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.     

Comparative biological activities of acellular pertussis vaccines produced by Kitasato.

The quality of 14 lots of acellular pertussis-diphtheria-tetanus (AC-PDT) vaccines manufactured by the Kitasato Institute during the period 1987-1990 were investigated. The geometric means of HSU, LPU, and BWDU were 0.078, 0.257, and 7.33 per ml respectively. The potency was higher than 14 IU per ml. These results indicated the consistency of the Kitasato AC-PDT vaccines. The antibody response to the AC-PDT vaccines was measured in primary and secondary vaccinated mice by ELISA. IgG antibody response to FHA and PT was obtained in all immunized mice (P less than 0.001) after the primary injection. In contrast, IgG antibody response to fimbriae 2 showed a significant titer rise (P less than 0.001) after the booster injection. The results indicated that the Kitasato AC-P vaccines consisted of protein, PT and FHA as the major antigens, and a little agglutinogen as the minor antigen.     

Sero epidemiology of Bordetella pertussis immune responses in a healthy population in northern Greece

A seroepidemiological study was conducted on a representative sample of the northern Greek population (healthy individuals, age range=1 day to 80 years) to assess the prevalence of antibodies to pertussis toxin (PT) and filamentous hemagglutinin (FHA). Antibody concentrations were significantly elevated with age (analysis of variance (ANOVA), P<0.001). In addition, a significant increase in antibody levels was detected in subjects >50 years old compared to children aged 5-10 years (post-hoc Scheffe analysis, P=0.007). These data suggest that pertussis occurs frequently in Greek adults, and that sometimes a fifth booster vaccine dose is not given after the second year of life. Routine revaccination with the acellular vaccine for children >4 years of age, adolescents, and adults should be considered in order to ensure effective protection of the whole population.     

Vaccine acquired pertussis immunity was weakened at 4 years of age and asymptomatic pertussis infection was suspected based on serological surveillance

Serological surveillance of pertussis antibodies was performed in 118 children aged 1–12 years. The positivity of pertussis toxin (PT) antibodies was low at 4–6 years and significantly higher at 8–9 years, compared with those at 6 years. Fimbriae 2 (Fim2) antibody showed similar response to the PT antibody. Higher antibody titers against Fim3 were observed among subjects ≥5 years and highest at 8 years. Data demonstrated that the vaccine-induced antibodies decayed by 4–5 years and subclinical pertussis infection was suspected thereafter, suggesting the need for additional dose at around 4–5 years.     

Specific humoral response to cows’ milk proteins and ovalbumin in children with atopic dermatitis

Summary Serum antibodies to four common food antigens, three cows’ milk proteins (casein, α-lactalbumin and β-lactoglobulin) and ovalbumin, were investigated in 21 children with atopic dermatitis (aged 3 months to 3 years) and in 15 age-matched healthy controls. Specific IgE was measured by radioallergosorbent test; an ELISA was developed to detect specific IgG, IgG subclasses and IgA. Specific IgE was found in 76% of patients, while antigen-directed IgG and IgA were present both in patients and healthy controls; IgG to ovalbumin and IgA to α-lactalbumin were significantly higher in children with atopic dermatitis. The analysis of the IgG subclass distribution showed different patterns of response, IgG1 and IgG4 being higher in patients (even though statistically significant only for ovalbumin), and IgG2 and IgG3 being lower in this group. The presence of food-specific IgE in the majority of atopic children and the different specific IgG subclass patterns observed in patients and controls may reflect an alteration in the immune response to dietary proteins in atopic dermatitis.     

Integrin-mediated localization of Bordetella pertussis within macrophages: role in pulmonary colonization

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough but the significance of bacterial attachment to macrophages has not been determined. Adherence to cilia and macrophages is mediated by two large, nonfimbrial bacterial proteins, filamentous hemagglutinin (FHA), and pertussis toxin (PT). PT and FHA both recognize carbohydrates on cilia and macrophages; FHA also contains an Arg-Gly-Asp (RGD) sequence which promotes bacterial association with the macrophage integrin complement receptor 3 (CR3). We determined that virulent B. pertussis enter and survive in mammalian macrophages in vitro and that CR3 is important for this uptake process. We then determined the relative contribution of CR3 versus carbohydrate-dependent interactions to in vivo pulmonary colonization using a rabbit model. B. pertussis colonized the lung as two approximately equal populations, one extracellular population attached to ciliary and macrophage surface glycoconjugates and another population within pulmonary macrophages. Loss of the CR3 interaction, either by mutation of FHA or treatment with antibody to CR3, disrupted accumulation of viable intracellular bacteria but did not prevent lung pathology. In contrast, elimination of carbohydrate-bound bacteria, either by a competitive receptor analogue or an anti-receptor antibody, was sufficient to prevent pulmonary edema. We propose that CR3-dependent localization of B. pertussis within macrophages promotes persistence of bacteria in the lung without pulmonary injury. On the other hand, the presence of extracellular bacteria adherent to cilia and macrophages in carbohydrate-dependent interactions is associated with pulmonary pathology.     

