IgG antibody in the sera of tuberculosis patients, and compared them with those of Mycobacterium avium-infected patients. Most of the serum samples obtained from the tuberculosis patients were highly reactive against M. tuberculosis (MTB) cord factor isolated from M. tuberculosis H37Rv, a human-type mycobacterial strain, whereas they were less reactive against M. avium (MAC) cord factor. Similarly, most of the serum samples of the MAC-infected patients were highly reactive against MAC cord factor and less reactive against MTB cord factor. These results suggest that anti-cord factor IgG antibody recognizes the mycolic acid subclasses as an epitope which comprises cord factor, since MTB and MAC cord factor differ in mycolic acid subclasses and molecular species composition. To clarify the exact antigenic epitope in cord factor and to find out a more sensitive and specific diagnostic test antigen, we examined the reactivity of patients' sera to glycolipids containing trehalose (cord factor and sulfolipid) obtained from various mycobacterial species. Furthermore, the reactivity of human antisera to various mycolic acid subclasses (alpha-, methoxy and keto mycolic acids) of MTB cord factor was compared. We found that anti-cord factor IgG antibody in the sera of human tuberculosis patients most strikingly recognized methoxy mycolic acid in the cord factor of M. tuberculosis, whereas it recognized alpha- and keto mycolic acids weakly. Pre-absorption studies of antibody with MTB cord factor or methoxy mycolic acid methyl ester showed that anti-cord factor antibody was absorbed partially, but consistently. This is the first report describing that the specific subclass of mycolic acid from mycobacteria is antigenic in the humoral immune system of human tuberculosis infection.     

Affinity of human IgG subclasses to mouse Fc gamma receptors

Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to FcγR-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse FcγRs is available. The orthologous mouse and human FcγRs share roughly 60–70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse FcγR. Human IgGs bound to mouse FcγR with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3>IgG1>IgG4>IgG2 and FcγRI>>FcγRIV>FcγRIII>FcγRIIb. This suggests human IgG subclasses to have similar relative FcγR-mediated biological activities in mice.     

Production of rabbit precipitating antisera to subclasses of human IgG]

Precipitating antisera to human subclasses IgG were obtained by immunization of rabbits by whole molecules IgG2, IgG3, IgG4 and gamma 1-chains derived from IgG1H (Pr). Analysis of the antisera obtained demonstrated that rabbits produced specific antibodies to the antigenic subclass determinants IgG3 well, to IgG2, IgG4--much worse, and failed to produce specific antibodies to subclass IgG1 (in immunization with whole molecules of this protein). Antisera contained antibodies to the antigenic determinants common of IgG, and antibodies to light chains which were removed by immunosorption, for which purpose a sorbent on the basis of BrCN sepharose conjugated with IgG of the three other subclasses and Fab-fragment was used.     

Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.     

Study of the subclasses of immunoglobulin G. II. Qualitative and quantitative characteristics of IgG subclasses using antisera systems]

Bispecific antisera, or "antisera-systems", containing class- and subclass-specific antibodies to IgG were obtained from rabbits, goats and guinea pigs after brief courses of immunization with purified G1, G2, G3 and G4 paraproteins. After the elimination of antibodies to light chains by adsorption these antisera were tested in immunoelectrophoresis and radial immunodiffusion in gel with sera containing G paraproteins of different subclasses. In immunoelectrophoresis double lines and in radial immunodiffusion with G paraproteins of heterologous subclasses double rings were obtained: the external lines (or the external rings) were formed as a result of interaction between G paraproteins and antibodies to class-specific IgG determinants, the inner lines (or the inner rings) were formed as a result of interaction between the corresponding subclass of normal IgG and subclass-specific antibodies. The identification of different G paraprotein subclasses gave similar results when carried out with "antisera-systems" and with monospecific antisera to the corresponding IgG subclasses. "Antisera-systems" proved to be suitable for use in the identification of G paraprotein subclasses, as well as in the quantitation of different subclasses in normal IgG.     

