Changes in the segmental flexibility and conformation stability of side groups in spin-labeled molecules of 2 IgG subclasses in the serum of normal cows and cows in various pathological states

The segmental flexibility of molecules from two IgG subclasses of cows' blood serum was first observed. It was shown that IgG1 molecule has a more flexible hinge region than IgG2 molecule and lower local conformational stability. At the same time the states of IgG1 and IgG2 molecules determined by dynamic-steric characteristics for ill and healthy cows practically did not differ.     

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs, and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.     

Quantitative evaluation of serum IgG subclass levels by an immunoenzyme method

We developed a enzyme linked immunosorbent assay (ELISA) for measuring IgG subclasses concentration in serum. For this we used monoclonal antibodies. The specificity of these antibodies was evaluated with a panel of myeloma proteins belonging to the 4 IgG subclasses. The ELISA was sensitive (allowing the detection of subclasses at ng level) and accurate (inter-assay coefficient of variation of 14%). Using the WHO serum 67/97 as reference, we determined the concentration of IgG subclasses in a pool of sera. In addition concentrations were measured in 69 healthy adults to study the distribution of each IgG subclass. A good correlation (r = 0.78) was obtained between the sum of the subclasses measured by ELISA and total IgG measured by immunonephelometry.     

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.Key words: antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, autoantibodies     

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.     

Seroepidemiological survey for antibody to Borrelia burgdorferi in cows.

Antibody to Borrelia burgdorferi was examined in 380 healthy and 38 clinical cases of cows from Hokkaido and Shizuoka in Japan. In healthy animals, IgG and IgM antibody to B. burgdorferi HO14 strain were found in 44 cows (14.6%) and 24 cows (8.0%) from Hokkaido. In contrast, antibody-positive case was not observed except for only 1 case which was IgM positive (1/79: 1.3%) in cows from Shizuoka. Mean antibody levels of healthy animals in Hokkaido and Shizuoka were 0.651 and 0.263 (IgG antibody to HO14 strain), 0.642 and 0.169 (IgG to HP3 strain), 0.613 and 0.367 (IgM to HO14 strain) and 0.582 and 0.286 (IgM to HP3 strain). The differences of the antibody levels between cows from Hokkaido and Shizuoka were significant. Seasonal difference was found in seropositive cows from Hokkaido. The rate of seropositive cows was high in summer (23.4% in June and 11.8% in July) but low in winter (0% in January and February). The pattern was discussed to be associated with activation of ticks. One of 4 cows with arthritis showed significantly higher IgG antibody level than that of healthy cows and cows with some disease, although the serum was collected from Shizuoka where antibody-positive animals for B. burgdorferi were rare among healthy cows. This high IgG antibody may suggest that the arthritis of such cows was caused by infection with B. burgdorferi. Two of 7 cows with unclassified abortion showed positive antibody reaction in Hokkaido. These cases, however, may not be related to the B. burgdorferi infection because the positive rate was similar to those of healthy cows in the same season.     

Crystallohydrodynamics for solving the hydration problem for multi-domain proteins: open physiological conformations for human IgG.

Hydrodynamic methods provide a route for studying the low-resolution conformation--in terms of time-averaged spatial orientation of the Fab' and Fc domains relative to each other--of the human IgG subclasses, IgG1, IgG2, IgG3 and IgG4 in the environment in which many exist naturally---a solution. Representative modelling strategies are now available using 'shell-bead' or 'shell' modelling of the surface of the molecules with the size-independent programme SOLPRO [J. Garcia de la Torre, S.E. Harding, B. Carrasco, Eur. Biophys. J. 28 (1999) 119-132]. The shell model fits to the equivalent inertial surface ellipsoids of the published crystal structures for the Fab' and Fc domains of IgG are made and an apparent hydration delta(app) of 0.51g/g for Fab' and 0.70 g/g for the glycoprotein Fc are obtained, which yield an average value of (0.59+/-0.07) g/g for the intact antibody (2 Fab'+1 Fc). The relative orientations of these domains for each of the IgG subclasses is then found (using where appropriate a cylindrical hinge) from SOLPRO by modelling the Perrin function, P (i.e. 'frictional ratio due to shape') using this delta(app) and experimentally measured sedimentation coefficients. All the IgG subclasses appear as open, rather than compact structures with the degree of openness IgG3>IgG1>(IgG2, IgG4), with IgG3 and IgG1 non-coplanar. The hingeless mutant IgGMcg, with s degrees (20,w) approximately 6.8 S yields a coplanar structure rather similar to IgG2 and IgG4 and consistent with its crystallographic structure. The extension of this procedure for representing solution conformations of other antibody classes and other multi-domain proteins is indicated.     

