Sensitive immunoradiometric assay for the detection of Paracoccidioides brasiliensis antigens in human sera.

In the present study we report the standardization of an immunoradiometric assay (IRMA) for detection of Paracoccidioides brasiliensis circulating antigens that could be useful in the diagnosis and prognosis of paracoccidioidomycosis. For this purpose we studied the reactivities of P. brasiliensis and other mycotic antigens with rabbit polyclonal anti-P. brasiliensis antibodies (immunoglobulin G) in order to evaluate the sensitivity and specificity of an IRMA for detecting P. brasiliensis antigens. The results were compared with those obtained by the double immunodiffusion test, the standard technique for the serodiagnosis of paracoccidioidomycosis. By using the immunoglobulin G fraction of rabbit antisera (900 ng per well), it was possible to detect up to 3.6 ng (0.12 micrograms/ml) of cellular antigen and 360 ng (12 micrograms/ml) of metabolic antigen in contrast to the double immunodiffusion test that could detect only 12 micrograms (1.2 mg/ml) of both antigens. IRMA was shown to be feasible and very sensitive and may therefore help, together with clinical data, in establishing early diagnosis and assessing disease activity. It could also allow the study of relationships between P. brasiliensis circulating antigens and host defense mechanisms during the disease.

Diagnosis of systemic candidosis by immunodiffusion test with locally prepared antigen.

Locally-made C. albicans, C. tropicalis, C. krusei antigens and antisera were made and compared with reference C. albicans and C. krusei antigens and antisera from the Center for Disease Control (CDC), Atlanta, Georgia, U.S.A., and the Provincial Laboratory of Public Health, University of Alberta, Edmonton, Alberta, Canada, respectively. The local antigens and antisera showed 3 precipitin bands when reacted with locally-made and reference antisera. When tested with human sera from 171 normal persons (aged 17-24 years), 30 Candida vaginitis, 30 Candida balanitis, 30 mucocutaneous candidosis, 30 pityriasis versicolor and 10 aspergillosis patients revealed no precipitin band. When tested with cryptococcosis, 2 out of 6 sera showed a precipitin band but only at 1:1 dilution. Four candidosis sera offered by CDC showed precipitin bands at dilutions up to 1:4. Three Thai candidosis sera also showed precipitin bands at dilutions up to 1:2. Therefore, we judged the baseline titer for Thai population to be 1:2.

[Aspergillus fumigatus: evaluations of the standardization of diagnostic extracts]. / Aspergillus fumigatus: Ansätze zur Standardisierung von Extrakten für die Diagnostik.

Extracts of Aspergillus fumigatus used for serodiagnosis of allergic diseases are of unknown and nonstandardized composition. Applying immunoprint technique subsequent to isoelectric focusing commercial and self-prepared extracts are compared. By use of an ABPA-pool serum and monoclonal antibodies recognizing antigens of this fungus, different immunological reactivities of the extracts can be demonstrated. It is suggested to use monoclonal antibodies as secondary standards as well as to improve standardization.

Production of a proposed international reference standard Alternaria extract. II. Results of a collaborative trial.

A lyophilized Alternaria extract prepared from a defined eight-strain source material was compared with three other Alternaria extracts to assess its potential suitability as an international standard (IS). Twenty-two laboratories in 12 different countries collaborated. Assay methods included RAST inhibition, quantitative immunoelectrophoresis, thin-layer isoelectric focusing, skin testing, leukocyte histamine release, and various other methods for thorough characterization of the extracts. In addition, three laboratories quantitated specific allergen in each extract. The proposed IS extract could be used to assign a relative potency to other test extracts. In separate studies, the proposed IS extract was demonstrated to be stable for at least 21 months when it is stored at -70 degrees C, -20 degrees C, and 4 degrees C.

A novel Candida albicans skin test antigen: efficacy and safety in man.