Autoimmune MRL/lpr mice sera contain IgG with interleukin 3-like activity

The spleen cells, thymocytes, and bone marrow cells of autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice do not constitutively produce interleukin 3 (IL-3), but these mice had IL-3-like activity in their sera. MRL/lpr sera supported the growth of the IL-3-dependent cell lines FDC-P2 and DA-1 but not the growth of IL-2-dependent T-572 cells. This IL-3-like activity increased with age. Biochemical analysis of the MRL/lpr sera by anion-exchange chromatography, gel filtration on a Superose 12 column, the binding to protein-A and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the serum factor with the IL-3-like activity was not IL-3 itself but was associated with IgG. Flow cytometric analysis also showed that the serum level of the Ig capable of binding FDC-P2 cells was high in MRL/lpr but not in MRL/+ mice. We suggest that IL-3 is not responsible for lymphoid hyperplasia, contrary to a previous report; rather auto-antibodies directed toward the IL-3 receptor may act pathogenically in MRL/lpr mice.     

Differential binding of histidine-rich glycoprotein (HRG) to human IgG subclasses and IgG molecules containing kappa and lambda light chains.

In previous studies we showed that the plasma protein histidine-rich glycoprotein (HRG) binds strongly to pooled human IgG. In the present work myeloma proteins consisting of different human IgG subclasses were examined for their ability to interact with human HRG. Using an IAsys optical biosensor we found initially that IgG subclasses differ substantially in their affinity of interaction with HRG. However, the most striking finding was the observation that the kinetics of the HRG interaction was dramatically affected by whether the IgG subclasses contained the kappa or lambda light (L)-chains. Thus, the on-rate for the binding of HRG to the kappa L-chain containing IgG1 and IgG2 (IgG1kappa and IgG2kappa) was approximately 4- and approximately 10-fold faster than that for the binding of HRG to lambda L-chain containing IgG1 and IgG2 (IgG1lambda and IgG2lambda), respectively, with the dissociation constants (K(d)) in the range 3-5 nM and 112-189 nM for the kappa and lambda isoforms, respectively. In contrast, the on-rate for the binding of HRG to IgG3kappa and IgG4kappa was found to be 9- and 20-fold slower than that for the binding of HRG to IgG3lambda and IgG4lambda, respectively, with the K(d) in the range 147-268 nM and 96-109 nM for the kappa and lambda isoforms, respectively. The binding of HRG to immunoglobulins containing the kappa L-chain (particularly IgG1kappa) was generally potentiated in the presence of a physiological concentration (20 microM) of Zn(2+) (K(d) decreased to 0.60 +/- 0.01 for IgG1kappa), but Zn(2+) had no effect or slightly inhibited the binding of HRG to immobilized IgG subclasses possessing the lambda L-chain. Interestingly, HRG also bound differentially to Bence Jones (BJ) proteins containing kappa and lambda L-chains, with HRG having a 14-fold lower K(d) for BJkappa than for BJlambda when 20 microM Zn(2+) was present. HRG also bound to IgM (IgMkappa), but the affinity of this interaction (K(d) approximately 1.99 +/- 0.05 microM) was markedly lower than the interaction with IgG, and the affinity was actually decreased 4-fold in the presence of Zn(2+). The results demonstrate that both the heavy (H)- and L-chain type have a profound effect on the binding of HRG to different IgG subclasses and provide the first evidence of a functional difference between the kappa and lambda L-chains of immunoglobulins.     

CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7

The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7.     

Laser nephelometer evaluation of factor IX.

Laser Nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetic of the reaction between antigen and antibody. In a few instances the method was also applied in the characterization of abnormal molecules. We developed assays for the measurement of Factor IX antigen and the results were compared with those obtained by conventional immunological methods such as rocket immunoelectrophoresis. Plasmas from patients with haemophilia B, on coumarin treatment, with liver cirrhosis were studied. A standard reference curve was obtained using pooled normal plasma. The factor IX levels obtained by laser nephelometer correlated fairly well with those obtained by electroimmunoassay.     

The binding of human factor IX to endothelial cells is mediated by residues 3-11.