Biological characterization of Campylobacter fetus lipopolysaccharides

Abstract Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.     

Relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained placenta

A study was conducted to investigate the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in cows that experienced a dystocia or retained their placenta. Fifteen healthy cows, 31 cows with retained placenta (RP) and 13 cows that had dystocia were clinically examined 1 or 2 days after parturition when a uterine swab for bacteriological examination was taken. In addition, plasma and uterine lochia samples were collected to determine lipopolysaccharide (LPS) and the plasma IgG anti-LPS concentrations. Subsequently, 15 RP and 6 dystocia cows were initially left untreated and another uterine swab was collected at 2 and 4 wk postpartum. Immediately after calving, RP cows had significantly higher LPS levels in uterine lochia (average of 2.24 x 10(4) Endotoxin Units (EU)/mL) as compared to dystocia and healthy postpartum cows (average of 0.10 and 0.26 EU/mL, respectively). However, plasma LPS levels were below the detection limit (<0.036 EU/mL platelet-rich plasma) in all groups of cows. IgG anti-LPS levels in plasma were not significantly different between the 3 groups immediately postpartum (average of 26, 16 and 44 Median Units (MU)/mL) for healthy, dystocia and RP cows, respectively), but they were significantly lower when compared to plasma IgG anti-LPS levels of healthy cows at more than 2 months postpartum (mean 83 MU/mL). High LPS levels in lochia at 1 or 2 days postpartum were significantly related to abnormal cervical discharge, the presence of Escherichia coli, black pigmented gram-negative anaerobes and Clostridium spp. shortly after calving, and Arcanobacterium pyogenes and gram-negative anaerobes in the uterus at 14 days postpartum. These results suggest that the presence of E. coli and LPS (endotoxins) in lochia early postpartum favor the development of uterine infections by A. pyogenes and gram-negative anaerobes later postpartum. LPS were not observed in plasma, suggesting that either they are not absorbed into the blood, or they are efficiently detoxified by IgG anti-LPS or other detoxification mechanisms.     

Immunoglobulin G subclasses of anti-thyroid peroxidase autoantibodies in human autoimmune thyroid diseases

A distribution of immunoglobulin G (IgG) subclass of anti-thyroid peroxidase (TPO) autoantibodies was studied to know whether anti-TPO autoantibodies are closely implicated in the pathogenesis of human autoimmune thyroid diseases. As a result of analyzing 14 patients' sera, 7 with Graves' disease and 7 with Hashimoto's thyroiditis, anti-TPO autoantibodies were found to consist of mainly IgG1 subclass. Percentages of both IgG1 and IgG2 subclasses in IgG class of autoantibodies corresponded to those in the normal serum composition, whereas IgG3 subclass was scarcely contained in anti-TPO autoantibodies and IgG4 subclass markedly increased. It was thought that anti-TPO autoantibodies had a capability to lyse thyroid follicular cells by the mechanism of antibody-dependent complement-mediated cytolysis, because IgG1 and IgG2 subclasses of antibodies can fix complement and TPO locates in apical membrane surface of thyroid follicular cells. Comparing Graves' disease with Hashimoto's thyroiditis, mean percentages of both IgG1 and IgG2 subclasses of 2 groups were statistically different. Namely, sera of patients with Graves' disease had higher and lower mean percentages of IgG1 and IgG2 subclasses of autoantibodies, respectively, than those with Hashimoto's thyroiditis, though no plausible explanation for these differences can be offered at the present time.     

