Immunoglobulin G subclass responses to cytomegalovirus in seropositive patients after transfusion

The immunoglobulin G subclass responses to cytomegalovirus (CMV) after red cell (RBC) transfusion were studied in 26 seropositive surgery patients and 34 transfused seropositive oncology patients. Also included as controls were 18 surgical patients who received no RBCs during surgery. None of the 78 patients studied had IgG2 to CMV before or after transfusion. The absence of a total IgG response to CMV after transfusion could not be attributed to preexisting deficiencies in one or more subclasses, because all 78 patients had similar levels of IgG1, IgG3, and IgG4 to CMV before transfusion. Discriminant analysis was used for statistical evaluation of the combined CMV subclass responses in each patient and the individual subclass responses. Individual patients responded to CMV antigens with an increase in concentration in any of the three subclasses or any combination of the subclasses, excluding IgG2. IgG subclass analysis showed that 10 of 27 patients who did not respond with at least a fourfold total IgG titer rise had a significant increase in IgG subclass antibodies to CMV. Three of 33 patients with at least a fourfold total IgG titer rise lacked a subclass response. These results suggest that the measurement of IgG subclasses may be a sensitive indicator of immune response to CMV.     

Direct quantitation of IgG subclasses 1, 2, and 3 bound to red cells by Rh1 (D) antibodies

The authors developed quantitative radioimmunoassays to allow direct measurement of total human IgG and individual IgG subclasses among antibodies bound to cell surfaces. The assays use four mouse monoclonal radioiodinated antibodies, one that reacts equally well with all four human IgG subclasses and three that are specific for human IgG subclasses 1, 2, or 3. The assays were used to analyze IgG subclass composition in 21 high-titer anti-D samples from Rh-negative volunteers immunized for Rh immunoglobulin production. Anti-D activity was restricted primarily to the IgG1 and IgG3 subclasses. Eleven of 21 sera demonstrated red cell antibodies with a marked predominance of IgG1 (87 +/- 3.6% of total IgG antibody, +/- SEM) and low levels of IgG3 (1.4 +/- 0.73%). In the remaining 10 sera, IgG3 made up a greater proportion of total IgG antibody (32 +/- 3.8%), although IgG1 was still predominant (61 +/- 4.1%). This observed dichotomy in the IgG subclass profiles of different anti-D sera may be a consideration in the selection of anti-D sera for the production of the immunoglobulin used in the prophylaxis of Rh-incompatible pregnancies.     

Coexistence of (partial) immune defects and risk of recurrent respiratory infections

BACKGROUND: Respiratory infections are major causes of morbidity and mortality, but determinants of susceptibility are poorly defined. We studied whether and to what extent immunologic and genetic factors are associated with increased susceptibility to respiratory infections. METHODS: We evaluated the prevalence of IgA, IgM, IgG, and IgG subclass deficiencies, impairment in the antibody response against pneumococcal polysaccharides, G2m(n) allotypes, Fc gamma RIIa polymorphisms, partial C2 and partial C4 deficiency, promoter polymorphisms in MBL2, and lymphocyte subset deficiencies in a control population and in consecutive children with recurrent respiratory infections. RESULTS: IgA and/or IgG subclass deficiency was found in 27 of 55 patients (49%) and 6 of 43 controls (14%) (P = 0.0006). An impaired antibody response to polysaccharides was found in 7 patients (19%) and in 0 of 37 controls (P = 0.002). The Gm(n)marker was absent in 25 of 55 patients (45%) and 6 of 42 controls (14%) (P = 0.009). The MBL2 variants O/O, A/O, and A/A occurred in 9, 14, and 32 of the 55 patients, respectively, and in 1, 19, and 23 of the 43 controls, respectively (P = 0.05). There was no increase in the prevalence of partial C4 deficiency, C2 deficiency, lymphocyte subset deficiency, or Fc gamma RIIa polymorphism in the patients compared to the controls. A combination of at least 2 immune defects was found in 31 of 55 patients (56%) and in 4 of 42 controls (11.6%) (P <0.0001). CONCLUSION: Specific antipolysaccharide antibody deficiency, IgA and/or IgG subclass deficiency, Gm(n) allotype, and MBL2 genotype are susceptibility factors for recurrent respiratory infections, and coexistence of several immune defects is the strongest risk factor in this study.     

