Affinity Purification of IgG Subclasses and the Distribution of Thyroid Auto-Antibody Reactivity in Hashimoto''s Thyroiditis

To delineate accurately the IgG subclass distribution of thyroid auto-antibodies, sera from nine patients with Hashimoto's thyroiditis were fractionated into IgG subclasses by complete depletion of the other IgG subclasses on affinity columns. All IgG subclass fractions contained thyroglobulin and microsomal (or thyroid peroxidase) antibody activity, although when compared to the total serum concentrations of IgG subclasses, IgG4 antibodies were overrepresented. However, in contrast to recent studies, this particular subclass never predominated--IgG4 antibody levels being exceeded by those of the IgG1 and IgG2 subclasses; it seems likely that these differences relate to varying sensitivity for different subclasses in previously used assay methods. This pattern of subclass activity differed from that of tetanus toxoid antibodies, which were found in six subjects. There was no light chain restriction within any subclass, showing that the overproduction of IgG4 thyroid antibodies is not of monoclonal origin. The functional affinity of subclasses for both thyroid antigens varied between patients, but IgG2 subclass fractions showed the highest functional affinity in the majority of samples. We also found that IgG2 subclass thyroid antibodies were ineffective in eliciting antibody-dependent cell-mediated cytotoxicity, as distinct from the other three subclasses. Our results show that thyroid antibodies are less restricted in their IgG subclass distribution and patients are less heterogeneous than previously described. Moreover, IgG2 thyroid antibodies are quantitatively important and differ in relative functional affinity and effector function from IgG1 and IgG4 thyroid antibodies.     

Pepsin digestion of human G-myeloma proteins of different subclasses. II. Immunochemical investigations of the products of peptic digestion

Immunochemical comparisons have been made of the F(ab'')₂ and the pFc'' fragments of each of the four subclasses of human IgG. The F(ab'')₂ fragments of IgG 3 proteins were found to exhibit anomalous gel-filtration behaviour compared with similar fragments from the other subclasses. This difference was unrelated to carbohydrate content and may indicate a unique conformation for IgG 3 molecules.The pFc'' fragments of all four subclasses were similar in size and antigenically indistinguishable by gel-diffusion tests but on both starch gel electrophoresis and immunoelectrophoresis the IgG 4 pFc'' fragment was of faster mobility.Some minor products of pepsin digestion arising from the IgG 2, IgG 3 and IgG 4 subclasses were also investigated. These fragments were derived from the Fab regions of the respective parent molecules.     

The significance of blood levels of IgM,IgA, IgG and IgG subclasses in Sudanese visceral leishmaniasis patients

We developed an ELISA test using leishmania antigenic extracts to detect antigen-specific antibody responses, including subclass and isotype analysis, in visceral leishmaniasis (VL) patients from the Sudan. A total of 92 parasitologically proven patients were compared with cutaneous leishmaniasis, schistosomiasis, malaria, onchocerciasis and tuberculosis patients, as well as with healthy endemic and non-endemic controls. Some VL patients were examined before and after chemotherapy. VL patients showed significantly higher IgG responses compared with all other groups (93·4% sensitivity, 93·7% specificity), and higher (but not significantly) IgM responses. All groups showed low IgA levels. All groups showed low IgA levels. All IgG subclasses, IgG1, 2, 3, and 4, showed higher levels in patients than all other groups, with IgG1 and IgG3 levels being significantly reduced following treatment. The rank order for specificity and sensitivity for IgG subclasses was IgG3 > IgG I> IgG2> IgG4.     