IgG subclass distribution among antibodies to varicella-zoster virus in human varicella/zoster immunoglobulin preparations and the corresponding donor plasma

IgG subclasses to varicella-zoster virus (VZV) were detected in plasma from different sources used for the production of varicella/zoster immunoglobulin (VZIG). IgG1 and IgG3 were the principal virus antibodies in plasma from healthy donors as well as from convalescents after primary and reactivated disease. Anti-VZV IgG3 antibodies were predominant among varicella convalescents while IgG1 antibodies dominated among zoster convalescents. IgG4 antibodies were present in zoster convalescents and healthy donors but were rarely detected in varicella convalescents. Antiviral IgG2 antibodies were found only in a few cases. Studies of plasma samples collected from one varicella convalescent during a period of seven months following an outbreak of disease, demonstrated a rapid fall in antiviral IgG1 and IgG3, while IgG4 increased to reach a maximum six months after the onset of symptoms. The relative distribution of VZV-specific subclasses in a plasma pool was conserved during a fractionation procedure combining polyethyleneglycol 6000 precipitation with ion exchange chromatography, thus suggesting that the protective efficacy is maintained in the resulting immunoglobin preparations.     

Phagocytosis by mouse peritoneal macrophages plated on monoclonal antibody-coated immune complex-substrates: effects of complexes of different IgG subclasses on Fc receptor functions

Macrophages plated on immune complex-coated substrates of different mouse IgG subclasses were examined for their capacities to phagocytose sheep red blood cells (SRBC) coated with monoclonal antibodies (MAb) of various IgG subclasses. IgG2a-and IgG2b-coated substrates abrogated macrophage phagocytosis of particles coated with any of the four mouse IgG subclasses. These results were confirmed by the use of two MAb of each of the IgG2a and IgG2b subclasses, with one of the MAb specific for dinitrophenyl groups and the others for SRBC. IgG3-coated substrates reduced the macrophage uptake of IgG2a-but not IgG2b-coated particles. Rabbit IgG-coated substrates ablated the uptake of SRBC coated with all mouse IgG subclasses. Resident and thioglycollate-elicited macrophages showed similar phagocytosis reduction when plated on these immune complexes. The phagocytosis of complement-coated particles was not affected by these IgG-coated substrates. Macrophages plated on both IgG2a-and IgG2b-coated substrates showed reduced immunofluorescence staining by an anti-IgG2b Fc receptor (FcR) Ab, 2.4G2 and reduced E(IgG2a) and E(IgG2b) binding. The results show that substrates coated with various IgG subclasses can abrogate phagocytosis mediated by FcR that do not have binding specificity for the substrate-immobilized Fc ligand, and suggest that the three classes of mouse FcR co-modulate.     

The effect of pre-partum milking on the transfer of immunoglobulin into mammary secretion of cows.

The significance of local effects associated with mammary involution on transfer of immunoglobulin and especially on the selective transfer of IgG1 into mammary secretion of cows approaching parturition has been determined. This was carried out by measuring the changes in the concentration of IgG1, IgG2, IgM and IgA in serum and mammary secretion of 5 cows in which two mammary glands were milked continuously (twice daily) during the period preceding parturition, while the other two glands were allowed to undergo normal involution. In the secretion of unmilked glands of all cows there was a substantial increase in the concentration of IgG1 as cows approached parturition. In contrast, the increases in the concentration of IgG1 and in the selective index for IgG1 of milked glands were either virtually non-existent (1 cow) or generally reduced in magnitude and delayed in time of onset (4 cows). It is clear from the results that continued milking of a mammary gland throughout pregnancy tends to maintain milk production in the milked gland and at the same time reduces the massive selective transfer of IgG1 into secretion of that gland.     