Humoral and cellular immune dysfunction in a patient with Bloom's syndrome and recurrent infections

Immunological evaluation of a patient with Bloom's syndrome (BS) who suffered from recurrent bacterial and fungal infections, revealed low serum levels of IgG and high levels of IgM accompanied by an elevated proportion of surface membrane IgM positive B-lymphocytes and a decreased proportion of IgG positive B-cells. In vitro IgG secretion was also reduced whereas IgM production was normal. Although proportions of T-cell subsets were normal and proliferative responses to T-cell mitogens were adequate, a defective regulatory T-cell function for the generation of IgG was observed. Natural killer (NK) cell activity against K562 tumor cells was also decreased in this patient. The findings in this patient may suggest a maturation arrest of lymphocytes at an early developmental stage, and this may explain in part the increased susceptibility to infections.     

Human immunoglobulin G and immunoglobulin G subclasses: biochemical, genetic, and clinical aspects

Human IgG consists of two identical heavy (H) chains and two identical light (L) chains joined by interchain disulfide bridges. Heterogeneity in the amino acid sequences of the H and L polypeptides results in at least three types of IgG variants at the structural and genetic levels. The four isotypic forms are IgG1, IgG2, IgG3, and IgG4, which share extensive homologies in the primary structure of their H chains. As a result, the subclasses cross-react antigenically, but they can be differentiated on the basis of subtle architectural dissimilarities. The biological and effector properties of the IgG isotypes have been associated, in part, with their structural differences. Genes determining the synthesis of human IgG heavy chains are located on chromosome 14. In several clinical situations the isotypes appear to be regulated or expressed in patterns reflecting the gene arrangement. The numeric designations of the subclasses correspond to the order of their proportional amounts in healthy adult serum: IgG1 greater than IgG2 greater than IgG3 greater than IgG4. Awareness of the importance of the roles of the four IgG isotypes in human health has steadily increased since they were first described in the 1960s. The recognition that deficits or increases in selected IgG subclasses may have clinical consequences has prompted considerable interest in quantifying the four isotypes in clinical specimens. In particular, deficiencies of IgG2, IgG3, and IgG4, singly or combined, are associated with chronic infections which may not be readily recognized in otherwise healthy people with normal serum total IgG concentrations. Different assay methods using polyclonal or monoclonal antisera with various calibrants have been applied; however, no standardized method exists at the present. IgG deficits are associated with gene defects and are acquired in secondary immunodeficiencies in conjunction with other disorders. IgG isotype selectivity has been recognized in autoimmune diseases and in response to carbohydrate and protein antigens derived from pathogenic microorganisms and common allergens.     

Significance of IgG-subclass antibody determinations in bullous pemphigoid

The presence of circulating basement membrane zone (BMZ) antibodies is characteristic of patients with bullous pemphigoid (BP) and are routinely employed in making the diagnosis. The positive tests, however, occur in 50–70% of patients with BP, thus necessitating consideration of other tests in a significant number of patients. The purpose of this study was to examine the efficacy of indirect immunofluorescence (IF) tests specific for various IgG subclasses antibodies to BMZ in BP, especially in patients who are seronegative on routine indirect IF tests with fluorescein-conjugated antibodies to IgG. BMZ antibodies primarily are of IgG4 subclass and are present in all BMZ antibody positive BP cases. Of BP patients negative for BMZ antibodies, 72% were found positive when tested for IgG4 subclass antibodies. In conclusion, testing for IgG4 subclass BMZ antibodies enhances the sensitivity of serum tests from 68.5% to 91%. This may be due in part to the inherent increased sensitivity of the assay and for detecting subclasses of IgG and in part, due to the subclass distribution of the BP antibodies. © 1996 Wiley-Liss, Inc.     

Distribution of IgG subclasses after anti-hepatitis B virus immunization with a recombinant vaccine

To assess whether a different IgG subclass distribution was elicited in “low” and “high responders” after vaccination with recombinant hepatitis B virus surface antigen, we selected from 360 vaccine recipients 30 “lowresponder” subjects, with anti-HBs levels of 10–160 mIU/ml, and 40 “high-responder” subjects, with anti-HBs levels greater than 10,000 mIU/ml. In both groups all IgG subclasses were elicited in the anti-HBs response and the greatest contribution was that of IgG 1, followed by IgG2. IgG l was significantly less represented after the second (58%) and third doses (61%) of vaccine in “low responders” compared with “high responders” (65% and 69%). The relative percentage of IgG2 was significantly higher after the second (33%) and third (30%) doses of vaccine in “low responders” than in “high responders” (29% and 26%). In “low responders” the age of vaccine recipients significantly influenced the anti-HBs IgG subclass distribution: IgG2 and IgG4 production was positively correlated with age, whereas the opposite was observed for IgGl. These data support the evidence that: (1) IgGl and IgG2 subclasses are mainly involved in the specific anti-HBs response both in “high” and “low responders”; (2) the relative contribution of specific IgG2 to vaccination is higher in low responders and progressively increases with age.     

IgG subclass distribution and relative functional affinity of thyroid microsomal antibodies in postpartum thyroiditis

An association between the development of postpartum hypothyroidism and high levels of IgG1 subclass microsomal (M) antibodies has been reported. Using an assay designed to detect reasonable levels of all the four IgG subclasses, we found no differences in the proportion of each IgG subclass in M antibodies of patients with postpartum hyperthyroidism or hypothyroidism compared with control postpartum patients with M antibodies but no thyroid dysfunction. However the total amount of M antibody of each IgG subclass was elevated above the controls in the patients with thyroid dysfunction. The relative functional affinity of M antibodies did not differ between controls and patients with hypothyroidism but declined 5 and 10-12 months after delivery compared to values at 2 months. These results do not support the suggestion that the amount of IgG1 subclass M antibodies particularly determines the course of postpartum thyroiditis. Rather, the total M antibody level, in all four subclasses, is associated with clinical outcome. Resolution of the disease, despite persisting M antibodies, may occur in part because the relative functional affinity of these antibodies declines after delivery.     

An enzyme-linked immunosorbent assay for the quantitation of IgG anti-D and IgG subclasses in the sera of alloimmunized patients

BACKGROUND: IgG subclass composition of maternal alloantibodies to the D antigen seems to play a role in the severity of hemolytic disease of the newborn.The subclassing of IgG anti-D is usually performed by hemagglutination techniques, but the results are not quantitative and sometimes are difficult to interpret. Thus, there is a need for quantitative methods. STUDY DESIGN AND METHODS: The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for the quantitation of specific IgG anti-D and IgG subclasses in the sera of alloimmunized patients. Group O R1R2 red cells were sensitized with anti-D. Red cell membranes were solubilized with nonionic detergent. IgG and IgG subclasses were measured by a sensitive and reproducible immunocapture ELISA. A serum calibrated for its IgG subclass content was used as a reference, and the anti-D preparation 68/419 was used as an internal control. Optimal conditions for the detection of IgG anti-D and IgG subclasses by ELISA were studied. The absolute concentration and the proportions of IgG subclasses were determined in the sera of 14 pregnant women. RESULTS: A close parallelism was observed between dilutions of the IgG reference serum and the IgG anti-D solubilized from sensitized RBCs. The sum of IgG anti-D subclass concentrations, determined by the ELISA, correlated well with other quantitative methods. CONCLUSION: The method described is sensitive and can be used routinely for the quantitative determination of specific IgG anti-D and IgG subclasses in sera.     

Analysis of the IgG subclass profile and IgG sum-total discrepancy in COVID-19 convalescent plasma donors: A single-centre prospective cohort study

IntroductionAlthough IgG1 and IgG3 have been shown to be the dominant subclasses in the acute phase of SARS-CoV-2 infection, little is known about the distribution of IgG subclasses during the recovery phase of COVID-19. The aim of the study was to analyze the profile of IgG subclasses in COVID-19 convalescent plasma donors.MethodsA total of 36 convalescent plasma donors were included in the analysis. IgG and IgG subclass levels were measured using a nephelometric assay in plasma samples obtained directly from the plasma container.ResultsAlthough there was no significant difference in the concentration of IgG subclasses between the study and control groups, the contribution of IgG1 to the total IgG pool between the study and control groups was statistically significant (p = 0.0478). In addition, there was a discrepancy between the total IgG and IgG sum values in the study group, exceeding 15 % in 19,4 % of samples (n = 7), while in the control group no samples with a sum/ total IgG difference > 15 % were observed.ConclusionsThe selective affinity of the IgG1 subclass for the polyclonal anti-IgG reagent may interfere with the determination of total IgG and should be considered when interpreting the results of enzyme immunoassaysData AvailabilityThe data that support the findings of this study are available on request from the corresponding author.     

Serum IgG subclass concentrations before and after administration of intravenous immunoglobulin in common variable immunodeficiency.

IgG subclass levels were measured before and after administration of intravenous immunoglobulins (IVIGs) in patients with common variable immunodeficiency (CVI). Six patients were treated with IVIG at the dose of 100-150mg/kg to maintain a trough level of 200mg/dl every 4 or 5 weeks, except for patient 5 who was given IVIG only 3 or 4 times per year. Three kinds of IVIGs, polyethylene-glycol (PEG)-treated IVIG, alkylated IVIG and sulfonated IVIG were used for replacement therapy. Although serum IgGl levels were low before administration of IVIGs, they increased to the normal levels after each administration of IVIGs in four patients. IgG2 preserved normal levels before and after administration of IVIGs in all patients. IgG3 was present at low normal concentration in patient 1, low concentration in patients 2, 3 and 6, and undetectable in patients 4 and 5 before infusion. Although increases in IgG3 levels were shown after infusion of PEG-treated IVIG, there were no increases after infusion of sulfonated or alkylated IVIG. However, there have been several reports that IgG3 is detected in sulfonated or alkylated IVIG preparations by another method. IgG4 levels were somewhat low before administration, but four patients achieved normal serum levels with treatment. In light of the above results of replacement therapy with IVIGs, we should consider the IgG subclass levels for patients such as CVI or selective IgG subclass deficiency.     

Red cell autoantibodies, multiple immunoglobulin classes, and autoimmune hemolysis

The effects and interrelationships of multiple immunoglobulin coating (i.e., increased red cell [RBC]-bound IgM and/or IgA in addition to IgG) were investigated in 404 patients with warm-reactive RBC autoantibodies on 590 occasions. Multiple immunoglobulins were detected by enzyme-linked direct antiglobulin tests in 218 samples (37%), but in only 87 (15%) by agglutination methods. Differences in populations were examined by chi-square, with p less than 0.005 being required for significance because of the multiple tests. Compared with IgG coating alone, multiple immunoglobulins were significantly associated with larger quantities (greater than 800 molecules/RBC) of IgG, multiple IgG subclasses, IgG3 and C3d bound to the cells, and with serum haptoglobin levels of less than 0.1 g per L. The latter association was still significant when higher levels of RBC-bound IgG and subclass pattern were taken into account. In samples with multiple immunoglobulin coating, there was no significant relationship (p greater than 0.05) between haptoglobins of less than 0.1 g per L and either C3d or multiple IgG subclasses. It was concluded that multiple immunoglobulin coating, even when undetected by agglutination methods, is a major cause of hemolysis: it is part of a more generalized autoimmune response and acts with other factors such as the quantity of bound IgG, the IgG subclass pattern, and complement; it also has an important hemolytic effect in its own right.     