IgG1 and IgG2 are the predominant subclasses of antiphospholipid antibody in women with the lupus anticoagulant

We studied the sera of 36 patients with lupus anticoagulant and IgG antibodies against both phosphatidylserine and cardiolipin. Most sera also had IgG antibodies against other phospholipids: 97% against phosphatidylinositol, 91% against phosphatidylglycerol, and 82% against phosphatidylethanolamine. IgG2 was the predominant subclass against cardiolipin and phosphatidylserine; 35 of 36 patients (98%) had IgG2 against both phospholipids. Most patients also had the IgG1 subclass; 32 of 36 (89%) against cardiolipin and 25 of 36 (69%) against phosphatidylserine. IgG3 and IgG4 subclasses were present at very low concentrations and in only a minority of the sera. The antibody response against phosphatidylserine was characterized by significantly less IgG1 than was the response against cardiolipin (P less than 0.01), although the IgG2 responses against each phospholipid were not different. IgG subclasses were unrelated to any other aspect of the patients' history, including a history of thrombocytopenia or thrombosis, a positive antinuclear antibody test, or a diagnosis of systemic lupus erythematosus.     

Bovine IgG1, but not IgG2, binds to human B cells and inhibits antibody secretion

We previously observed that milk-derived bovine IgG, but not serum-derived bovine IgG, strongly inhibits antibody secretion by pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC). Bovine milk contains a greater percentage of IgG1 (90%) than does bovine serum (53%). To determine whether bovine IgG subclasses have different functional capabilities, we have examined the effects of bovine IgG1 and IgG2 subclasses upon not only antibody secretion but also mitogenesis by human PBMC. Both bovine IgG subclasses markedly inhibited PWM-stimulated mitogenesis. However, only bovine IgG1, and not IgG2, inhibited antibody secretion during a 14-day in vitro culture period. Also, antibody secretion was inhibited following a 24-hr preincubation of human PBMC with bovine IgG1, but not with IgG2. To determine whether these differences corresponded to specificities of human Fc gamma receptors on subsets of mononuclear cells, fluorescence-activated cell sorter (FACS) analyses were performed. Both bovine IgG subclasses bound to human monocytes. However, only bovine IgG1 bound to human B cells, and bovine IgG1 bound more avidly to human B cells than did human IgG. One model to explain these findings is that inhibition of mitogenesis may be due to the binding of both bovine IgG1 and IgG2 subclasses to monocytes; whereas subclass-specific inhibition of antibody secretion may result from the selective binding of bovine IgG1, but not bovine IgG2, to B cells. The observation that bovine IgG1 has a greater avidity for human B lymphocyte Fc receptors than human IgG may have important implications for future studies of Fc gamma receptors on human leucocytes.     

Human constant regions influence the antibody binding characteristics of mouse-human chimeric IgG subclasses

Although antibody affinity is primarily determined by immunoglobulin variable region structure human IgG antibodies of the four subclasses specific for the same antigen have been shown to differ in their affinity. To explore the influence of the immunoglobulin constant region on functional antibody affinity, a set of V region identical mouse–human chimeric IgG subclasses specific for TAG72 (tumour-associated glycoprotein) were studied. Biomolecular interaction analysis (BIA) was used to determine the binding kinetics of whole IgG subclasses and F(ab')₂ fragments. Despite identical V regions, binding kinetics differed for the four subclasses. The apparent dissociation rate constants of the intact immunoglobulins ranked IgG4 < IgG3 < IgG2 < IgG1. In contrast, analysis of the binding characteriztics of the F(ab')₂ fragments derived from IgG1, IgG2 and IgG4 revealed identical binding kinetics. The structure of the constant regions of the humanized IgG subclass antibodies clearly influenced functional antibody affinity, as has been described for the murine IgG subclasses. The exact mechanism for this phenomenon remains obscure but such differences should be taken into account when designing or choosing antibodies for therapeutic use.     

A monoclonal antibody may show cross-reactivities in Ouchterlony assays but not in other assays

A monoclonal antibody to human IgG was tested with myeloma proteins of the four IgG subclasses. When tested by immunofluorometric assay, enzyme-linked immunosorbent assay, hemagglutination and hemagglutination inhibition assays, the antibody reacted with IgG3 but not with the other three IgG subclasses. When tested by Ouchterlony assays in the presence of polyethylene glycol, the antibody formed lines with all four IgG proteins. The line with IgG3 was sharp and stable, but the lines with the other three IgG subclasses tended to blur with time and with the lower PEG concentrations. These findings show that Ouchterlony assays can reveal cross-reactions of a monoclonal antibody that can be missed by more sensitive assays.     