We have used chimeras and point mutations of recombinant coagulation factor IX to examine factor IX's specific interaction with bovine endothelial cells. Previously (Toomey, J. R., Smith, K. J., Roberts, H. R., and Stafford, D. W. (1992) Biochemistry 31, 1806-1808), we restricted the region of factor IX responsible for binding to endothelial cells to its Gla domain. Molecular modeling of the Gla domain of factor IX using the coordinates of the Gla domain of bovine prothrombin-(1-145) (Soriano-Garcia, M., Padmanabhan, K., deVos, A. M., and Tulinsky, A. (1992) Biochemistry 31, 2554-2566) reveals two major surface determinants whose sequences differ among factors IX, X, and VII. A chimeric protein comprised of the Gla domain of factor VII with the remainder of the molecule of factor IX did not bind to the endothelial cell binding site. We changed residues 33, 34, 35, 39, and 40 to those of factor IX without restoring endothelial cell binding. Replacement of amino acid residues 3-10 with those of factor IX restored normal binding. With the knowledge that specific binding was localized to the first 11 amino acids, point mutations were made at residues predicted to be on the surface in this region of the factor IX molecule. Changing lysine 5 to alanine (K5A) or valine 10 to lysine (V10K) resulted in loss of binding with total retention of in vitro clotting activity. The lysine 5 to arginine (K5R) mutation also was fully active in vitro but displayed 3-fold tighter binding. In addition to defining the sequence of factor IX necessary for binding to endothelial cells, these results suggest that the binding site is not phospholipid but instead is specific, and in all likelihood, protein.     

IgGs containing light chains of the λ and κ type and of all subclasses (IgG1‐IgG4) from sera of patients with multiple sclerosis hydrolyze DNA

We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1–IgG4) are catalytically active in the hydrolysis of DNA and on average their relative activity (nM supercoiled DNA/1mg IgG/1 h) increases in the order: IgG1 (0.58) < IgG2 (0.94) < IgG3 (1.4) < IgG4 (4.1), while their approximate relative contribution to the total activity of abzymes increases in the order: IgG1 (6.9%) < IgG3 (9.3%) < IgG2 (18.2%) < IgG4 (65.6%). On average IgGs containing light chains of the λ‐type are severalfold more active in the hydrolysis of DNA than IgGs with light chains of the κ‐type. Using different physicochemical methods of antibody analysis we have shown that the immune system of multiple sclerosis patients generates a variety of anti‐DNA abzymes of different type and with different catalytic properties, which can play an important role in multiple sclerosis pathogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

Conformation of human IgG subclasses in solution. Small-angle X-ray scattering and hydrodynamic studies

The structure of six human myeloma proteins: IgG1(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgG1(Bal) and IgG3(Het) the experimental data, including radius of gyration (Rg degree), radii of gyration of the cross-section (Rq1, Rq2), intrinsic viscosity [eta], sedimentation coefficient (S degree 20,w) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances. The values Rg degree = (6.00 +/- 0.05) nm, S degree 20,w = (6.81 +/- 0.10) S and [eta] = 0.0062 +/- 0.0005 cm3/mg obtained for IgG1(Bal) are compatible with a planar model in which the angle between the Fab arms is about 120 degrees. For IgG3(Het) the following data were obtained: Rg degree = (4.90 +/- 0.05) nm, S degree 20,w = (6.32 +/- 0.01) S and [eta] = (0.0065 +/- 0.0005) cm3/mg. The apparent contradiction between the higher molecular mass and lower Rg degree and S degree 20,w values for IgG3(Het) in comparison to IgG1(Bal) can be resolved by proposing a 'non-planar' (tetrahedral) molecular shape, in which the long hinge peptide is in a folded conformation and the two Fab and Fc parts are in a closely packed arrangement. In this model the angle between the two Fab arms is about 90 degrees, in the average position. The X-ray scattering and hydrodynamic behaviour of the IgG2 and IgG4 types of antibodies appeared to be similar to IgG1(Bal). The parameters of the two IgG3 proteins are similar while they are different to the others.     

Evaluation of production and characterization of monoclonal antibodies to human IgG of four subclasses

Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.