Isotype concentrations of human antibodies to Haemophilus influenzae type b polysaccharide (Hib) in young adults immunized with the polysaccharide as such or conjugated to a protein (diphtheria toxoid)

Antibody responses of young adults to Haemophilus influenzae type b polysaccharide (Hib) or its protein conjugate were studied with special attention to the isotype composition of the antibodies. Three conclusions of interest can be made: 1) Immunoglobulin G (IgG) antibodies in polysaccharide-immunized volunteers displayed the subclass pattern previously found in antibodies to meningococcal type A polysaccharide. IgG1 was the predominant subclass in IgG antibodies of some individuals, IgG2 in others. Still others had the two subclasses in varying but more even proportions. 2) The conjugate vaccine induced a geometric mean response 2 to 3 times higher and an IgG response 4 times higher to Hib than the polysaccharide vaccine. 3) Anti-Hib antibodies induced by the conjugate vaccine still had essentially the same IgG subclass composition as anti-Hib antibodies induced by the polysaccharide. This composition was strikingly different from the composition of the anti-diphtheria toxoid response induced by the same conjugate vaccine.     

Ig allotype-linked regulation of class and subclass composition of natural antibodies to group A streptococcal carbohydrate

To determine the importance of genes located in or near the Ig constant regions in regulating the human antibody response, we correlated Ig allotypic markers with total Ig concentrations and natural antibody concentrations to the streptococcal group A carbohydrate (A-CHO) in 193 healthy adult blood donors. The major correlations between Ig allotypes and total Ig and specific antibody concentrations were observed with the Gm(f;n;b) haplotype. When compared with Gm(f;n;b) negative individuals, Gm(f;n;b) positives had significantly higher concentrations of total IgG2 (p less than 0.001) and IgG2 anti A-CHO (p less than 0.05), lower concentrations of total IgG1 (p less than 0.001) and IgG1 anti A-CHO (p less than 0.001), and lower concentrations of total IgM (p less than 0.001) and IgM anti A-CHO (p less than 0.05). We conclude that individuals with the Gm(f;n;b) haplotype respond preferentially with IgG2 rather than IgG1 subclass antibodies. This increased capacity to respond with IgG2 antibodies may be reflected in the magnitude of the total antibody response when the IgG2 subclass comprises a major proportion of the response, as occurs in the adult response to many polysaccharide Ag.     

Isotype concentrations of human antibodies to group A meningococcal polysaccharide

Antibodies to meningococcal group A polysaccharide (MenA) in the sera of 34 vaccinated adults were quantitated by an isotype-resolving solid-phase RIA (IgA, IgM, IgG1, IgG2, IgG3, and IgG4). All individuals had antibodies before vaccination. The geometric mean concentration was 2.9 micrograms/ml. Two weeks after vaccination the mean antibody concentration had trebled. Average proportions of the three isotypes were then as follows: IgA 15%, IgM 48%, IgG 37%. No differences were found between individuals who had been immunized with the polysaccharide 7 to 8 yr earlier and "primary responders." The subclass composition of IgG antibodies was determined in the 24 postvaccination samples with a definite IgG response (greater than 2-fold increase). IgG1 was the predominant subclass in antibodies of some sera and IgG2 in others, but the average proportions of both subclasses were nearly the same. IgG3 and IgG4 were only found in occasional sera, but when present, each subclass accounted for up to 6%. Although the ratio of kappa and lambda chains could not be determined, there was evidence to suggest that it was higher in anti-MenA antibodies than in antibodies to protein antigens.     

Subclass distribution of human antibodies to Haemophilus influenzae type b capsular polysaccharide

The polysaccharide (PS) capsule of Haemophilus influenzae type b (Hib) is a "simple" antigen, polyribosylribitolphosphate. Although similar carbohydrate antigens have been reported to elicit IgG antibodies relatively restricted to the IgG2 subclass in man, we report here that Hib PS elicits substantial quantities of both IgG1 and IgG2 serum antibodies in most individuals. Because the determination of IgG subclass distribution can be technically difficult, we used four different approaches to establish our finding. First, we used an IgG subclass-specific, antigen-specific "sandwich assay." Second, we measured IgG subclasses of purified antibodies to Hib PS. Third, we showed that significant amounts of IgG anti-PS can be absorbed with a monoclonal anti-IgG1 affinity column. Fourth, we showed that IgG1 and IgG2 fractions of immune sera have clonally restricted anti-Hib PS antibodies that are easily distinguishable by their isoelectric points. The data indicate that both IgG1 and IgG2 contribute substantially to the IgG antibody response of most adults to immunization with Hib PS.     