Erythrocyte autoantibodies, subclasses of IgG and autoimmune haemolysis

The subclass pattern of red cell bound IgG autoantibody was studied in 304 patients on 426 occasions. Subclass interrelationships with time, other cell bound immunoglobulins (IgM, IgA), amount of bound IgG and serum haptoglobin levels were investigated using population proportions; because of the multiple statistical tests, P less than 0.01 was required for significance. IgG1 was most common, being found in 98% of cases and as the sole subclass in 64%; multiple subclasses occurred in 34.5%. The IgG subclass pattern possibly changed with time (P less than 0.02, greater than 0.01), populations being compared at 6 and 12 months. There was a highly significant and important correlation between multiple IgG subclasses and multiple immunoglobulin coating; in our further studies, this necessitated the use of samples where only cell bound IgG was increased. Multiple IgG subclasses strongly correlated with larger amounts of cell bound IgG, groups with greater than 2 and less than 1 OD units by the enzyme-linked direct antiglobulin test being compared (approximately greater than 800 and less than 400 molecules IgG per red cell respectively). Multiple subclasses (P less than 0.05, greater than 0.01), but not IgG3, were possibly associated with low haptoglobin levels; significance was reached, however, if the multiple immunoglobulin effect was ignored. IgG subclass interrelationships are clearly complex and require strictly defined populations for their study.     

Lung humoral response toPseudomonas species

Immunoglobulin G (IgG) is quantitatively the predominent immunoglobulin protein in the distal airway of the human host. IgG enters the distal lung from circulating pools and plasma cells located largely in the interstitium. Although all four IgG subclasses are present in human lung secretions, only subclasses G3 and, to a lesser degree, G1 attach to pulmonary macrophage membrane receptors. IgG opsonic antibodies are essential for the optimal clearance of a very troubling pathogen,Pseudomonas aeruginosa. Nosocomial pneumonia caused by this gram-negative bacillus responds poorly to potent antimicrobial agents and is associated with a 70 % mortality. To a great extent the morbidity and mortality resulting from nosocomial pneumonia caused byPseudomonas spp. are attributable to ineffective humoral immune response to this bacterium. IgG2 antibodies in response to pseudomonal lipopolysaccharide are poorly opsonic in the macrophage system, and derepression of the potent pseudomonal elastase, an enzyme with broad substrate specificity, contributes to the disruption of other IgG antibodies.     

Distribution of Surface, Cytoplasmic and Secreted IgG Subclasses in Human Lymphoblastoid Cell Lines and Normal Peripheral Blood Lymphocytes

Twenty-two IgG-positive human lymphoblastoid cell lines and normal peripheral blood lymphocytes were studied for surface and cytoplasmic IgG, IgG subclasses, IgD and IgM, using monospecific fluorescein-and rhodamine-conjugated F(ab')₂ antibody fragments, and for secretion by double antibody radioimmunoassay. Several parallel observations and several differences in IgG subclass expression were noted between cell lines and normal lymphocytes. Surface IgG2 was frequently expressed in normal IgG-positive lymphocytes but was seldom expressed in cell lines. Cell lines resembled normal IgG-positive lymphocytes in the frequent expression of cytoplasmic IgG3 and lgG4, often without secretion. Cell lines and normal lymphocytes both showed more frequent distribution of IgG and IgG sub-classes in cytoplasm than in surface immunoglobulin, and often a discrepancy of surface versus cytoplasmic IgG subclass. A good correlation was noted between surface, cytoplasmic and secreted IgG1. Despite a predominance of IgG2 and IgG4 surface IgG subclasses, and IgG3 and IgG1 in cytoplasm, secreted immunoglobulins from normal lymphocytes in short-term culture showed a similar distribution of IgG subclasses to that seen in normal sera. Multiple expression of IgG subclasses was much more frequent in IgG-positive cell lines than in normal peripheral blood lymphocytes, both in surface and cytoplasmic IgG.     