Serological response to plasmid-encoded antigens in children and adults with shigellosis

The immunological response to plasmid-encoded antigens of virulent Shigella was determined in Thai children less than 4 yr of age and in Thai adults by immunoblot analysis and ELISA. Forty-two percent (8/19) of Thai children and 4% (1/22) of Thai adults with shigellosis developed a greater than or equal to 4-fold rise in IgG antibody titer to water-extracted antigens of Shigella flexneri M90T by ELISA (p = 0.006). Two children and one lactating mother with shigellosis developed a 4-fold rise in serum IgA antibody titers to water-extracted antigens of M90T. The results of the ELISA were confirmed by immunoblot analysis in all of the 41 paired sera examined. Five patients developed IgA, and four developed IgM, antibodies as detected by immunoblot analysis, that were not detected by ELISA. The reciprocal log2 geometric mean titers of antibodies to plasmid-encoded antigens in acute sera was higher in Thai adults than Thai children: IgG 7,265 versus 1,659; IgM 879 versus 480; and IgA 662 versus 60 (p less than 0.001). Thai adults had high titers of antibodies to plasmid-encoded antigens in their acute sera, but were susceptible to Shigella infections, although they were historically less susceptible than Thai children.     

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.     

Diagnostic performance of commercial serological assays measuring Bordetella pertussis IgG antibodies

Due to their specificity to B. pertussis antigens, immunoglobulin G (IgG) antibodies should be measured primarily for diagnosing pertussis. We compared the diagnostic performance of commercially available enzyme-linked immunosorbent assays (ELISAs) and chemiluminescent immunoassays (CLIAs) measuring IgG to B. pertussis antigens. An in-house ELISA with purified pertussis toxin (PT) was used as reference system. Commercial assays using PT only as coating antigen showed better performance as compared to those using a mixture of different antigens. The best diagnostic performances were achieved by CLIAs. Results were analyzed using a dual cutoff of either ≥125 IU/mL anti-PT IgG or ≥62 IU/mL anti-PT IgG for the in-house ELISA and accordingly to package inserts for commercial assays. Using the in-house ELISA at a 62 IU/mL cutoff, as the gold standard for interpretation of results from the commercial kits, resulted in lower sensitivity and higher specificity as compared to 125 IU/mL, thus, it may be especially useful in outbreak situations when high specificity is required.     

Isotype distribution and serial levels of antibodies reactive with dietary protein antigens in dermatitis herpetiformis

Using a sensitive enzyme-linked immunoassay (ELISA), a significantly increased prevalence (p less than 0.001) of serum antibodies reactive with wheat gliadin, bovine milk or ovalbumin has been demonstrated in 75% (33/44) of adult patients with dermatitis herpetiformis (DH), compared with healthy adults. There was no significant difference in the prevalence of antibodies (79%) in patients on a gluten-free diet or not on a gluten-free diet (72%). These serum antibodies reactive with gliadin, milk and ovalbumin were of the IgG isotype. However, IgA anti-gliadin antibodies were also detected in DH patients, but only in patients who were not on a gluten-free diet. In contrast, IgA anti-milk antibodies were also detected in DH patients irrespective of whether the patient was on a gluten-free diet. In DH patients, antibodies reactive with ovalbumin were often restricted to the IgG4 subclass and antibodies reactive with bovine milk antigens (notably casein) were distributed predominantly in both IgG2 and IgG4 subclasses, a similar IgG isotype distribution to that observed in healthy individuals. However, anti-gliadin antibodies in DH patients showed no predominant IgG4 subclass restriction. IgG4 anti-ovalbumin antibodies and IgG4 and/or IgG2 anti-casein antibodies persisted for up to 4 yr without fluctuation, irrespective of whether DH patients were on a gluten-free diet.     

Subclass restriction of murine antibodies. III. Antigens that stimulate IgG3 in mice stimulate IgG2c in rats

The IgG subclass distribution of rat antibodies to 13 different antigens was measured. Antibodies to protein and hapten-protein conjugates were predominantly IgG2a. Antigens labeled thymus-independent type 1, based upon responses in mice, stimulated both IgG2b and IgG2c antibodies, but little IgG2a. Polysaccharide and hapten-polysaccharide antigens (thymus-independent type 2) as well as phosphocholine-keyhole limpet hemocyanin, stimulated predominantly IgG2c antibodies. A division of antigens into essentially the same categories has been made on the basis of subclass restriction in mice. Antigens that stimulate IgG2c in rats stimulate IgG3 in mice. Thus, by comparing subclass preference with a variety of antigens, functional analogues among subclasses in different species can be identified.     