Yeast phase Candida albicans (ATCC No. 10231) was grown in a nonantigenic medium, harvested and lyophilized. Ammonium sulfate fractions of an aqueous extract of the lyophilized cells were evaluated and the fraction yielding the highest specific delayed cutaneous reactivity in sensitized guinea-pigs was used to prepare a C. albicans skin test antigen (CASTA). The safety of the antigen was evaluated by measuring immediate and delayed (0.25, 6, 24, 48 and 72 h) cutaneous reactions in atopic and nonatopic human subjects. The outcome of three repetitive monthly Mantoux skin tests with 0.01-1 microgram antigen doses was used to test for booster effects in 14 subjects and to estimate a safe initial test antigen dose. The utility of a single skin test as a measure of cell-mediated immunity was evaluated in 40 healthy subjects. Reactor rates (greater than or equal to 2 mm, 48 h) of 40% and 85% were detected, respectively, with doses of 0.0316 and 1 microgram. Using a skin test reaction diameter greater than or equal to 5 mm at 48 h, the reactor rate was 50% for the 1-microgram dose. The only adverse reaction (45 mm, 0.25 h) was detected with the 1-microgram dose in an atopic subject who also exhibited exquisite scratch test reaginic hypersensitivity to C. albicans allergen. The prevalence of other adverse reactions to this antigen compared favorably with that to other antigens used for recall antigen testing. These studies suggest the 1-microgram CASTA dose can be used for effective, safe recall antigen skin tests.

Standardization of fungal allergens.

The ability of various fungi to induce human IgE-mediated disease, especially allergic asthma, has been well established. The relative clinical importance of fungal aeroallergens varies considerably in different geographic areas. The magnitude of exposure to fungal aeroallergens can be quantitated more precisely by newer immunochemical assays than by more traditional fungal cultures or quantitation of fungal spores by microscopy. Although the wide variations in biologic potency among commercial mold allergenic extracts have been appreciated for decades, only recently have laboratory technologic advances and the production of international reference extracts permitted the development of standardized fungal extracts of defined potency. Future efforts should be directed toward documenting the expected diagnostic and therapeutic advantages of such standardized fungal extracts.

Studies on alternaria allergens. VI. Stability of the allergen components of Alternaria tenuis extracts under a variety of storage conditions.

The allergenic potencies of samples of two commercial Alternaria tenuis extracts stored under various conditions were compared at 3 months intervals over a period of 1 year. Aliquots of the extracts at 5.12 mg/ml were maintained in: saline; 50% glycerol-saline mixture; 0.4% phenol-saline; 0.4% phenol-saline containing 0.03% human serum albumin, and samples were also kept in lyophilized form. The samples were stored at -20, 4, 24, 37 and 56 degrees C. Their potency was measured by radioallergosorbent inhibition assay and by mouse IgE passive cutaneous anaphylaxis (PCA) tests. The lyophilized extracts fully maintained their activity at all temperatures for 12 months. Aqueous extracts with or without preservatives also maintained their potency if stored at -20, 4 and 24 degrees C, but showed a significant loss of PCA activity at 56 degrees C, after 3 months. A deleterious effect of phenol was observed in one of the extracts stored at -20 degrees C for 3 months, by PCA tests. The use of preservatives to retain potency over a period of 12 months is therefore unnecessary and may be deleterious. The results indicate that, while storage of A. tenuis in a lyophilized form is best at all temperatures, aqueous extracts are also remarkedly stable when stored at -20, 4 and 24 degrees C, over a 12-month period.

Antigens and allergens of Aspergillus fumigatus. I. Characterization by quantitative immunoelectrophoretic techniques.

Both the IgG (precipitinogen) and IgE combining components of Aspergillus fumigatus have been studied by quantitative immunoelectrophoretic techniques with sera of patients with allergic bronchopulmonary aspergillosis and specific rabbit antisera. Considerable batch-to-batch variation in biochemical properties and in immunologic (both antigenic and allergenic) activity was revealed in identically prepared culture-filtrate antigens of the same strain of A. fumigatus. Profiles of crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis appeared to vary qualitatively; however, the differences in antigenicity and allergenicity were demonstrated by crossed-line immunoelectrophoretic techniques to be essentially quantitative rather than qualitative in nature. Both types of activity were concentrated in the protein fraction obtained by salt precipitation, and little activity remained in the predominantly polysaccharide supernatant that accounted for greater than 30% (dry weight) of the antigenic extract. Heat-stability studies demonstrated a varied pattern of denaturation. Certain allergenic components were stable to considerable heating, and the extracts so treated were still active on skin testing.

[Characterization of exoantigens of Aspergillus fumigatus. Study of factors influencing their quality]. / Caracterización de exoantígenos de Aspergillus fumigatus. Estudio de factores que influyen en su calidad.