Serum immunoglobulin IgG subclass distribution of antibody responses to Yop proteins and lipopolysaccharide of Yersinia enterocolitica in patients with yersiniosis.

To assess the humoral immunological responses at the IgG subclass level in yersiniosis specific antibody responses against lipopolysaccharide of Yersinia enterocolitica 03 (LPS) and Yersinia Yop proteins were analyzed by ELISA. Thirty five patients with arthritis and forty nine patients with uncomplicated yersiniosis were included in the study. Analysis of the IgG subclass responses to the LPS revealed that the subclass distribution for both groups of patients was IgG2>IgG1>IgG3. The concentration of IgG4 was below detection level. The predominant antibody responses to Yop proteins were IgG1>IgG3>IgG2>IgG4 but the frequency of detection of particular IgG subclass antibodies were dependent on the age of patients. Generally, the frequency of occurrence of IgG2 antibodies for Yop proteins of Yersinia together increased with age reaching its peak among individuals aged above 40 years. On the other hand, IgG1 for Yop proteins and IgG3 for Y. enterocolitica LPS were diagnosed more often in serum samples obtained from children than from adults. We also found significantly higher frequency of IgG4 to Yop proteins of Y. enterocolitica in men than in women.     

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.     

Th1-type immune response to a Coccidioides immitis antigen delivered by an attenuated strain of the non-invasive enteropathogen Vibrio cholerae

The antigen-2 or proline rich antigen (Ag2/PRA) from Coccidioides immitis, known to protect mice against experimental Coccidioidomycosis, was expressed in the genetically attenuated cholera vaccine candidate Vibrio cholerae 638 and its thymine auxotrophic derivative 638T. Intranasal immunization of mice with strains producing Ag2/PRA induced serum vibriocidal antibody and Ag2/PRA-specific total IgG responses in outbred Swiss Webster and inbred BALB/c mice. Analysis of IgG subclasses showed a predominance of IgG2a subclass antibodies. Lymphocytes from immunized mice stimulated with pure Ag2/PRA showed a significant proliferative response with production of interferon-gamma. Positive selection for plasmid maintenance in vivo did not enhance immune response to Ag2/PRA. These results demonstrate that genetically attenuated strains of the non-invasive pathogen V. cholerae can be used to express and deliver foreign antigens to stimulate a Th1 type of immune response.     

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

Subclass distribution of antigen-specific IgA antibodies in normal donors and individuals with homozygous C alpha 1 or C alpha 2 gene deletions

To analyze the subclass restriction of Ag-specific IgA, sera and saliva from healthy blood donors and from IgA class or subclass deficient individuals were studied. The latter included donors with or without C alpha 1 or C alpha 2 gene deletions. Monoclonal human IgA1 and a genetically engineered IgA2 antibody, normal human serum and colostrum IgA were used as standards to estimate serum and saliva levels of Ag-specific antibodies. In normal individuals, there was a strong IgA1 preference of naturally acquired antibodies in serum against both polysaccharide Ag (PPS 6A, PPS 23, pneumococcal C-polysaccharide, and LPS from Escherichia coli) and protein Ag (Staphylococcus aureus alpha-toxin and HSV). Specific IgA2 in serum against the tested Ag were frequently not measurable. In contrast, most of the individuals with homozygous C alpha 1 gene deletions displayed substantial amounts of specific IgA2 against protein as well as polysaccharide Ag. The median levels of specific IgA in serum against protein Ag were approximately one-third as compared to normal individuals and one-fifth, or less, against polysaccharide Ag. Normal serum levels of IgA against the tested Ag, restricted to the IgA1 subclass, were noted in two individuals with IgA2 deficiency, one of whom carried a homozygous C alpha 2 gene deletion. Median values of specific IgA, against the tested Ag S. aureus alpha-toxin, HSV, and pneumococcal C-poly-saccharide, from normal healthy donors were approximately four to eight times higher in serum as compared to saliva. Individuals with homozygous C alpha 1 gene deletions displayed increased levels of the various specific IgA2 antibodies in saliva. In conclusion, the individuals with homozygous C alpha 1 gene deletions displayed decreased median levels of specific IgA antibodies in serum despite normal levels of total IgA. Normal levels of both specific IgA and total IgA in saliva were found.     

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.     

Control of allergic reactivity in human filariasis. Predominant localization of blocking antibody to the IgG4 subclass.

