Evaluation of a rapid enzyme-linked immunosorbent assay for IgG antibodies to Aspergillus fumigatus in the serological diagnosis of allergic aspergillosis

A range of 6 somatic and culture filtrate antigens of Aspergillus fumigatus were evaluated in a rapid ELISA procedure for anti-A. fumigatus IgG where the component incubation times had been reduced to 10 min. Sera from patients with allergic aspergillosis, patients with suspected allergic aspergillosis, and asthmatic patients with or without A. fumigatus precipitins were tested. For all antigens, levels of anti-A. fumigatus IgG were higher in patients with allergic aspergillosis than in the other 3 groups. Low levels of specific IgG were, however, detected in asthmatic patients who had no precipitins against A. fumigatus. None of the antigen preparations enabled all patients with proven or suspected allergic aspergillosis to be separated from the other 2 groups of asthmatic patients. Positive-negative discrimination in ELISA was achieved by the inclusion of 10 pools of precipitin test-negative sera from the 50 asthmatics without A. fumigatus precipitins. The number of sera that were classed as positive in ELISA ranged from 9 to 15 in the allergic aspergillosis group, depending on the antigen used; in the suspected aspergillosis group, the number of positive reactions ranged from 1 to 8, while in the asthmatics with precipitins, the number ranged from 0 to 2.     

Serological tests in the diagnosis of pulmonary aspergillosis

Sera from 44 patients with clinically suspected pulmonary aspergillosis (mainly of an allergic type) were examined for antibodies to Aspergillus fumigatus using an enzyme-linked immunosorbent assay (ELISA) and counter-immunoelectrophoresis (CIE). Of the sera, 15 were considered to contain Aspergillus antibodies using ELISA; 11 of these also contained precipitins by CIE. In no instance was a CIE-positive serum negative by ELISA. The serum of 1 patient with autopsy-verified invasive pulmonary aspergillosis was negative in both tests. Protein-enriched antigens derived by ammonium sulphate precipitation of crude hyphal homogenates seemed of most use in ELISAs. In addition, 4 of 8 sera obtained from patients with possibly invasive candidosis also revealed significant levels of Aspergillus antibody by ELISA (but not CIE). All of the 8 sera contained readily detectable Candida precipitins by CIE. Problems of potential cross-reactivity obviously need careful consideration with ELISAs. Our results suggest that ELISA procedures under appropriately controlled conditions are more sensitive than CIE for detecting Aspergillus antibodies. However it seems that some patients with invasive aspergillosis will be antibody-negative even with sensitive tests such as ELISA.     

Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillus fumigatus antibodies.

A rapid enzyme-linked immunosorbent assay (ELISA) where component incubation periods were shortened to one hour, was compared with agar gel double diffusion (AGDD) and a standard ELISA procedure for detecting antibodies to Aspergillus fumigatus in 28 asthmatic patients with suspected allergic aspergillosis. Using two A fumigatus antigens the rapid ELISA compared well with AGDD and the standard ELISA method. Eleven sera that reacted with both antigens in AGDD were all positive against antigen 1 in both forms of ELISA, but two failed to react with antigen 2 in the standard ELISA and three failed to react with this antigen in the rapid method. Thirteen AGDD-negative sera were also negative in both forms of ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of allergic aspergillosis.     

Evaluation of a recombinant antigen-based enzyme immunoassay for the diagnosis of noninvasive aspergillosis

Antibody detection is a key diagnostic tool for noninvasive aspergillosis (NIA) such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. Specific immunoprecipitin detection (IPD) is considered as the reference but lacks standardization and is time-consuming. To evaluate the performance of a new anti-Aspergillus fumigatus IgG enzyme immunoassay (EIA) kit using a recombinant A. fumigatus antigen (Bio-Rad), a retrospective study was performed on 551 sera collected from patients with a definite diagnosis of NIA (group 1; n = 64), bronchial Aspergillus colonization (group 2; n = 26), and probable aerial Aspergillus contamination (group 3; n = 44); from patients suspected of NIA with negative serological and mycological investigations (group 4; n = 49); and from a group of 222 patients not suspected of NIA (group 5). The EIA exhibited excellent reproducibility with coefficients of variation below 10%. Agreement with IPD was calculated between 62.5 and 84.4% according to the group of patients with Cohen's kappa coefficient at 0.6196 ± 0.077. Taking as reference a composite status including clinical, radiological, mycological, and serological data, sensitivity (group 1) and specificity (other groups) were calculated between 90.2 and 93.8% and 54.3 and 100%, respectively. Lower specificity was observed for patients with Aspergillus colonization. However, Yule Q coefficients estimating the correlation between EIA result and the definite diagnosis of NIA were calculated between 0.97 and 0.98. The method is a highly useful screening tool for the diagnosis of NIA, reducing the need for confirmatory IPD tests.     

Clinical and serological survey of pulmonary aspergillosis in patients with cystic fibrosis

A 5.5-year survey revealed 9 patients with allergic bronchopulmonary aspergillosis (ABPA) and 1 patient with a highly probable diagnosis among 200 Danish patients with cystic fibrosis (CF). The incidence of ABPA was 0.9 (0.4-1.7, 95% confidence limits) per 100 patients per year. All the patients with definite or probable ABPA had serum antibodies to the Aspergillus fumigatus catalase antigen, and in 5 patients the appearance of catalase antibodies during the incipient phase of ABPA was documented. 29 patients without ABPA also had catalase antibodies, but in lower levels than ABPA patients (p = 0.006). The 6-month prevalence rate of A. fumigatus in sputum was 80 and 72% in the 2 groups, respectively. The finding of catalase antibodies in some CF patients without ABPA may indicate the occurrence of a symptom-poor form of pulmonary aspergillosis.     