Correlation between G2m(n) immunoglobulin allotype and human antibody response and susceptibility to polysaccharide encapsulated bacteria

To determine whether genetic factors influence the human antibody response to polysaccharides, we correlated Ig allotypes with the concentrations of antibody to 14 bacterial capsular antigens in 130 actively immunized Caucasian adults. The 88 individuals possessing G2m(n), an allotype antigen of IgG2 subclass heavy chains, had significantly higher postimmunization antibody levels to Haemophilus influenzae type b (Hib) and 8 of 11 pneumococcal types (P less than 0.05) than the 42 lacking this antigen. For Hib, pneumococcus type 14, and meningococcus group C, an increased response was observed in IgG class but not in IgM or IgA classes of antibody. The G2m(n) positive individuals also had higher preimmunization antibody levels to most polysaccharide antigens. Total IgG2 concentrations were correlated with the mean postimmunization antibody concentrations to pneumococci (P = 0.005), but this correlation was independent of G2m(n) by multiple regression analysis. To determine if the lack of G2m(n) was associated with increased susceptibility to infection, we compared the frequencies of various Ig allotypes in 98 children infected with Hib and 98 matched controls. Caucasian children with Hib infections other than epiglottitis were significantly more likely to lack the G2m(n) allotype than controls (P less than 0.05). G2m(n) negative Caucasian children less than or equal to 18 mo old have a 5.1-fold higher risk of nonepiglottitic Hib infections than G2m(n) positive children (P less than 0.01). We conclude that allotypic variants of the gamma-2 heavy chain genes, or genes in linkage equilibrium with them, exert a regulatory influence on the caucasian antibody response to a variety of immunologically distinct bacterial polysaccharide antigens. Young Caucasian children of the low responder phenotype, i.e., those lacking the G2m(n) allotype, are genetically predisposed to Hib and perhaps other bacterial infections.     

Seroprevalence of IgG antibodies to pertussis toxin in the Slovene population

BACKGROUND: The use of pertussis vaccines has reduced the morbidity and mortality of whooping cough. Immunity following the natural disease or vaccination is not life-long and reinfections causing an increase of pertussis antibodies can occur. In this study, the distribution of IgG antibodies to pertussis toxin (anti-PT IgG) among different age groups in Slovenia was determined. METHODS: The seroprevalence of anti-PT IgG antibodies to Bordetella pertussis was investigated in 3418 persons (49.1% males). The population under study was stratified into 27 age groups. The serological results were assigned to five groups, according to their titer levels. The geometric mean titers (GMT) were calculated. RESULTS: In 11.5% sera tested, no IgG antibodies to pertussis toxin were detected. High titers (> or =125 U/ml) were confirmed in 2.3% sera. There were no statistically significant differences between age groups in the proportion of antibody levels. Pre-school children from three to five years of age had the lowest anti-PT IgG GMTs (9.6-10.7 U/ml). Vaccinated children (aged from one to two years) and adolescents from 17-18 years of age had the highest GMTs (>20 U/ml). GMTs were not statistically significantly different between males and females. CONCLUSIONS: The study demonstrated an early decline of anti-PT IgG after vaccination. According to the serological profile, school-age children and adolescents have the highest rate of infection. The large proportion of seropositive adults indicates that reinfection with B. pertussis is relatively common.     

Effect of Heat Inactivation of Serum on Bordetella pertussis Antibody Determination by Enzyme-linked Immunosorbent Assay

The effect of heat inactivation on Bordetella pertussis antibodies determined by enzyme-linked immunosorbent assay (ELISA) was studied. Sera were heated at increasing temperatures (from 30 to 50°C at 5°C increments and from 52 to 70°C at 2°C increments). Between 30 and 50°C, no significant differences were observed in immunoglobulin G (IgG) antibodies to pertussis toxin (PT). From 50 to 56°C the antibody values were twofold higher than those of uninactivated sera; at 64°C the values were 3.6- to 9.1-fold higher. The increase in PT IgG antibody values was more pronounced in sera with low antibody values. ELISA antibody values of sera from a vaccine trial were determined in unheated and heat inactivated sera. The geometric mean value (GMV) of the heat inactivated samples was 3.2 times the geometric mean value of the uninactivated sera. ELISA IgG antibodies to filamentous hemagglutinin, fimbriae-2, and pertactin were studied, and the values of heat inactivated sera did not differ significantly from the values of the uninactivated sera. Our findings indicate that heat inactivation of sera leads to higher, variable, and false-positive PT IgG values.     