Patients with chronic helminth infections, despite having abundant basophils and mast cells specifically sensitized with antiparasite IgE and often exposed repeatedly to parasite Ag, rarely manifest allergic symptoms. This control of clinical allergic reactivity likely results from Ag-specific IgG "blocking antibodies" shown previously to be abundant in the sera of such patients. In the present study we used two approaches to determine in which of the four IgG subclasses this blocking activity was localized. First, specific antifilarial antibodies of each of the four IgG subclasses were quantified in the sera of 28 patients with Bancroftian filariasis and correlated with the levels of blocking activity in these sera (determined by histamine release assays). A significant correlation with blocking activity was seen only for antibodies of the IgG4 subclass, and, indeed, the correlation was especially strong in the group of totally asymptomatic patients (but with microfilariae circulating in the blood) in whom blocking antibody levels were highest. Interestingly, however, if the analysis excluded these asymptomatic microfilaremic patients and focused instead on those with lymphatic inflammatory pathology (who had relatively low levels of both serum blocking activity and specific IgG4 antibodies), then the small amount of blocking activity found in these sera correlated only with the levels of IgG1 subclass antibodies. The second approach utilized selective depletion of IgG4 (by anti-IgG4 affinity columns) from the sera of three microfilaremic patients with high levels of blocking activity and demonstrated clearly that removal of IgG4 abolished the majority of the blocking activity in these sera (53, 78, and 81%). These two sets of findings demonstrate a predominant role for specific IgG4 antibodies in blocking IgE-mediated allergic responses to the parasite Ag in vitro, but they also indicate that in some situations IgG1 antibodies can block such reactions. Furthermore, the correlation demonstrated between patients' clinical presentations and the levels of both their specific IgG4 antibodies and serum blocking activity suggests that these antibodies play a similar role in vivo as well.     

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.     

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.     

IgG subclass expression by human B lymphocytes and plasma cells: B lymphocytes precommitted to IgG subclass can be preferentially induced by polyclonal mitogens with T cell help

The human IgG subclasses expressed by circulating B lymphocytes, tissue plasma cells, and plasma cells generated from B cell precursors in response to the polyclonal mitogens LPS and PWM were examined by immunofluorescence using subclass-specific monoclonal antibodies. The subclass distribution observed for circulating B lymphocytes was IgG2 (48%) greater than IgG1 (40%) greater than IgG3 (8%) greater than IgG4 (1%), while the distribution among IgG plasma cells in bone marrow, blood, spleen, and tonsils was IgG1 (64%) greater than IgG2 (26%) greater than IgG3 (8%) greater than IgG4 (1%). Multiple IgG isotypes were not observed on B cells or in plasma cells. Although IgG plasma cell responses to both LPS and PWM were T cell dependent, the distributions of IgG subclasses elicited were strikingly different. In control and LPS-stimulated cultures of blood mononuclear cells, the induced plasma cells expressed the IgG subclass distribution: IgG2 greater than 80%, IgG1 less than 20%, IgG3 less than 1%, IgG4 less than 1%. In PWM-stimulated cultures, the subclass distribution, IgG1 approximately 65%, IgG2 approximately 25%, IgG3 approximately 7%, IgG4 approximately 1%, was in perfect concordance with the in vivo subclass distribution of IgG plasma cells. Selective inhibition of suppressor T cell activity by x-irradiation and mitomycin C treatment did not alter the IgG subclass distribution pattern induced by LPS and PWM. Monoclonal antibodies were used to deplete selectively the B cell precursors bearing IgG1, IgG2, or IgG3 before PWM stimulation of blood mononuclear cells. In each instance, a reduction was observed only in the subpopulation of plasma cells producing the homologous IgG subclass. The results indicate that T cells can preferentially influence the terminal differentiation of B cells that are precommitted to different IgG subclasses.     

IgG1 subclass dominates autoimmune response to tyrosine phosphatase-like molecule IA-2 in Chinese type 1 diabetes patients.