Binding of macrophages and phospholipid flip-flop in supported lipid bilayers

Subclass-specific antibody-dependent interactions (binding and triggering) between macrophages and supported lipid bilayers have been studied. Percentages of mouse macrophage binding (J774 cell line) to the lipid bilayers were dependent on mouse monoclonal IgG subclasses. The efficiencies were as follows: IgG1 = IgG2a greater than IgG2b greater than IgG3. Furthermore, macrophage triggering (spreading) was more efficient on IgG2a- or IgG1-coated lipid bilayers than on IgG2a, IgG3, or non-specific rabbit IgG. The present experiments show also that phospholipid molecules are able to flip-flop from one side of a supported planar bilayer membrane to the other with a half-life of 10 h-1 day at 25 degrees C.     

Identification of a unique IgG Fc binding site in human intestinal epithelium

In experiments to determine whether serum antibodies in patients with Crohn's disease could be used as probes for detecting potentially etiologic Ag in the patients' tissues, we found that peroxidase (HRP)-labeled IgG from healthy persons, as well as from the patients, bound to normal colonic and small intestinal epithelium, mostly or entirely to goblet cells. The binding was due to a reaction involving the Fc region of IgG because HRP-labeled Fc fragments of IgG bound, but HRP-Fab, HRP-IgA, and HRP-bovine albumin did not, and because binding of HRP-IgG was inhibited competitively by unlabeled IgG or Fc fragments but not by IgG Fab fragments or IgA. These immunohistochemical results were confirmed by ELISA with microtiter wells coated with a sonicated homogenate from human colonocytes. The epithelial IgG Fc binding site was characterized by SDS-PAGE as consisting of a high Mr (greater than 200,000 Da) and a 78,000-Da component. It bound all four subclasses of human IgG and bound aggregated as well as monomeric IgG. It is distinct from known human Fc-gamma R by lack of recognition by mAb to those receptors and differences in affinity for various subclasses of human and murine IgG. This unique IgG Fc binding site might be involved in immunologic defense of the gut, perhaps by mediating reactions between foreign Ag and the contents of goblet cells.     

Factors associated with the concentration of immunoglobulin G in the colostrum of dairy cows

Transfer of sufficient immunoglobulin G (IgG) to the neonatal calf via colostrum is vital to provide the calf with immunological protection and resistance against disease. The objective of the present study was to determine the factors associated with both colostral IgG concentration and colostral weight in Irish dairy cows. Fresh colostrum samples were collected from 704 dairy cows of varying breed and parity from four Irish research farms between January and December 2011; colostral weight was recorded and the IgG concentration was determined using an ELISA method. The mean IgG concentration in the colostrum was 112 g/l (s.d. = 51 g/l) and ranged from 13 to 256 g/l. In total, 96% of the samples in this study contained >50 g/l IgG, which is considered to be indicative of high-quality colostrum. Mean colostral weight was 6.7 kg (s.d. = 3.6 kg) with a range of 0.1 to 24 kg. Factors associated with both colostral IgG concentration and colostral weight were determined using a fixed effects multiple regression model. Parity, time interval from calving to next milking, month of calving, colostral weight and herd were all independently associated with IgG concentration. IgG concentration decreased (P < 0.01) by 1.7 (s.e. = 0.6) g/l per kg increase in the colostral weight. Older parity cows, cows that had a shorter time interval from calving to milking, and cows that calved earlier in spring or in the autumn produced colostrum with higher IgG concentration. Parity (P < 0.001), time interval from calving to milking (P < 0.01), weight of the calf at birth (P < 0.05), colostral IgG concentration (P < 0.01) and herd were all independently associated with colostral weight at the first milking. Younger parity cows, cows milked earlier post-calving, and cows with lighter calves produced less colostrum. In general, colostrum quality of cows in this study was higher than in many previous studies; possible reasons include use of a relatively low-yielding cow type that produces low weight of colostrum, short calving to colostrum collection interval and grass-based nutritional management. The results of this study indicate that colostral IgG concentration can be maximised by reducing the time interval between calving and collection of colostrum.     

