Selective IgG subclass deficiency: quantification and clinical relevance

Each of the four human IgG subclasses exhibits a unique profile of effector functions relevant to the clearance and elimination of infecting microorganisms. The quantitative response within each IgG subclass varies with the nature of the antigen, its route of entry and, presumably, the form in which it is presented to the immune system. This results in antibody responses to certain antigens being predominantly or exclusively of a single IgG subclass. An inability to produce antibody of the optimally protective isotype can result in a selective immunodeficiency state. This is particularly apparent for responses to certain bacterial carbohydrate antigens that are normally of IgG2 isotype. A failure to produce the appropriate specific antibody response may result in recurrent upper and/or lower respiratory tract infection. Careful patient investigation can identify such deficiencies and suggest appropriate clinical management. In this review we outline the biology and clinical relevance of the IgG subclasses and summarize current rational treatment approaches.     

Clinical efficacy of intravenous immunoglobulin in patients with severe inflammatory chest disease and IgG3 subclass deficiency

To investigate the efficacy of i.v. IgG treatment in pediatric patients with inflammatory lung disease, a prospective, controlled clinical trial was carried out over a 2-year study period. Patients were enrolled on the basis of severe clinical symptomatology. After 1 year of conventional treatment, the patients received 400 mg/kg per month of an i.v. IgG product containing only trace amounts of IgG3 in addition to their regular treatment throughout the second year. Significant clinical improvement, as documented by duration of hospital stay (first year 27.8 days, second year 4.9 days), use of antibiotics (132.8 versus 30.9 days) and use of steroids (21.4 versus 0.7 days) could be observed. Data obtained on a subgroup of patients with IgG3 deficiency were analysed separately. These results indicate that patients with severe chest disease who have IgG3 deficiency will also benefit from i.v. IgG treatment. The mode of action cannot be attributed to replacement of the respective isotypes, but is probably due to the effect of i.v. IgG in preventing repeated viral infections.     

Clinical and immunological evaluation of patients with mild IgG1 deficiency.

Serum IgG subclass concentrations were determined in patients visiting, the pulmonology out-patient clinic with chronic respiratory tract problems. A total of 24 patients with a serum IgG1 concentration < 4.9 g/l (i.e. below the reference range) and normal values for IgG2, IgM and IgA were included. Patients with a selective IgG1 deficiency were vaccinated with a 23-valent pneumococcal polysaccharide vaccine. There were nine patients with a poor antibody response to pneumococcal capsular polysaccharide antigens. Responsiveness to protein antigens was intact in all patients. Patients with pneumonia showed a significantly lower anti-polysaccharide response in the IgG2 subclass than patients without pneumonia. Patients with recurrent sinusitis showed a significantly lower response in the IgA isotype after vaccination with pneumococcal polysaccharide vaccine compared with non-sinusitis patients. It can be concluded that patients with recurrent sinopulmonary infections and a mild IgG1 subclass deficiency have an impaired IgG1 anti-polysaccharide response, which can extend to decreased IgG2 and IgA anti-polysaccharide responses.     

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.     

IgG subclass response to Helicobacter pylori and CagA antigens in children

Specific serum IgG subclass antibodies against Helicobacter pylori antigens and recombinant CagA were analysed in 75 symptomatic children with histologically confirmed H. pylori infection. H. pylori stimulated an IgG1 predominant response, and IgG3 titres showed a positive association with peptic ulcer disease, chronicity of antral inflammation and density of H. pylori colonization. Two methods used for assessing serum IgG CagA antibody status, i.e. Western blotting and enzyme-linked immunosorbent assay (ELISA), were concordant. CagA stimulated an IgG1 and IgG3 predominant humoral response. Total CagA IgG titres were higher in children with active and more severe chronic antral inflammation. These findings suggest that in children the systemic humoral immune response to H. pylori infection may reflect gastroduodenal pathology.     