BACKGROUND AND AIMS: Islet autoantibodies are known markers for type 1 diabetes with an immune-mediated basis; their isotype or subclass profiles may also provide clues to changes in immune response during disease or after intervention. For ICAs and GADab, the IgG1 subclass consistently dominates in recent-onset disease. The aims of our study were to determine the isotype patterns for IA-2ab in Asian Chinese patients with autoimmune diabetes. MATERIALS AND METHODS: From an initial screening of over 400 diabetes patients, 40 subjects (mean age 22.2 +/- 15.8 years) with IA-2ab were enrolled for this study. IA-2ab was detected by radioimmunoassay of [35S]-labelled recombinant human IA-2 ic(605 - 979). Of them, 31 (median age 15 years, range 2 - 57 years; 16 children) had clinical type 1 diabetes (that is, they required insulin at onset or within 1 year) with the majority having been recently diagnosed (< 1 year). The other 9 patients had clinical type 2 diabetes phenotype. RESULTS: IA-2ab IgG subclasses determined with monospecific secondary antibodies showed that both type 1 diabetic adults and children had similarly non-restricted isotype patterns with a strong presence of IgG1-IA-2ab. The rank order was IgG1 > 3 > 2 > 4; 15 subjects had detectable IgG4-IA-2ab. Clonality of immune response determined with kappa/lambda chain-specific antibodies also showed a non-restricted pattern. Patients aged 38.2 +/- 15.2 years with type 2 diabetes had broad patterns of isotypes - IgG1/3 was detected more frequently (n = 8) than IgG2/4 (n = 5). Of three patients on insulin treatment, one was also positive for GADab. The remaining 6 patients were on oral hypoglycaemic treatment. IA-2ab in type 2 diabetes showed a low titre compared to type 1 diabetes. CONCLUSIONS: Isotype responses to IA-2 had a strong IgG1 presence, similar to ICAs and GADab. With IgG3 subclass representation, a predominant Th1 milieu in the systemic environment is likely. There is no suggestion of differences in immune response to IA-2 between adults and children with type 1 diabetes.     

Subclass restriction of murine anti-carbohydrate antibodies.

Examination of the subclass distribution of murine antibodies directed against groups A and C streptococcal carbohydrate, alpha-(1 leads to 3) dextran and phosphocholine yields the surprising observation that these carbohydrate antigens stimulate IgG responses largely restricted to the rare IgG3 subclass. This subclass restriction is particularly impressive in light of the low circulating levels of IgG3 in nonimmune mouse serum and the failure of a variety of other antigens including proteins and aromatic haptens to stimulate IgG3 antibody production. Attempts to alter the subclass restriction of antibodies with carbohydrate specificity by immunization with carbohydrate-coupled protein have been unsuccessful and indicate that immunoregulation of subclass expression probably occurs at the level of the antibody forming (B) cell. It is therefore conceivable that VH regions of murine immunoglobulins may be restricted to particular IgG subclasses. A similar type of subclass restriction has been reported in human and rat anti-carbohydrate antibodies. This recruitment of a minor immunoglobulin isotype by carbohydrate antigens in several species further supports the concept of immunoregulation at the level of subclass, and suggests that these and other mammals may share a structurally similar isotype with perhaps a common evolutionary origin.     

Heritability of Antibody Isotype and Subclass Responses to Plasmodium falciparum Antigens

Background

It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters.

Methodology/Principal Findings

This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE) naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types) and MSP3 (two allelic types). Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h ²) estimates for 48% (20/42) of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested) among the adults, 48% (20/42) among the children in the dry season and 31% (13/42) among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20) of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution.

Significance

Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation.     

IgG subclass responses in childhood Helicobacter pylori duodenal ulcer: evidence of T-helper cell type 2 responses

BACKGROUND: Duodenal ulcer in adults chronically infected with Helicobacter pylori is associated with a polarized T-helper cell type 1 (Th1) mucosal immune response, with a predominantly immunoglobulin G2 (IgG2) systemic specific response. It has been suggested that children colonized by H. pylori also produce a mucosal Th1 response, but there are few studies that have measured IgG subclass responses in children with duodenal ulcer. MATERIALS AND METHODS: Seven children with endoscopically proven duodenal ulcer and H. pylori infection and 18 children with biopsy proven H. pylori infection but no duodenal ulcer had relative concentrations of IgG subclass responses (IgGsc) against H. pylori antigens measured by ELISA. Eighteen IgG seropositive adults acted as controls. The range of antigens recognised by IgG1 and IgG2 subclass responses were investigated by Western blots. RESULTS: There were no differences in mean IgGsc responses between children with or without duodenal ulcer. Adults produced an IgG2 predominant response. Western blots showed no qualitative differences in antigens recognised by IgG1 or IgG2. CONCLUSION: Children with duodenal ulcer, in contrast to adults, produce an IgGsc response consistent with a mucosal Th2 response to H. pylori regardless of the presence of duodenal ulceration. This suggests that disease causation amongst children with H. pylori associated duodenal ulceration may not be dependant upon a mucosal Th1 biased response.     

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.