Quantitation of IgG subclass antibodies to pneumococcal capsular polysaccharides by ELISA, using Pneumovax-specific antibodies as a reference

A quantitative enzyme-linked immunosorbent assay (ELISA) method has been developed to assay the levels of IgG subclasses to pneumococcal capsular polysaccharides (PCP) by using a reference standard. This standard solution containing specific antibodies to a polyvalent pneumococcal vaccine (Pneumovax) was purified from the serum of an immunized healthy adult by affinity chromatography. In order to determine the predominant response to Pneumovax in the four IgG subclasses, specific IgG subclasses in preimmune and postimmune sera from six healthy adults were assessed quantitatively by the ELISA. With regard to peak concentrations after immunization, there was a marked increase in the IgG2 subclass, compared with those of IgG1 and IgG3. Such a quantitative assay of Pneumovax-specific IgG subclass antibodies is useful for the direct evaluation of immune responses to immunization with a polyvalent pneumococcal vaccine, and at the same time, for estimating the IgG2 response to PCP antigens in individuals.     

Human IgG subclass measurements in the clinical laboratory

Complement activation, cell surface-receptor binding, blocking activity, and possibly placental transfer are among the biologically important functional differences that have been detected between the four human IgG subclasses by use of polyclonal antisera. In 1985, a IUIS/WHO panel of immunologists, using eight immunological methods, documented the specificity of select monoclonal antibodies for the IgG subclasses. Clinical assays have been developed involving these monoclonal antibodies that allow quantification of the concentration of IgG subclass protein and distribution of the IgG subclass antibodies in human immune responses. This review addresses issues of concern to investigators who are evaluating and (or) developing quantitative human IgG subclass assays in the clinical laboratory. Unique physical (structural) and biological (functional) properties of human IgG subclasses are summarized, with a focus on aspects pertinent to their clinical importance and in vitro quantification. The HP-series monoclonal antibodies with documented specificity are examined within the context of their application to several immunological methods. I describe unique technical aspects of total and antigen-specific IgG-subclass immunoassays involving these monoclonal antibodies. Finally, this report outlines clinical applications and indications for IgG-subclass measurements in the study of human health and disease.     

IgG heavy-chain subclass restriction of thyrotropin-binding inhibitory immunoglobulins in Graves'' disease

The IgG subclass composition of antibodies is an important determinant of their function. Thyrotropin receptor antibodies cause the hyperthyroidism of Graves' disease but their subclass distribution has been incompletely investigated. We have therefore purified IgG subclasses from Graves' sera by passage over affinity columns designed to deplete all but a single subclass, and then assayed those pure subclass fractions for their ability to displace radiolabelled thyrotropin from its solubilized receptor as a measure of thyrotropin receptor antibody activity. Sufficient activity was recovered for analysis in nine of 10 Graves' patients, in five of whom activity was almost completely (97-100%) restricted to the IgG1 subclass; in the remaining four patients the response was predominantly IgG1 and IgG4 with marked under-representation of the IgG2 subclass. This contrasts with the unrestricted subclass response, in the same fractions, for autoantibodies against thyroglobulin and microsomes. These results suggest that there may be a primary defect at the B-cell level in Graves' disease.     

Thyroid-stimulating antibody activity between different immunoglobulin G subclasses

To investigate the distribution of thyroid-stimulating antibody (TSAb) activity between IgG subclasses, sera from 11 patients with Graves disease (including the National Institute of Biological Standards and Control (NIBSC) Research Standard, long acting thyroid stimulator-B) were fractionated by chromatography on affinity columns of monoclonal IgG subclass antibodies or protein A to deplete all but a single subclass. The resulting fractions were 98% or more pure for a single subclass. In all 11 patients, TSAb activity appeared to be confined to the IgG1 fraction as determined by cAMP production on addition of the fractions to the FRTL-5 rat thyroid cell line. In all of eight specimens from seven patients so tested, the whole serum activity was recovered in the IgG1 fraction, after adjusting for the recovery of the isotype from the column. TSAb activity in one serum comprised both lambda and kappa light chains but was IgG1 restricted. This IgG subclass restriction was not found when the same fractions were tested for thyroglobulin, microsomal/thyroid peroxidase, or tetanus toxoid antibody activity. Together with previous results showing marked restriction of both light chain usage and isoelectric point of TSAb, these results support the idea that Graves' disease may be the result of an oligo- or possibly monoclonal response at the B cell level.     