Molecular and functional characterization of cynomolgus monkey IgG subclasses

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.     

Lymphocyte mitogenesis induced by monoclonal antibodies to the T3 complex. Differential modulation by human IgG

Murine monoclonal antibodies OKT3 (IgG2), 64.1 (IgG2), and Leu 4 (IgG1) react with a common membrane antigen on human T cells and induce potent mitogenesis at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml, respectively. Human serum inhibits the mitogenic effect of antibodies OKT3 and 64.1, but not that of Leu 4. The inhibitor in serum has been identified as immunoglobulin G (IgG) as evidenced by the ability of anti-human IgG-Sepharose affinity columns to retain the inhibitory activity. Various immunoglobulin classes and subclasses obtained from human myelomas differ in their ability to inhibit the OKT3-induced activation. The best inhibition is obtained with the IgG subclasses IgG1 and IgG3, followed by IgG2; IgG4, IgM, and IgA have little if any effect. None of the IgG subclasses inhibit the Leu 4-induced mitogenesis. Indomethacin as well as supernatants containing interleukin 2 (IL-2) can reverse the inhibitory effects of IgG. Prostaglandins (PGE1 and PGE2) inhibit both the OKT3- and Leu 4-induced mitogenesis, thus lacking the selectivity seen with IgG. Since stimulation by the monoclonal antibodies requires the participation of monocytes, an interpretation consistent with the present data is that IgG stimulates monocytes via its Fc portion to release prostaglandins and/or other suppressor factors via an indomethacin-sensitive pathway. The inability of IgG to inhibit Leu 4-induced mitogenesis may therefore relate to an inability of the monocyte subpopulation, which mediates the Leu 4 response, to secrete suppressor factors. These data suggest a potential value of the mitogenic monoclonal antibodies as probes in studying monocyte heterogeneity and T-cell-monocyte interactions.     

H chain C domains influence the strength of binding of IgG for streptococcal group A carbohydrate

We have produced a panel of murine anti-streptococcal mAbs, expressing identical V domains and different H chain C domains, corresponding to the IgG3, IgG1, and IgG2b subclasses. We have used these mAb to evaluate the role of IgG subclass-specific C region determinants in modulating the interaction between antibody and the bacterial surface. We report, for the first time, that V region-identical murine IgG of different subclasses exhibit substantial differences in binding to specific Ag; IgG3 mAb binds more strongly to streptococci than the IgG1 and IgG2b mAb or IgG3-derived F(ab')2 fragments. Furthermore, the IgG3 mAB binds cooperatively to the bacteria, whereas the IgG1, IgG2b, and IgG3-derived F(ab')2 fragments do not exhibit significant cooperativity, which suggests that differences in Fc region structure can affect antibody binding to multivalent Ag by modulating the potential for cooperative binding. These results suggest a plausible mechanism by which murine IgG3 could be more effective, than other antibodies bearing identical V domains, but of different gamma-subclass, in mediating bacterial immunity.     

Separation and purification of IgG1 and IgG2 from IgG-Cohn-II fraction of human plasma by pseudobioaffinity chromatography on histidyl-AH-sepharose.

L-histidine coupled to aminohexyl-sepharose (H-AH) has been used as an affinity sorbent to separate IgG from human plasma. Two subclasses IgG1 and IgG2 were specifically bound to histidyl-AH-sepharose at pH 7.4 and eluted using 0.2 M and 1M NaCl. The specificity of the two subclasses were determined by immunoelectrophoresis. Quantitative determination of IgG1, IgG2 was carried out using radial immunodiffusion technique.