Methodologic problems in establishing normal values for IgG subclass concentrations in a pediatric population; comparison of radial immunodiffusion and ELISA methods

The aim of this study was to establish an enzyme-linked immunosorbent assay (ELISA) to measure IgG subclasses by means of monoclonal antibodies. The distribution of IgG subclass protein concentrations in sera from 227 healthy Danish children and 90 adults was measured. Furthermore, this newly established ELISA was compared with different assay systems for determination of IgG subclasses: two radial immunodiffusion methods (RID), one using polyclonal and one using monoclonal antibodies, as well as a commercially available ELISA kit. There was good agreement of results obtained by the different methods of measuring IgG3 and IgG4 concentrations. There was good correlation between results obtained by both RID methods. Despite good correlation between the assays, the ELISA kit showed higher levels of IgG1 in all investigated sera, and the ELISA kit showed no correlation with the other methods, when IgG2 was measured. Analysis of the normal ranges measured by ELISA developed in our laboratory and by RID with polyclonal antibodies showed that the levels obtained by RID were higher than those obtained by our ELISA in sera with low levels of both IgG1 and IgG2, and lower in sera with high concentrations of these two immunoglobulins. Our results emphasize the importance of establishing age-related normal limits for any novel assay measuring IgG subclass concentrations.     

Relationship between serum IgG2 concentrations and antibody responses to pneumococcal polysaccharides in children with chronic chest symptoms

We measured IgG-class antibodies to 12 pneumococcal antigens pre- and post-immunization with polyvalent pneumococcal vaccine in 31 children who had experienced chronic chest symptoms. The purpose of the study was to determine the relation of IgG subclasses, especially IgG2, to the subjects' antibody responses to bacterial polysaccharide antigens, to see if measuring IgG subclasses would predict these responses. Twenty-nine children (90%) had low or low-normal levels of one or more IgG subclasses, including 20 out of 31 (65%) with low or low-normal levels of IgG2. Children studied had a relatively poor increase in levels of antibody to 10 of the 12 pneumococcal vaccine antigens investigated. Both pre- and post-immunization antibody levels were related to pre-immune serum concentrations of IgG2. Pre-immunization antibody levels were strongly related to post-immunization levels; when post-immunization antibody levels were adjusted for pre-immunization levels by partial correlation, the correlation between anticapsular antibody level post-immunization and IgG2 was no longer significant. Thus, in children with chronic chest symptoms, levels of antibody measured at a random interval after natural exposure to these bacterial polysaccharide antigens are related to levels of IgG2 subclass, but antibody increases after vaccination appear to be affected more by other factors.     

IgG Subclasses in Bronchoalveolar Lavage Fluid from Patients with Asthma

We have measured Immunoglobulin G (IgG) subclasses in serum and bronchoalveolar lavage fluid (BALF) from 12 non-smoking patients with stable asthma and 9 non-smoking healthy volunteers to obtain information on their possible role in local immunological reactions. The quotients (concentration of IgG subclass in BALF)/(concentration of IgG subclass in serum) were calculated. In controls QIgG3 were lower than QIgGI, QIgG2 and QlgG4.
The IgG subclasses in BALF and epithelial lining fluid (ELF) from patients with asthma were significantly higher than in controls, mainly due to increased leakage from the blood. Again QIgG3 were lower than Q of other subclasses. In the analysis of local production of IgG, albumin or ceruloplasmin was used as reference protein. Several patients showed a local production or a preferential accumulation of one or more IgG subclasses.
We conclude that in healthy persons the IgG subclasses in ELF originate from the systemic circulation by passive permeation. In patients with asthma, the permeability of the respiratory membrane may be increased resulting in increased concentrations of subclasses in lung-lining fluid. In some patients with asthma, an additional local production of IgG subclasses occurs.     