IgG subclass distribution of anti-ADAMTS13 antibodies in patients with acquired thrombotic thrombocytopenic purpura

Summary.  Background : ADAMTS13-neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). Objective : To analyze the IgG subclass distribution of anti-ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. Methodology : An enzyme-linked immunosorbent assay-based method was used to quantify the relative amounts of IgG subclasses of anti-ADAMTS13 antibodies in acquired TTP plasma. Results : IgG₄ (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG₁ (52%), IgG₂ (50%), and IgG₃ (33%). IgG₄ was found either alone (17/52) or with other IgG subclasses (35/52). IgG₄ was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG₄ and IgG₁ antibodies ( P  < 0.01). Patients with high IgG₄ levels and undetectable IgG₁ are more prone to relapse than patients with low IgG₄ levels and detectable IgG₁. Conclusions : All IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG₄, followed by IgG₁, antibodies dominating the anti-ADAMTS13 immune response. Levels of IgG₄ could be useful for the identification of patients at risk of disease recurrence.     

IgG2 subclass deficiency: IgG subclass assays and IgG2 concentrations among 8015 blood donors

Among the four IgG subclasses in humans, IgG2 is preferentially expressed in antibodies to carbohydrate antigens whereas IgG1 subclass is commonly associated with antibodies to protein antigens. Because of this association with carbohydrate antigens, values for IgG2 in serum are often used as an index of immunocompetence against carbohydrate antigens. To investigate the value of IgG2 measurements in a general population, we have developed a convenient IgG subclass assay, using monoclonal antibodies and particle concentration fluorescence immunoassay. Our assay is specific, precise, convenient, and accurate. When IgG2 concentrations were determined in the serum samples from 8015 adult blood donors, there were more individuals with low IgG2 concentrations than predicted by the log-normal distribution. The observed distribution suggested the presence of a subpopulation with low IgG2 concentration. Because apparently healthy individuals in a general population have low IgG2 concentrations, IgG2 measurements alone may have a limited clinical usefulness as an index of immune function against carbohydrate antigens.     

Widespread Unilateral Pain Associated With Herpes Simplex Virus Infections

We describe a patient group with unexplained widespread pain on one side of the body and pain exacerbations during active labial or genital herpes and during herpetic central nervous system infections. The patients had no visible lesion of the central nervous system on magnetic resonance imaging or abnormality in electrophysiological studies. To understand the nature of the pain and its possible relation to herpes simplex virus (HSV) infections, a clinical neurological examination was performed and quantitative sensory testing and skin biopsies were assessed in 17 patients. The levels of serum total immunoglobulins and IgG subclasses and the frequencies of the immune response genes at the IGH@, HLA-A, -B, -DRB1, C4A, and C4B loci were analyzed in the patients and in control subjects. The patients manifested a uniform clinical syndrome with unilateral pain that was best described as neuropathic and that was exacerbated by HSV reactivations. Low plasma IgG3 concentrations, the presence of either low plasma IgG1 or IgG3 or both, and high anti-HSV-2–IgG titers were more common in the patients than in the control subjects, which rendered the patients more vulnerable to HSV recurrences.PerspectiveWe suggest that low immunoglobulin subclass levels and certain MHC alleles render the patients susceptible to recurring HSV infections. HSV reactivations and the accompanying inflammatory process cause dysfunction of the central nervous system that manifests as neuropathic pain. Studies using functional brain imaging are needed to clarify this syndrome.     

The quantification of human IgG subclasses in reference preparations

Three laboratories took part in an investigation about the possibilities of quantitative determination of IgG subclasses. Polyclonal monospecific antisera were used in combination with different kinds of calibrators to assess the IgG subclass levels in three W.H.O. reference preparations. IgG 1 could be determined with satisfactory precision; for the other subclasses the quantification was less precise. The precision was dependent on the antisera for IgG 1, IgG 2 and IgG 4 but mainly on the calibrators for IgG 3. Mass units are to be preferred to relative units. W.H.O. reference preparation 67/97 is proposed as a reference for determination of IgG subclasses in mass units. The target values are 5.0 g/l for IgG 1, 2.6 g/l for IgG 2, 0.4 g/l for IgG 3 and 0.5 g/l for IgG 4. These values add up to a total IgG value close to earlier W.H.O. estimates.