Several methods are available for exoantigen preparation and standardization, but until now there is no agreement about which is the best of them. Moreover an exhaustive analysis of the influence of several experimental conditions on the antigenic composition of exoantigens of Aspergillus fumigatus is badly needed. In this paper the analysis of the influence of the following conditions on this antigenic composition is described (origin of strains, culture media composition, incubation time of mycelial stationary growth in culture media and immunological techniques used to standardize the antigens. Antigenic extracts were obtained from six strains of A. fumigatus from different origin (pathogenic, collection and contaminants) growing in different broth culture media (glucose-asparagine, Sabouraud, dialyzed neopeptone, and Czapek Dox). The culture media were separated from mycelium, dialyzed and freeze-dried. These extracts were used at concentrations of 100 mg/ml. Immunoprecipitation techniques (immunodiffusion, immunoelectrophoresis and crossed-immunoelectrophoresis) were used in this work to evaluate the quality of the extracts. All these methods were performed with human sera from patients with aspergilloma and animal sera (rabbit and goat) experimentally immunized. Averages and standard deviations were calculated for each group and the possible significance of differences between groups was studied using the Student-t test. Analysis of results shows that immunological characteristics of the exoantigens used for diagnosis are independent of the different incubation times studied. It has been confirmed in this study that results depend to a certain degree on the origin of the serum used. The strains kept in collection for several years showed less reactivity than those recently isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of ELISA-inhibition to Aspergillus antigen standardization for immunodiagnosis.

An inhibition ELISA method was used to test culture filtrate antigens of Aspergillus fumigatus for the avidity of antibodies to them. Four different culture media were used to check the ability of this assay to distinguish between similar antigenic preparations. Differences between antigens exhibiting similar crossed immunoelectrophoresis patterns were observed by inhibition ELISA.

[Production and standardization of Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus antigens to be used in immunodiagnosis. Comparison between immunodiffusion and immunoelectrophoresis]. / Producão e padronização dos antígenos de Paracoccidioides brasiliensis (Pb), Histoplasma capsulatum (Hc) e Aspergillus fumigatus (Af) para uso no imunodiagnóstico. Comparação entre as técnicas de imunodifusão e imunoeletroosmoforese.

Soluble antigens (Ag) from Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus were prepared and standardized by double immunodiffusion (DID) and immunoelectroosmophoresis (IEOP). No difference in sensitivity was observed between the two techniques; 100% of standard patient sera were positive with P. brasiliensis and A. fumigatus Ag and 83.3% were positive with H. capsulatum Ag. The specificity of the tests was verified testing 96 sera from patients with paracoccidioidomycosis, histoplasmosis, systemic candidiasis, sporotrichosis, tuberculosis, lung cancer, visceral or cutaneous leishmaniasis and 18 sera from healthy individuals. All the three antigens were 100% specific with the DID (using the identification pattern indicated by the confluence of test serum with standard serum precipitin lines as a positive criterium). However in the IEOP, the specificity varied with each Ag. Positive reactions with P. brasiliensis Ag were observed in 16.7% of histoplasmosis sera and in 10% of cutaneous leishmaniasis sera. On the other hand 31.8% of paracoccidioidomycosis and 10% of cutaneous leishmaniasis sera reacted with H. capsulatum Ag. The high sensitivity and specificity of the DID test, its easy reproducibility and low cost, led us to consider it highly appropriate as a routine procedure for the screening of patients with respiratory infections.

Standardization of allergenic extracts of Aspergillus fumigatus. Liberation of IgE-binding components during cultivation.

During cultivation of Aspergillus fumigatus a rapid liberation of IgE-binding components was found reaching maximum values during the logarithmic phase of growth (phase I). After a fall in IgE-binding titers during phase II, appearance of additional IgE-binding components was noted during the period of lysis of the microorganism (phase III). These latter allergenic components are different from the phase I IgE-binding components, as was shown by crossed-inhibition studies. The number of precipitating antigenic components was not related with the corresponding IgE-binding titers and showed an increase during all phases of growth. The rapid changes in both IgE- and IgG-binding properties and the discrepancies between precipitating properties and IgE binding are discussed in relation to standardization and quality control of aspergillus extracts.

Standardization of antigens from Aspergillus fumigatus.

Batches of soluble antigen prepared by mechanical disintegration of Aspergillus fumigatus reproducibly contained identical protein components on polyacrylamide gels. This consistency was ascribed to standardization at the procedure for disruption: there was little variation in the protein patterns obtained from A. fumigatus mycelium grown for times varying from one to seven days at temperatures from 27 to 37 degrees C. Rabbit antisera to different A. fumigatus antigen preparations varied in their reactivity in crossed immunoelectrophoresis tests, but line immunoelectrophoresis indicated that all the antisera contained the same qualitative antibody components to the fungus. All but one of these components appeared to be present in serum samples from two patients with invasive pulmonary aspergillosis and high precipitin titres to A. fumigatus. The data suggest that many antibodies with different specificities are formed in response to aspergillosis in vivo, so that most preparations of A. fumigatus antigens are likely to contain at least one component capable of detecting antibodies to the fungus for diagnostic purposes.

Delayed cutaneous hypersensitivity testing.

The topic of delayed cutaneous hypersensitivity testing is reviewed. When skin tests are used to determine whether an individual is anergic, T cell immunity is evaluated. Skin testing can be used to determine the causative organisms of infection or to discover an immunologic deficiency state. Characteristic skin lesions usually develop after intradermal injection of the antigen. Skin testing uses the most ubiquitous environmental antigens and those that are safe, cheap, and effective. Important issues dealing with skin tests include false-positive and false-negative reactions, which antigens to use in an antigen "battery," their lack of standardization, and the method of administration. Despite the problems, skin testing remains an easy, relatively safe method of assessing the immune system. Some investigators are developing more reliable testing methods and alternative antigens. Further research is needed to develop a reliable method for assessing delayed cutaneous hypersensitivity.

Antibodies against Aspergillus fumigatus. I. Standardization of the antigenic composition.

Three different stages were found during growth of Aspergillus fumigatus as characterized by the changes in pH of the medium: (1) An acidic phase (phase I); (2) a second phase (phase II) during which the pH rises till values around pH = 8.5; (3) a third phase (phase III) during which the pH remains constant or drops slightly. During phase I a fast appearance of certain antigenic components was found that is ascribed to an active process of excretion. Additional antigenic components appeared in the culture medium after lysis of the microorganisms (phases II and III). Lysis of the microorganisms and appearance of antigenic components are dependent on the glucose concentration of the medium.

Experimental murine candidiasis: cell-mediated immunity after cutaneous challenge.

Attempts were made to isolate an antigen(s) from Candida albicans suitable for detecting hypersensitivity in a murine model of candidiasis. Using footpad reactivity in cutaneously infected animals as the assay, comparisons were made of two commercial extracts and cell wall and cytoplasmic preparations made in the laboratory. An extract of the cell wall, a glycoprotein (GP) removed with ethylenediamine, and an extract prepared from the membrane fraction of disrupted C. albicans blastospores proved most useful in demonstrating delayed hypersensitivity in the murine model. The activity of the GP fraction was considerably reduced by oxidation with periodate and was abrogated entirely by digestion with proteolytic enzymes. The extract from the membrane fraction was obtained by incubating the insoluble membrane fraction with phosphate-buffered saline, pH 7.4, at 50 degrees C, and the proteins in the extract were subsequently precipitated with ammonium sulfate to yield a test preparation that was approximately 75% protein and 25% carbohydrate. The precipitated extract was designated ppt-HEX. Footpad reactivity to ppt-HEX could be transferred with cells and not with serum if the cells were taken from animals at the appropriate time after sensitization. Since the membrane and GP fractions appear to elicit true delayed hypersensitivity reactions, further investigations into their specificity and biochemistry seem warranted.

Blastomycosis: specificity of antigens reflecting the mating types of Ajellomyces dermatitidis.

In a study of sera from patients with proven or suspected blastomycosis, positive immunodiffusion tests were obtained in all active cases when fresh sera were tested with a cell sap (CS) antigen. False negatives occurred on occasion when an ethanol precipitate (EPF) antigen was used alone. No false positives were found. The CS antigen from the (+) mating type had in common two lines of identity with the (CS) antigen of the (-) type. In addition, other lines were present when the patient was infected with the same mating type as was used for the preparation of the antigen. No differences in the electrophoretic patterns of the enzymes leucine amino peptidase or phosphatase were noted when preparations from the two mating types were compared. However, a distinct pattern was noted when the alpha esterases of the (+) and (-) mating types were examined. Specific alpha esterase antibodies were present in patients' sera.