Immunoglobulin G (IgG) and IgA subclass pattern of human antibodies to Shigella flexneri and Salmonella serogroup B and D lipopolysaccharide O antigens

The subclass distribution of human serum antibodies to the O-antigenic lipopolysaccharides of Salmonella serogroups B and D and to Shigella flexneri serotypes 1b, 2a, and 4a lipopolysaccharide antigens were analyzed in an enzyme-linked immunosorbent assay with monoclonal antibodies to the immunoglobulin subclasses. The patients had culture-verified Salmonella (17 Swedes) or Shigella flexneri (23 Vietnamese; 11 children and 12 adults) infections. Consecutive samples drawn during 1 year postinfection were investigated. Antibodies to the Salmonella antigens were mainly of the immunoglobulin G1 (IgG1), IgA1, and IgA2 subclasses. For the Salmonella serogroup B O polysaccharide, the IgA1 and IgA2 subclasses had peak values earlier than (6/9) or coinciding with the IgG1 (3/9) peak value. Furthermore, the IgA2 response to Salmonella serogroup B was positively correlated to the duration of the carrier state (P less than 0.001); the corresponding IgA1 response was less well correlated but was still significant (P less than 0.02). In the case of the Shigella flexneri O polysaccharide, specific antibodies appeared mainly in the IgG1 and IgA1 subclasses. Some IgG2 was also found, surprisingly even in very young patients. No subclass shift with time within the immunoglobulin classes was noted in any of the groups.     

IgG subclass antibodies to Pseudomonas aeruginosa in sera from patients with chronic Ps. aeruginosa infection investigated by ELISA

ELISAs using subclass-specific monoclonal antibodies were developed for the quantification of human IgG1, IgG2, IgG3 and IgG4 antibodies to Ps. aeruginosa. We investigated the pattern of IgG subclass antibodies against Ps. aeruginosa in serum from patients with cystic fibrosis (CF), other patients with chronic Ps. aeruginosa infection, and healthy controls. Healthy controls and patients with CF but without Ps. aeruginosa infection showed no or very low titres of antibodies against Ps. aeruginosa. In the early stage of chronic Ps. aeruginosa infection, antibody titres in all four subclasses were significantly higher than either normals or CF patients without infection. Other patients with Ps. aeruginosa infection showed the same increased level of IgG subclass antibodies as CF patients in an early stage of infection. Sixteen patients (eight in good and eight in poor clinical condition) have been followed for an average of 13 years with multiple serum samples covering the pre-infection, early and late stages of chronic infection. Patients in a poor clinical condition showed significantly higher levels of IgG3 antibodies in the first year of infection and 2 years later also had significantly higher IgG2 antibody levels. We conclude that elevated levels of IgG2 and IgG3 antibodies to Ps. aeruginosa are a sign of poor prognosis in CF.     

Distribution of IgG subclasses in antimonial unresponsive Indian kala-azar patients

Sodium antimony gluconate (SAG) is the mainstay of treatment for visceral leishmaniasis (VL) or kala-azar. In view of the increasing incidence of refractoriness to SAG in India, we compared the levels of parasite-specific IgG and IgG subclasses in 20 longitudinally followed up kala-azar patients. In both SAG-responsive (n = 10) and unresponsive patients (n = 10), the levels of total IgG, IgG1, IgG2, IgG3 and IgG4 were increased, the rank order being IgG1 > IgG2 > IgG3 = IgG4. Following treatment, a significant decrease in total IgG and the four subclasses occurred in the SAG-responsive group, whereas in the SAG-unresponsive group these levels were unchanged or slightly increased. Therefore, monitoring of IgG1 and IgG2 levels in Indian kala-azar patients is a good serologic alternative to monitoring the disease status.     

Interaction between rat peritoneal macrophages and sensitised erythrocytes: dependence on IgG subclass, antibody density and the degree of hapten conjugation.

The interaction between macrophages (M phi) and antibody sensitised target cells was studied by the use of rat peritoneal macrophages, TNP hapten conjugated sheep red blood cells (SRBC) and homologous antibodies of subclasses IgG1 and IgG2a. Under optimal conditions, the great majority of the M phi formed rosettes with IgG1-sensitised antibodies while a maximum of 50% was achieved when target cells were sensitised by IgG2a. Using a double rosette technique, the major part of rosette-forming cells was found to bind both of the isotypes. IgG1-mediated rosette formation was observed at very low degrees of sensitisation as opposed to IgG2a-mediated target cell binding. Not only the amount of bound antibody but also the degree of hapten conjugation (epitope density) appear to influence the ratio of rosette-forming cells. IgG1-mediated rosette formation was partially inhibited by monomeric IgG1 and more efficiently by soluble ovalbumin (OVA)-anti-OVA complexes involving IgG1-type antibodies, while IgG2a mediated rosette formation was inhibited by OVA-anti-OVA complexes containing IgG2a type antibodies, and less efficiently by complexes involving IgG1. No inhibition was found by monomeric IgG2a. Based on the present data, we propose that two types of receptors are involved in the interaction of M phi and target cells coated by IgG1 and/or IgG2a type antibodies. One requires a multiple antibody-receptor interaction, binding both subclasses at overlapping binding sites; the other is able to interact with IgG1 and does not depend on the multiplicity of interactions.     

Differences in titres of IgE, IgG4 and other IgG subclass anti- Der p 2 antibodies in allergic and non-allergic patients measured with recombinant allergen

Background The mite allergens are recognized as major causes of allergic disease such as bronchial asthma, allergic rhinitis and atopic dermatitis. The functions of allergen-specific IgG subclass antibodies are not defined.
Objective In order to clarify the relationship between IgE and IgG subclasses, we examined scrum levels of the Dermatophagoides pteronyssisus group 2 (Der p 2)-specific antibodies of IgH. IgG total and IgG subclasses in children with mite allergy.
Methods We prepared a recombinant Der p 2 fusion protein and examined serum levels of Der p 2 antigen-specific antibodies by enzyme-linked immunosorbent assay (FLISA) systems developed in our laboratory using a recombinant Der p 2 as target antigen. Sera from 240 children with mite allergy and 25 controls were measured.
Results The serum levels of specific IgE and, to lesser degree, lgG4 were higher in allergic children than non-allergic controls, while in the levels of the other IgG subclasses there was no difference between the two groups. There was no correlation between levels of specific IgF and IgG4 or in those between specific IgG4 and other IgG subclasses.
Conclusion Results indicate that the induction of Der p 2-specific lgG4 in allergic diseases is independent to IgE as well as other IgG subclasses.     

A quantitative determination of IgG anti-D subclasses by Elisa in hemolytic disease of the newborn]

The quantification of IgG anti-D subclasses is one of the most important parameters considered in the assessment of the severity of hemolytic disease of the newborn. Traditionally IgG subclassing is performed using qualitative haemagglutination methods, difficult to interpret. A quantitative enzyme-linked immunosorbent assay (Elisa) was implemented for measuring IgG anti-D subclasses in 20 sera collected from 14 RhD-immunized pregnant women. All 4 IgG subclasses were detected in the 20 sera tested. The mean proportion of IgG1 was 52.8%. The mean proportion of IgG3 was 30.7%. The mean proportions of IgG2 and IgG4 were 14.5 and 1.9% respectively. A good correlation between the sum of IgG subclasses and the severity of HDN was found. Severe HDN occurred when both IgG1 and IgG3 were present. IgG1 anti-D was the predominant subclass in 4 of the 8 severe cases.     

Serial studies on the affinity and heterogeneity of human autoantibodies to thyroglobulin

The functional affinity and heterogeneity of autoantibodies to thyroglobulin (Tg) were measured by an IgG subclass-specific solid-phase competition ELISA in patients with autoimmune thyroid disease. High-affinity IgG1 and IgG4 antibodies formed the major anti-Tg response. Both titre and affinity of IgG3 and IgG2 anti-Tg were generally low but in some Hashimoto's disease patients high-affinity IgG2 anti-Tg were found and IgG2 anti-Tg, unlike those of other subclasses, showed very restricted heterogeneity. The affinity of IgG4 anti-Tg was similar in patients with thyroid disease and their clinically euthyroid (normal) relatives. In contrast, a progressive increase in IgG1 anti-Tg affinity was seen in clinically euthyroid individuals compared with their relatives with thyroid disease and high titred Hashimoto's disease patients, suggesting that either rising titres of high affinity IgG1 anti-Tg or affinity maturation of IgG1 anti-Tg may be indicative of impending hypothyroidism.