Dissecting human T cell responses against Bordetella species

To identify the minimal structures that may be important for the creation of a synthetic and/or recombinant vaccine against whooping cough, human T cell clones were obtained against Bordetella antigens. Cloned peripheral blood T lymphocytes from an immune donor were grown in IL-2 and tested for proliferation in response to inactivated Bordetella species (B. pertussis, B. parapertussis, and B. bronchiseptica) and mutants deficient for the expression of virulence-associated antigens. All the T cell clones obtained were CD4+8- and recognized specifically the Bordetella antigens when presented by autologous B cells. On the basis of the responsiveness to the whole inactivated bacteria, it was possible to cluster the 12 clones obtained into four groups with the following specificity: (1) filamentous hemagglutinin (FHA); (2) B. pertussis-specific antigens; (3) virulence-associated Bordetella-specific antigens; and (4) nonvirulence-associated Bordetella-specific antigens. Using two new B. pertussis deletion mutants, clone 6 (representative of cluster 1) was found to recognize the COOH terminus of FHA. Furthermore, three out of four clones of cluster 3 were specifically stimulated by the soluble 69-kD protein from the outer membrane of B. pertussis. Surprisingly, none of the twelve clones obtained by stimulation in vitro with whole inactivated bacteria recognized pertussis toxin (PT), which is believed to be the most important protein to be included in an acellular vaccine. However, when a new generation of clones was obtained using soluble PT as the in vitro stimulus, it was observed that 11 clones of this group recognized this antigen. Thus, PT does not seem to be the most representative antigen on the whole inactivated bacteria, although T cell memory against PT exists in a donor who had the disease several years ago.     

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.     

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.     

HUMAN IMMUNITY TO THE MENINGOCOCCUS : II. DEVELOPMENT OF NATURAL IMMUNITY

Results of the present study suggest that natural immunity to meningococcal disease is initiated, reinforced, and broadened by intermittent carriage of different strains of meningococci throughout life. In young adults, carriage of meningococci in the nasopharynx is an efficient process of immune sensitization. 92% of carriers of serogroup B, C, or Bo meningococci were found to develop increased titers of serum bactericidal activity to their own meningococcal isolate, and 87% developed bactericidal activity to heterologous strains of pathogenic meningococci. The rise in bactericidal titer occurred within 2 wk of onset of the carrier state, and was accompanied by an increase in titer of specific IgG, IgM, and IgA antibodies to meningococci. In early childhood, when few children have antibodies to pathogenic meningococci, active immunization seems to occur as a result of carriage of atypical, nonpathogenic strains. Immunity to systemic meningococcal infection among infants in the neonatal period is associated with the passive transfer of IgG antibodies from mother to fetus. The antigenic determinants which initiate the immune response to meningococci include the group-specific C polysaccharide, cross-reactive antigens, and type-specific antigens.     

Pneumococcal immunity and response to immunization with pneumococcal vaccine in bone marrow transplant patients: the influence of graft versus host reaction

The aim of this study was to evaluate levels and subclass distribution of pneumococcal antibodies in 40 bone marrow transplant (BMT) and 42 autologous bone marrow transplant (ABMT) recipients during the first year after transplant, and response to vaccination with a polyvalent pneumococcal vaccine. Before transplantation, 35/40 recipients of allogeneic grafts, all 42 autologous BMT recipients and 38/39 donors had adult levels of anti-pneumococcal antibodies of the IgG2 subclass. During the first year after transplantation, antibody levels decreased in 29 BMT patients while 11 retained their pretransplant antibody levels. No change was noted among ABMT patients. In the 8 BMT patients who had chronic graft versus host disease (GVHD), none showed normal levels of anti-pneumococcal antibodies 1 year after BMT as compared to 11/32 without chronic GVHD. Three different response patterns were seen after vaccination of 29 BMT patients who lost immunity with a polyvalent pneumococcal vaccine. Ten patients responded with an increase in IgG2 antibodies, 8 responded with an increase in IgG1 and 11 patients did not respond at all. In the 8 patients with chronic GVHD, none responded with an increase in IgG2 antibodies and 6/8 did not respond at all. The results of this study suggest that chronic GVHD is the main factor contributing to loss of immunity to pneumococci and lack of responsiveness to vaccination with pneumococcal polysaccharides after BMT. Furthermore, the difference in capability, between BMT and ABMT recipients, of retaining anti-pneumococcal activity may explain the clinical experience of severe pneumococcal infections in these patients.     

Multiplex assessment of SARS-CoV-2 antibodies improves assay sensitivity and correlation with neutralizing antibodies

ObjectivesDetection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection.MethodsA cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61).ResultsSpecificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79–95% agreement with the presence of neutralizing antibodies.ConclusionsThe BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations.