A method to determine significant levels of immunoglobulin G to Aspergillus fumigatus antigens in an ELISA system and a comparison with counterimmunoelectrophoresis and double diffusion techniques

A method is described which assesses results obtained from an ELISA system for the determination of human serum levels of IgG class antibodies to Aspergillus fumigatus. The method is used to discriminate positive from negative samples, and significant antibody activity may be reported to the clinician, relative to a reference positive control serum monitored simultaneously under the same test conditions. Antibody content is expressed as the absorbance of a certain dilution of serum. Duplicate samples were analysed at a single serum dilution and their absorbtion values obtained from a semi-automated ELISA microplate reader. These were entered into a computer programmed to convert the data into units on a logarithmic scale. In parallel experiments, ELISA results were compared with those obtained by the techniques of counterimmunoelectrophoresis and double diffusion which measure precipitating antibody of all classes. A relatively good degree of correlation between tests was found only among sera with a high level of antibody.     

Detection of Aspergillus fumigatus precipitins: a comparison of counter immunoelectrophoresis and double diffusion.

A counter immunoelectrophoresis method was compared with a double diffusion test for detecting Aspergillus fumigatus antibodies. Of 70 patients, 23 gave positive results in both tests and the remainder gave negative results. Fifteen patients with proven aspergillosis gave positive results in both tests.     

Detection of antibodies against Aspergillus fumigatus: comparison between double immunodiffusion, ELISA and immunoblot analysis

The performance of ELISA to detect IgG and IgM antibodies to Aspergillus fumigatus has been evaluated in strongly precipitin-positive, weakly precipitin-positive and precipitin-negative patient sera, with immunoblot analysis as the confirmatory test. All strongly precipitin-positive sera contained increased IgG titers and showed clearly positive immunoblot patterns. Most of the weakly precipitin-positive sera contained ELISA titers within the normal range established with sera of healthy blood donors and showed normal immunoblot patterns. Increased titers of IgG and/or IgM were measured in one-sixth of the precipitin-negative patient sera. Immunoblot analysis confirmed the presence of antibodies to A. fumigatus in 55% of the precipitin-negative sera with increased antibody titers. ELISAs for A. fumigatus-specific IgG and IgM are sensitive tests for screening of patient sera. However, positive results with ELISA should be confirmed by means of immunoblot analysis.     

Measurement of ferritin levels: comparison of a commercial IRMA to an in-house ELISA method

The purpose of this study was a comparison of a commercial IRMA to an in-house ELISA method. Because IRMA methods are considered to be very sensitive and results of these assays are widely accepted, the ferritin levels of 378 school children were determined in parallel with a commercial IRMA kit (Becton Dickinson Co., Orangeburg, NY) and the ELISA. For organizing reasons plasma was used for the ELISA, whereas serum was used for the IRMA. Regression analysis of the pairs of values, taking the IRMA reading as the independent variable, yielded a coefficient of variation r=0,90 (Spearman, two-tailed, p=0,01) and the straight line y=0,99x+3,13. Using this ELISA technique described by DAKO, ferritin levels in plasma were, on average, 13,2% higher than in serum taken at the same time. The ELISA technique described was found to have good accuracy and the speed and ease with which it may be carried out makes it suitable for a large number of samples.     

Enzyme linked immunosorbent assay (ELISA) for IgG and IgE antibodies to protein and polysaccharide antigens of Aspergillus fumigatus

ELISA has emerged as a useful alternative to other more costly and complex tests. Polystyrene microhaemagglutination plates have been used as solid phase to absorb Aspergillus fumigatus protein and polysaccharide components for detection of specific antibodies in patients with various forms of pulmonary aspergillosis. IgG and IgE antibodies to the polysaccharide as well as the protein allergens have been found. For the IgE test a double antibody technique has been developed, which is more sensitive than the conventional indirect ELISA.     

Resistance to voriconazole due to a G448S substitution in Aspergillus fumigatus in a patient with cerebral aspergillosis

A voriconazole-resistant isolate of Aspergillus fumigatus was recovered from an immunocompetent patient receiving long-term antifungal therapy for cerebral aspergillosis. A G448S amino acid substitution in the azole target (Cyp51A) was identified as the cause of the resistance phenotype. This article describes the first isolation of a voriconazole-resistant A. fumigatus isolate from an immunocompetent patient in Spain.     

A Method to Determine Significant Levels of Immunoglobulin G to Aspergillus Fumigatus Antigens in an ELISA System and a Comparison with Counterimmunoelectrophoresis and Double Diffusion Techniques

A method is described which assesses results obtained from an ELISA system for the determination of human serum levels of IgG class antibodies to Asperglllus fumigatus. The method is used to discriminate positive from negative samples, and significant antibody activity may be reported to the clinician, relative to a reference positive control serum monitored simultaneously under the same test conditions. Antibody content is expressed as the absorbance of a certain dilution of serum. Duplicate samples were analysed at a single serum dilution and their absorbtion values obtained from a semi-automated ELISA microplate reader. These were entered into a computer programmed to convert the data into units on a logarithmic scale.

In parallel experiments, ELISA results were compared with those obtained by the techniques of counterimmunoelectrophoresis and double diffusion which measure precipitating antibody of all classes. A relatively good degree of correlation between tests was found only among sera with a high level of antibody.     

A comparison of different extracts of Candida albicans in agar gel double diffusion techniques

A preliminary comparative study, by 4 independent groups, of certain extracts of Candida albicans, showed variation in their antigenicity. Different agar gel double diffusion tests were used by each group; two groups used micro-methods and two used macro-methods. The number of positive precipitin reactions detected by these methods was shown to vary greatly.     

A sandwich enzyme immunoassay of rabbit immunoglobulin G with an enzyme labeling method and a new solid support

A method was devised for enzyme labeling goat antibody to rabbit immunoglobulin G with beta-D-galactosidase (EC 3.2.1.23) using a heterobifunctional cross-linker, N-(gamma-maleimidobutyryloxy)-succinimide. Labeling of the purified antibody was by a continuous 2-step process and including a chromatographic purification procedure could be completed within one day. The partially purified anti-rabbit IgG was coated on Amino-Dylark cylinders, a new solid support, using glutaraldehyde as the coupling reagent. With enzyme-labeled antibody and the solid-phase anti-rabbit IgG, a sandwich enzyme immunoassay for rabbit IgG was developed with a lower limit of detection at 3.5 pM (0.1 ng/tube). The specificity of the assay was excellent and all 4 types of IgG tested showed 0.0001% or less cross-reactivity with rabbit IgG.     

Effect of immunotherapy on immunoglobulin E and immunoglobulin G antibodies to ragweed antigens: a six-year prospective study

The effect of immunotherapy with aqueous short ragweed (SRW) extract on IgE and IgG antibodies was tested over a 6 yr period in 47 adults with ragweed hay fever. Sera were collected each year in July and October from 1973 through 1979. In May 1976, 23 patients began immunotherapy with a lyophilized standardized SRW extract. From 1976 through 1979, treated patients received an average total dose of 4.8 × 10³ protein nitrogen units (1039 μg of AgE). IgE antibodies to SRW and ragweed AgE were measured by the radioallergosorbent test (RAST) in antigen excess using allergens bound to Sepharose. Blocking antibodies primarily of the IgG class were measured by RAST interference. In response to inhalation of ragweed pollen, untreated patients showed seasonal rises (July to October) and postseasonal falls (October to July) of IgE antibodies during the entire study period. IgE antibody levels in the untreated patients decreased with time and from 1974 to 1979 fell 41% (p < 0.003) for an average halftime of 6.2 yr. Before immunotherapy, treated patients also showed seasonal rises and postseasonal falls. Treatment with SRW extract in 1976 produced an abrupt increase in IgE and IgG antibodies and a clear-cut suppression of seasonal rises of IgE antibodies without an effect on postseasonal falls through 1978. From 1974 to 1979, IgE antibodies to AgE and SRW decreased more in the immunized group than in the control group, and by 1979 these levels showed a mean fall of 73%. Blocking antibodies increased in the treated patients and reached maximal levels by July 1978. In 1978 and 1979, the levels of IgG blocking antibodies to AgE were inversely related to the IgE antibody levels to AgE. These results indicate that adults with ragweed hay fever show regular seasonal and postseasonal changes in IgE antibodies and that IgE antibodies spontaneously decrease with time. Immunotherapy magnifies these decreases by suppression of the seasonal rises, but it does not affect the postseasonal falls.     

Virulence of Aspergillus fumigatus double mutants lacking restriction and an alkaline protease in a low-dose model of invasive pulmonary aspergillosis.

To investigate the pathogenicity of Aspergillus fumigatus mutants lacking putative virulence factors, we have developed a new murine model of invasive pulmonary aspergillosis based on neutropenia, the major factor predisposing patients to this infection. Mice were treated with cyclophosphamide and inoculated by the intranasal route with 5 x 10(3) conidia, a significant reduction from inoculum levels used in previous models. Evidence for the production of the extracellular alkaline protease (Alp) in lung tissue was obtained by using a fungal transformant harboring an alp::lacZ reporter gene fusion. The pathogenicities of single mutant strains lacking either Alp or the ribotoxin restrictocin and of a double mutant strain lacking both proteins were assessed in this infection model. There were no significant differences between the mutant and the wild-type strains in terms of mortality or histological-features. Inoculations with mixtures of conidia showed that the double mutant strain is slightly less virulent than the wild-type strain. We conclude that Alp and restrictocin are not important virulence determinants in pulmonary infection.     

Development of a fluorescent-bead-based multiplex immunoassay to determine immunoglobulin G subclass responses to Neisseria meningitidis serogroup A and C polysaccharides

A fluorescent-particle-based multiplex flow cytometric immunoassay (MIA) for the detection of serum immunoglobulin G (IgG) and two IgG subclasses, IgG1 and IgG2, specific for Neisseria meningitidis serogroup A (MenA) and C (MenC) polysaccharides (PS) was developed. The assay comprised three separate duplex assays, one for the detection of the IgG response to MenA and MenC PS, another for the detection of the IgG1 response to MenA and MenC PS, and a third for the detection of the IgG2 response to MenA and MenC PS. Next, the three separate duplex assays were combined and analyzed as a hexaplex assay. No interference between monoplex, duplex, and hexaplex assays was observed, and the assay was found to have low intra- and interassay variation (<9.0% and <27%, respectively). Comparison of the meningococcal subclass MIA to the in-house enzyme-linked inmmunosorbent assays showed a good correlation (R >or= 0.85) for each of the subclasses. We conclude that the hexaplex meningococcal subclass MIA is an easy and specific assay for the determination of anti-MenA and anti-MenC PS subclass IgG, requiring minimal amounts of serum to study IgG subclass responses to vaccines.     

A comparison of the prevalence of sensitization to Aspergillus antigens among asthmatics in Cleveland and London

A survey of the frequency of sensitization to Aspergillus antigens was conducted in a group of asthmatics in Cleveland and compared with a group of asthmatics in London, using common antigens for testing purposes. The two groups were comparable except for earlier onset, longer duration of asthma, and a larger number of males in the London group. Twenty-eight per cent of the asthmatics from Cleveland and 23% from London had immediate skin reactivity to Aspergillus. Seven and one-half percent from the Cleveland group and 10.5% of the London group had Aspergillus precipitins in the serum. Aspergillus skin test reactivity was related to the severity of airways obstruction (p < 0.01) but was not influenced by other factors. We conclude that sensitization to Aspergillus antigens occur with equal frequency in both the United States and the United Kingdom.     

Sarcoidosis does not belong to or overlap with immunoglobulin G4-related diseases based on an assessment of serum immunoglobulin G4 levels in cardiac and noncardiac sarcoidosis

Although sarcoidosis may exhibit histopathologic features similar to those of a newly emerging clinical entity, immunoglobulin G4-related sclerosing disease, sarcoidosis is currently not considered to be associated with immunoglobulin G4-related immunoinflammation. Not many studies on this association have been reported. We investigated serum immunoglobulin G4 levels among patients with sarcoidosis with or without cardiac involvement (cardiac sarcoidosis and non-cardiac sarcoidosis patients). The mean serum immunoglobulin G4 level among the 65 patients with sarcoidosis was 56.8 ± 43.0 mg/dL, which did not significantly differ between patients with cardiac sarcoidosis (54 ± 48 mg/dL, n = 12) and patients without cardiac sarcoidosis (58 ± 42 mg/dL; n = 53). Serum level of soluble interleukin 2 receptor, a potent marker that may reflect sarcoidosis activity, was elevated in cardiac sarcoidosis (910 ± 683 U/L) and noncardiac sarcoidosis (689 ± 399 U/L) but did not significantly differ between the groups. Immunohistochemistry of cardiac or lymph node specimens from patients with cardiac sarcoidosis showed only sparse or no infiltration of immunoglobulin G4-positive lymphocytes, in contrast to the moderate to severe infiltration of CD68-positive macrophages and CD45-positive lymphocytes. Although the number of study subjects was small, these findings collectively suggest that regardless of the presence or absence of cardiac involvement, sarcoidosis does not belong to or overlap with immunoglobulin G4-related sclerosing disease.     

Regulation of antigen-specific immunoglobulin G subclasses in response to conserved and polymorphic Plasmodium falciparum antigens in an in vitro model

Cytophilic antibodies (Abs) play a critical role in protection against Plasmodium falciparum blood stages, yet little is known about the parameters regulating production of these Abs. We used an in vitro culture system to study the subclass distribution of antigen (Ag)-specific immunoglobulin G (IgG) produced by peripheral blood mononuclear cells (PBMCs) from individuals exposed to P. falciparum or unexposed individuals. PBMCs, cultivated with or without cytokines and exogenous CD40/CD40L signals, were stimulated with a crude parasite extract, recombinant vaccine candidates derived from conserved Ags (19-kDa C terminus of merozoite surface protein 1 [MSP1(19)], R23, and PfEB200), or recombinant Ags derived from the polymorphic Ags MSP1 block 2 and MSP2. No P. falciparum-specific Ab production was detected in PBMCs from unexposed individuals. PBMCs from donors exposed frequently to P. falciparum infections produced multiple IgG subclasses when they were stimulated with the parasite extract but usually only one IgG subclass when they were stimulated with a recombinant Ag. Optimal Ab production required addition of interleukin-2 (IL-2) and IL-10 for all antigenic preparations. The IgG subclass distribution was both donor and Ag dependent and was only minimally influenced by the exogenous cytokine environment. In vitro IgG production and subclass distribution correlated with plasma Abs to some Ags (MSP1(19), R23, and MSP2) but not others (PfEB200 and the three MSP1 block 2-derived Ags). Data presented here suggest that intrinsic properties of the protein Ag itself play a major role in determining the subclass of the Ab response, which has important implications for rational design of vaccine delivery.     

Development of a sandwich ELISA to detect IgG and IgG sub-class antibodies specific for a major antigen (Ag 7) of Aspergillus fumigatus

A sandwich ELISA has been developed, using an affinity purified monospecific antiserum as a capture antibody, to detect specific IgG and IgG sub-classes to a major antigen (Ag 7) of Aspergillus fumigatus in the sera of patients with allergic bronchopulmonary aspergillosis (ABPA). Significantly elevated levels of specific IgG to Ag 7 were detected in 97% of ABPA sera tested, as compared to control sera and to sera from A. fumigatus skin-prick test positive individuals. IgG sub-class antibody levels to Ag 7 were also determined in a similar sandwich ELISA, but using specific monoclonal antisera instead of the polyclonal anti-IgG. Both Ag 7 specific IgG1 and IgG4 levels were found to be significantly raised in the ABPA sera compared to controls. It is proposed that this antigen-specific ELISA may provide a more specific diagnostic test for IgG antibody detection in sera of ABPA patients.     

A comparison of two techniques for the detection of an antibody in human serum to Chang liver cell surface antigens

The antibody-dependent cell-mediated cytotoxicity technique (ADCC) and an isotopic antiglobulin technique have been compared for their abilities to detect human serum antibodies to Chang liver cell membrane antigens. The optimal conditions were established for the latter technique, which was found to be superior with respect to reproducibility and sensitivity. Also, unlike ADCC, the isotopic antiglobulin technique was unaffected by other factors, such as immune complexes present in pathological sera.     

High levels of immunoglobulin E and a continuous increase in immunoglobulin G and immunoglobulin M by age in children with newly diagnosed type 1 diabetes

The incidence of type 1 diabetes (T1D) is increasing, either because of environmental factors accelerating onset of the disease or because of inducement of autoimmune diabetes in children who previously were at lower risk. High levels of immunoglobulin (Ig), specifically, IgM and IgA, and a low level of IgG were reported in adult patients; however no studies have analyzed the increasing incidence in relation to Ig levels. Our aim was to describe Ig in children newly diagnosed with diabetes and in their healthy siblings. Children with T1D expressed significantly lower IgG (p < 0.01) and higher IgA levels (p = 0.045), whereas no differences in IgE or IgM (p > 0.5) levels were found. Age-specific levels were unchanged over a 9-year period. In patients and siblings IgG, IgA and IgE increased by age (p < 0.001); which was in contrast to IgM (p > 0.05). The continued increase in IgG levels by age indicates that adult levels are reached later than in previously studied cohorts, thereby indicating a slower maturation of the immune system.     

Killing of Aspergillus fumigatus spores and Candida albicans yeast phase by the iron-hydrogen peroxide-iodide cytotoxic system: comparison with the myeloperoxidase-hydrogen peroxide-halide system.

A new fungicidal system composed of ferrous ion, H2O2, and iodide is described and compared with the myeloperoxidase-hydrogen peroxide-halide system. Both systems had similar activity against Aspergillus fumigatus spores and the Candida albicans yeast phase, but only the ferrous ion-hydrogen peroxide-iodide system was inhibited by hydroxyl radical scavengers.     

A fast and efficient method for quantification of monoclonal antibodies in an ELISA using a novel incubation system

We have developed a sensitive, reliable, optimized ELISA to measure human IgM monoclonal antibodies using a novel shaking incubator system with short incubation periods of 15 min at 37 degrees C for all stages. The shaking incubator is compared with a static incubator over a range of incubation times and temperatures. For each stage using static incubation conditions the system does not reach a saturation level and the results are inconsistent, unlike the shaking incubator. No 'edge effects' are observed in the shaking system due to even heating from beneath and across the plate. The orbital shaking ensures optimal mixing of reagents which eliminates a diffusion limited reaction rate caused by a depletion of reactants at the solid phase as observed in the static system. The optimized shaking system permits economical use of reagents since the coating antibody can be used at high dilutions.