Clinical characteristics of autoimmune pancreatitis with IgG4 related kidney disease

PurposeTo clarify the clinical characteristics of autoimmune pancreatitis (AIP) in immunoglobulin (Ig)G4-related kidney disease (IgG4-RKD).Patients and methodsA total of 92 patients with AIP were divided into an IgG4-RKD-positive group (RKD-P group, n = 13) and an IgG4-RKD-negative group (RKD-N group, n = 79) on the basis of the diagnostic criteria for IgG4-RKD. Clinical characteristics, including: age; sex; the presence of extrapancreatic lesions other than renal lesions, proteinuria, and hematuria; serum concentrations of IgG, IgG4, IgE, and creatinine; and urinary concentrations of liver-type fatty acid binding protein, α1-microglobulin, β2-microglobulin, and N-acetyl-β-d-glucosaminidase were compared between the RKD-P and RKD-N groups. The clinical course of the RKD-P group was also characterized.ResultsThe prevalence of extrapancreatic lesions other than renal lesions was significantly higher in the RKD-P group (84.6% vs 43.0%,p < 0.01). Serum creatinine (1.19 mg/dl versus 0.74 mg/dl, p < 0.05), urinary β2-microglobulin (6609.8 μg/l vs 265.8 μg/l, p < 0.05), and the prevalence of proteinuria (30.7% vs 7.6%, p < 0.05) were significantly higher in the RKD-P group. Nine out of thirteen patients in the RKD-P group had multiple low-density renal lesions on enhanced computed tomography, 3 patients had multiple high-intensity lesions on diffusion-weighted magnetic resonance images, and 1 patient had diffuse thickening of the renal wall, with a smooth intra-luminal surface.ConclusionsPatients who had AIP with IgG4-RKD were more likely to have extrapancreatic lesions other than those in the kidney, and their serum creatinine and urinary β2-microglobulin concentrations were significantly higher than in those without IgG4-RKD.     

Autoimmune pancreatitis and IgG4-related systemic diseases

Autoimmune pancreatitis (AIP) is a rare form of chronic pancreatitis that is characterized by lymphoplasmacytic infiltrate, storiform fibrosis, obliterative phlebitis, and increased IgG4⁺ plasma cells. Serum IgG4 levels usually are elevated. Patients with AIP frequently have disease affecting other organs or sites; these tissues show similar histologic changes, including increased IgG4⁺ plasma cell infiltrate and response to corticosteroid therapy. A new clinicopathologic concept of IgG4-related systemic disease (ISD) has been proposed. These diseases often are not limited to the pancreas, and the pancreas may not be involved at all. In this article, we review the literature and our own experience to detail the clinicopathologic features of AIP and extrapancreatic lesions in ISD.     

Immunoglobulin class and IgG subclass distribution of anticardiolipin antibodies in patients with systemic lupus erythematosus and associated disorders.

The class and subclass distribution of an antibody response may give insight into the stimulating mechanism and likely effector functions. IgA, IgG and IgM anticardiolipin antibodies (aCL) were quantified in a consecutive series of 200 samples sent to an autoimmune serology laboratory to determine the relationships between aCL responses of each of these antibody classes and, in particular, whether there was any utility in the measurement of IgA aCL. Positive results for one of the three aCL isotypes were found in 105 samples (53%), and in 41 samples IgA aCL was detected (21%). However, amongst these unselected samples, little additional information was obtained by measurement of IgA aCL, which was found in conjunction with IgM or IgG aCL in all but five samples, and in these the isolated elevation of IgA aCL was only slight, and showed no disease specificity. The levels of each of the four IgG subclasses of aCL were measured in a subgroup of serum samples from 28 patients with autoimmune disease and from 29 patients with syphilis. Amongst the SLE patients IgG1 and IgG3 aCL were the predominant IgG subclasses, consistent with an antigen-driven, T cell-dependent antibody response. However, a subgroup of eight of the autoimmune subjects had predominant elevation of IgG2 aCL, possibly implying a role for T cell-independent antibody production to cardiolipin. Amongst the syphilis patients IgG1 and IgG3 aCL were also the predominant subclasses of aCL but IgG4 aCL were also detected in the majority of subjects, consistent with prolonged antigenic stimulation.     

The inductive effect of interleukin-4 on IgG4 and IgE synthesis in human peripheral blood lymphocytes

Using murine monoclonal antibodies against human IgG subclasses, specific and sensitive ELISAs assay to quantify the four human IgG subclasses in cell culture supernatants were established. The effect of human recombinant interleukin-4 (IL-4) on the regulation of IgG subclasses by normal peripheral blood lymphocytes was investigated. In addition to the enhancement of IgE synthesis, IL-4 preferentially induced IgG4 synthesis in vitro, whereas IL-4 had no effect on IgG1, IgG2, and IgG3 synthesis. IL-4-induced IgG4 production was blocked in a dose-dependent manner by recombinant interferon-gamma and anti-human IL-4 monoclonal antibody. Collectively, this data indicates that IL-4 plays an important regulatory role in both IgG subclass and IgE synthesis.     

Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults

The role of specific IgG2 antibody in the protection against serious infection with Streptococcus pneumoniae is unclear. We therefore decided to investigate the relationship between serum antibody levels and opsonization and phagocytosis of this microorganism. We have measured serum IgM, IgA and IgG subclass antibody specific for pneumococcal capsular polysaccharide and in vitro phagocytosis of serotype 14 pneumococcus by polymorphs, in healthy adults before and after immunization with Pneumovax II. IgM and IgG2 were the predominant anti-pneumococcal antibodies seen, IgA and IgGl being present at low titre. No significant relationship of phagocytosis with specific IgM and IgA antibodies was found. However, both specific IgG 1 and IgG2 antibodies in post-immunization sera correlated significantly with phagocytosis of the pneumococcus in the presence of complement (r= 0.57, P= 0.029 and r= 0.59, P= 0.022 respectively). After heat-inactivation, the remaining opsonic activity of sera correlated only with levels of specific IgG2 antibody (r= 0.61, P = 0.0006). Whereas phagocytosis supported by specific IgG 1 and IgG2 antibody to serotype 14 pneumococcus after immunization is mediated by complement activation, IgG2-specific antibody in high titre may also be able to function by complement-independent interaction with Fcγ receptors on polymorphs.     

IgG subclass and vaccination stimulus determine changes in antigen specific antibody glycosylation in mice

Immunoglobulin G (IgG) glycosylation can modulate antibody effector functions. Depending on the precise composition of the sugar moiety attached to individual IgG glycovariants either pro‐ or anti‐inflammatory effector pathways can be initiated via differential binding to type I or type II Fc‐receptors. However, an in depth understanding of how individual IgG subclasses are glycosylated during the steady state and how their glycosylation pattern changes during vaccination is missing. To monitor IgG subclass glycosylation during the steady state and upon vaccination of mice with different T‐cell dependent and independent antigens, tryptic digests of serum, and antigen‐specific IgG preparations were analyzed by reversed phase‐liquid chromatography‐mass spectrometry. We show that there is a remarkable difference with respect to how individual IgG subclasses are glycosylated during the steady state. More importantly, upon T‐cell dependent and independent vaccinations, individual antigen‐specific IgG subclasses reacted differently with respect to changes in individual glycoforms, suggesting that the IgG subclass itself is a major determinant of restricting or allowing alterations in specific IgG glycovariants.     

Immunoglobulin subclass distribution of specific antibodies in allergic patients

The IgG and IgA subclass distribution of specific antibodies against a variety of protein and polysaccharide antigens was determined in sera from individuals with high levels of IgE. No shift of the antibody pattern could be observed, suggesting that the aberrant regulation of responses against allergens noted in these patients is limited, encompassing selected antigens only. Antibodies against protein antigens are mainly of the IgG1 subclass. In addition, low levels of specific IgG3 or IgG4 antibodies may be formed. Our data suggest that a given antigen induces either IgG3 or IgG4 and that potential allergens, in addition to IgG1 and IgE, elicit a response restricted to IgG4.     

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.     

Isotype distribution of mucosal IgG-producing cells in patients with various IgG subclass deficiencies

The subclass distribution of IgG-producing immunocytes was examined by immunohistochemistry in nasal and rectal mucosa of infection-prone patients with untreated IgG subclass deficiencies. Biopsy specimens from the two sites were obtained in 18 clinically and serologically well-characterized adult subjects; only a nasal or rectal sample was available from nine similar patients. Chronic lung disease was common in the patient groups with selective serum IgG1 deficiency and combined IgG1 and IgG3 deficiency, whereas the other categories of patients had mainly upper airway and other mild infections. Serum IgG2 or IgG3 deficiency was usually expressed also at the cellular level in rectal mucosa, and the proportion of rectal IgG1 cells was significantly correlated with the IgG1 level (r = 0.90, P less than 0.001). Likewise, there tended to be a decreased expression of the actual subclass at the cellular level in nasal mucosa of patients with serum IgG1 or IgG2 deficiency. Conversely, the median nasal proportion of IgG3 cells was remarkably unaffected by a deficiency of this subclass in serum and rectal mucosa. Interestingly, these patients rather tended to have raised IgG3 and reduced IgG2 cell proportions in their nasal mucosa, although this apparent local IgG3 compensation was nevertheless strongly correlated with the serum IgG3 level (r = 0.87, P less than 0.002). These disparities may reflect different antigenic and mitogenic exposure of the two tissue sites; for example, a persistent protein bombardment of the nasal mucosa that could conceivably override locally a B cell maturation defect. The possible clinical consequences of such variable mucosal expression of IgG subclass deficiencies remain to be studied.     

Anti-myeloperoxidase IgG subclass distribution and avidity in sera from patients with propylthiouracil-induced antineutrophil cytoplasmic antibodies associated vasculitis

OBJECTIVE: Propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The aim of this study was to compare the IgG subclass distribution and avidity of MPO-ANCA in sera from patients with primary ANCA-associated vasculitis (AASV) and PTU-induced vasculitis. METHODS: Nineteen patients with primary AASV with MPO-ANCA and thirteen patients with PTU-induced vasculitis were enrolled in the current study. Sera in both active phase and remission were collected. Anti-MPO IgG subclasses were detected by antigen specific ELISAs using specific monoclonal antibodies as second antibodies, and MPO-ANCA avidity was assessed by antigen-inhibition ELISAs. RESULTS: In primary AASV, all four anti-MPO IgG subclasses could be detected in active phase with IgG1 (100%), IgG2 (73.7%), IgG3 (63.2%) and IgG4 (94.7%), and in remission, IgG1 and IgG4 subclasses in most patients remained positive. However, in PTU-induced vasculitis, anti-MPO IgG3 subclass could not be detected, the anti-MPO IgG subclasses in active phase were IgG1 (100%), IgG2 (61.5%) and IgG4 (46.2%). Furthermore, five out of the six patients (88.8%) with PTU-induced vasculitis with positive IgG4 subclass in active phase turned to negative in remission, however, only eight out of the fourteen patients (57.1%) with primary AASV turned to negative. The median avidity constant of MPO-ANCA was 56 (8.96 to >140) x 10(7) mol/l for patients with primary AASV and 0.7 (<0.28 to >140) x 10(7) mol/l for patients with PTU-induced vasculitis respectively. Furthermore, the relative levels of MPO-ANCA avidity were associated with elevation of ESR in primary AASV and were associated with BVAS scores in patients with PTU-induced vasculitis, respectively. CONCLUSION: MPO-ANCA IgG subclass distribution and avidity were different between patients with primary AASV and PTU-induced vasculitis. It was suggested that the mechanism of ANCA production in PTU-induced vasculitis was different from that in primary AASV, and the avidity of MPO-ANCA might be associated with disease activity.     

Ig class and IgG subclass responses toTreponema pallidum in patients with syphilis

The Ig class and IgG subclasses of anti-Treponema pallidum antibodies in human serum were quantified using solid-phase enzyme-linked immunosorbent assays. Development of these assays with monoclonal antibodies, each specific for a human immunoglobulin class or IgG subclass, provided quantitative data concerning the major antibody specificities. In patients with primary syphilis, anti-T. pallidum activity was limited almost exclusively to IgG1 and IgM. Coordinate, restricted expression of IgG1 and IgG3 responses inT. pallidum-specific assays was observed with sera from patients with active secondary syphilis. IgG1 and IgG3 accounted for roughly 53 and 43% of the total anti-treponemal IgG antibody activity, respectively. While IgM antibody levels were elevated in the patients with secondary syphilis, IgG2 and IgG4 levels, if present at all, represented less than 10 and 2% of the total IgG activity, respectively. Ig in sera from patients who had been treated adequately for secondary syphilis were restricted almost entirely to IgG3 and IgG1. Considering the low level of IgG3 in serum, disproportionately high percentages of antitreponemal antibodies were found in this subclass during and after treatment for secondary syphilis. The restricted, coexpression of the IgG1 and IgG3 isotypes may reflect the close genetic linkage of the 1 and 3 genes and possibly the impact of immunoregulatory mechanisms in response to the induction and expression of autoantibodies which arise during the course of secondary syphilis.     

Linkage of IgA deficiency to Gm allotypes; the influence of Gm allotypes on IgA-IgG subclass deficiency

IgA deficiency (IgAD) is the most common immunodeficiency, characterized by an arrest in B cell differentiation. It has a sporadic occurrence or variable inheritance pattern, and is also linked to the HLA genes. IgA deficiency is sometimes associated with IgG subclass deficiency. In this study the Gm allotypes, as genetic characteristics of the IgG1, IgG2 and IgG3, were analysed in 83 Caucasian IgAD individuals. Half of the patients presented with IgG4 < 0·01 g/l compared with 5% (P < 0·001) in a healthy population. Three of the 83 had significantly low IgG2 and four had significantly low IgG3 levels. Gm allotype frequencies in IgAD deviated compared with a normal population. Of the 83 patients, 44 (53%) showed homozygous G2m(“,”) expression on the IgG2 locus (33% in controls, P < 0·01). In IgAD the Gm(a, g) haplotype was more frequent (43%) compared with controls (31%, P < 0·01). The Gm homozygous phenotype Gm(a', g/a', g) was most common, found in 20 of 83 patients (24%, P < 0·05) compared with controls (14%). On the other hand the Gm(f,n,b) haplotype of IgAD was rare (28%) compared with controls (45%, P < 0·001). The low IgG4, < 0·01 g/l, found in 50% of the patients, was even more frequent (56–69%) among the G2m(“,”) phenotypes. IgG subclass levels were given for different Gm phenotypes of the IgAD group and compared with controls. Significantly low IgG4 was revealed in the Gm(a,“,g/a,”,g) phenotype (P < 0·01) and significantly low IgG2 in the Gm(a,“,g/f,”,b) phenotype (P < 0·01). The Gm(a,“,g/f,”,b) phenotype contained the three patients found with IgG2 levels < - 2 s.d., and the four patients with IgG3 levels < -2 s.d. were present among those with the homozygous Gm(a,“,g/a,”,g) phenotype; both phenotypes with G2m(“,”) on the IgG2 locus. The ‘compensatory’ increase of IgG was significant for both IgG1 and IgG3 in all Gm phenotypes, but in the Gm(a,“,g/f,”,b). Thus, the susceptibility of IgAD with the additional IgG antibody deficiencies, down-regulated IgG4 and IgG2/IgG3, is associated with Gm allotypes, especially the homozygous G2m(“,”) expression on the IgG2 locus.     

Utilizing self-prepared ELISA plates for a cross-population study of different anti-HBe IgG subclass profiles

Fourteen serum samples obtained from hepatitis B virus (HBV) chronic carriers and patients recovered from hepatitis B infection were used with four sodium dodecyl sulfate-treated enzyme-linked immunosorbent assay (ELISA) plates available commercially, and one self-prepared HBcAg analog for evaluation of anti-HBe subclass pattern absorbance. The self-prepared plates had the best performance and were thus used for samples obtained from 104 (60 male and 44 female) HBV chronic carriers and 439 (247 male and 192 female) recovered individuals. Tests for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also carried out in 21 of the subjects (>25 IU/ml). Statistical comparison of these patients with elevated ALT/AST levels with other ALT/AST-normal chronic carriers revealed no significant differences in the anti-HBe OD, although the mean optical density (OD) of patients with elevated ALT/AST levels was higher. The results suggest that the anti-HBe IgG subclass profiles in the chronic carriers did not change with inflammation of the liver, and were independent of sex and age. In contrast to previous anti-HBc findings, the distribution pattern of anti-HBe subclasses in HBV chronic carriers was IgG1 > IgG4 > IgG3 while in the recovered individuals it was IgG1 > IgG3 > IgG4, for both males and females. Subclasses IgG1 and IgG2 were the most and least prevalent isotypes, respectively, in both study groups. The results of the study suggest that induction of IgG1 and/or IgG3 antibodies is important for effective virus neutralization, while IgG2 antibodies are of limited importance. Significantly higher OD values for anti-HBe IgG4 were observed when comparing samples from the chronic carriers and recovered individuals, which may reflect the effects of persistence. Further, in contrast to previous anti-HBs results, the concentrations of total IgG and IgG1 were higher in the samples from chronic carriers relative to those from recovered individuals.     

16例伴生长激素缺乏的多种垂体激素缺乏患者的临床特点及生长激素治疗效果分析

目的探讨伴生长激素缺乏(GHD)的多种垂体激素缺乏症(MPHD)患者的临床特点以及生长激素(GH)治疗效果。方法回顾性分析16例伴有生长激素缺乏的多种垂体激素缺乏患者的临床资料。结果本研究纳入了16例MPHD的患者,其中伴甲状腺功能减退症9例、伴低促性腺激素性性腺功能低减13例和伴肾上腺皮质功能减退6例。臀位、足先露和难产等不良生产史患者10例。骨龄(11.0±3.5)岁,明显落后实际年龄。L-Dopa-GH激发试验GH峰值为(0.14±0.17)ng/m L,GH治疗平均剂量(0.11±0.02)IU/kg。治疗后IGF-1水平及生长速度均明显增加。结论排除下丘脑、垂体占位等病变后,伴GH缺乏的MPHD患者在纠正其他轴系激素缺乏后,使用GH治疗可明显改善身高,并且无严重不良事件发生。     

IgG2 subclass restriction of anti-β2 glycoprotein 1 antibodies in autoimmune patients

The IgG subclass and light chain distribution of antiphospholipid antibodies (aPL) occurring in autoimmune patients were determined by means of two radioimmunoassays using either cardiolipinor β₂ glycoprotein 1 (β₂GP1)-coated microtitre plates and mouse MoAbs. Of 50 sera selected for positivity of anticardiolipin antibodies (ACA) of the IgG isotype, 32 (64%) possessed anti-β₂GPl antibodies and their presence was closely associated with clinical features of the antiphospholipid syndrome. Good correlations were found between ACA and anti-β₂GP1 antibodies when considering antibody level and patterns of light chain and IgG subclass, suggesting that, overall, the same antibodies were being measured. Light chain analysis showed the polyclonal origin of these antibodies and, in most sera, a trend towards use of λ chain. Among sera positive for anti-β₂GP1 antibodies, IgG2 was the major subclass reactive with β₂GP1 and cardiolipin (87% and 74β₂ of the IgG antibody activity, respectively). In contrast, in the group of 18 sera lacking anti-β₂GP1 antibodies, ACA were largely restricted to lgG3, with a lesser contribution by IgGl. A few selected sera from the anti-β₂GP1-positive group were shown to contain mixtures of antibodies that required β₂GP1 (restricted to IgG2 present in large amounts) and did not require this cofactor (restricted to IgG3 and/or IgG1 present in low amounts) for their reactivity with cardiolipin. There was no contribution of glycosylation to the epitopes recognized by anti-β₂GP1 antibodies, even though human anti-carbohydrate antibodies are restricted to the IgG2 subclass. These findings further emphasize the intra- and interindividual heterogeneity of aPL, and should help to discriminate clinically relevant specificies.