一种快捷、定量检测烟曲霉GM抗原双mAb夹心ELISA的方法

目的 :建立一种快速诊断烟曲霉病的双mAb夹心ELISA法。方法 :应用 4株抗烟曲霉GM单克隆抗体 (mAb) ,分别包被和制备HRP mAb ,用双mAb夹心ELISA法配对试验 ,选择捕获及检测mAb。结果 :经筛选得到捕获及检测mAb的最佳组合 ,并建立了双mAb夹心ELISA法。该法检测GM抗原的灵敏度为 0 .1μg/L ,测出范围在 0 .1～ 10 μg/L之间。连续 6d用ELISA法检测同一份样品 ,所获CV的平均值为 ( 7.2± 3.8) %。结论 :建立了一种可快速、定量检测烟曲霉GM抗原的双mAb夹心ELISA法 ,灵敏度高、重复性好 ,对研制试剂盒应用于烟曲霉病早期诊断和防治 ,具有重要的临床应用价值     

Detection of cell wall galactomannoprotein Afmp1p in culture supernatants of Aspergillus fumigatus and in sera of aspergillosis patients

Mannoproteins are important and abundant structural components of fungal cell walls. The AFMP1 gene encodes a cell wall galactomannoprotein of Aspergillus fumigatus. In the present study, we show that Afmp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Afmp1p was capable of detecting this protein from the cell culture supernatant of A. fumigatus. The anti-Afmp1p antibody is specific since it fails to react with any protein from lysates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium marneffei, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum by Western blotting. In addition, this Afmp1p antigen-based ELISA is also specific for A. fumigatus since the cell culture supernatants of the other eight fungi gave negative results. Finally, a clinical evaluation of sera from invasive aspergillosis patients indicates that 8 of 15 (53%) patients are Afmp1p antigen test positive. Furthermore, an Afmp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for invasive aspergillosis carry a sensitivity of 86.7% (13 of 15). The specificities of the tests are high since none of the 138 control sera, including 100 from normal blood donors, 20 from patients with penicilliosis marneffei, 6 from patients with candidemia, 8 from patients with typhoid fever, and 4 from patients with melioidosis, was positive by either test. In conclusion, the combined Afmp1p antibody and antigen tests are highly sensitive and specific for A. fumigatus invasive aspergillosis.     

Prevalence of aspergillosis in bronchogenic carcinoma

Bronchoalveolar lavage of 42 patients of bronchogenic carcinoma was studied to find out the prevalence of aspergillosis. Sera of the patients were also analysed for presence of anti-Aspergillus antibodies by Immunodiffusion (ID), Enzyme linked immunosorbent assay (ELISA) and dot blot assay (DBA). Aspergillus was isolated in culture from 6 (14.2%) patients of bronchogenic carcinoma. Aspergillus fumigatus was the predominant species isolated. All the strains of Aspergillus were sensitive to itraconazole, ketoconazole and amphotericin B while resistance (33.3%) was found with fluconazole. Anti-aspergillus antibodies were detected equally by ID, ELISA and DBA in 9 (21.4%) cases. The present study revealed prevalence and seroprevalance of Aspergillus in bronchogenic carcinoma to be 14.2% and 21.4% respectively. Consistent reactivity against 18 kDa Aspergillus fumigatus antigen was noted in serologically positive cases. Antibodies against 18 kDa protein antigen in western blotting may be used as a reference marker for diagnosis of aspergillosis in bronchogenic carcinoma. It is also suggested that the simplest serological technique like ID may be performed along with culture for diagnosing Aspergillosis in patients of bronchogenic carcinoma since ID, ELISA and DBA showed similar sensitivity.     

Fractionation of Aspergillus fumigatus antigens by hydrophobic interaction chromatography and gel filtration

Aspergillus fumigatus somatic antigen prepared from young hyphae was fractionated by hydrophobic interaction chromatography followed by gel filtration. The antigenic activity of fractions was investigated by enzyme-linked immunosorbent assay, crossed immunoelectrophoresis, and immunodiffusion. By enzyme-linked immunosorbent assay, high levels of IgG antibodies were detected against an antigen fraction of approximate molecular weight 470,000 both in patients with aspergillosis (n = 3) and in healthy controls (n = 3). High levels of antibodies to four fractions of MW 250,000 160,000, 160,000-50,000, and 50,000-25,000 were found only in patients. The MW 250,000 fraction exhibited catalase activity, and the MW 50,000-25,000 fraction had neutral protease activity. These purified aspergillus antigens may be useful in serodiagnosis of aspergillosis.     

Aspergillus fumigatus: identification of 16, 18, and 45 kd antigens recognized by human IgG and IgE antibodies and murine monoclonal antibodies.

The immunochemical properties of antigens produced by Aspergillus fumigatus were investigated with biochemical purification techniques in conjunction with the production of murine monoclonal antibodies (MAbs) and binding studies with human IgG and IgE antibodies. A. fumigatus antigens were partially purified by gel filtration and hydrophobic interaction chromatography on phenyl-Sepharose. Two fractions that eluted with either 2 mol/L or 0.15 mol/L of NaCl demonstrated strong binding to human IgG and IgE antibodies. Immunoprecipitation analysis with IgG antibodies from six patients with different Aspergillus-related diseases demonstrated that the 2M and 0.15M fractions contained major antigens of molecular weight 18 kd (Asp f I) and 45 kd, respectively. The 125I-labeled 2M fraction was used to compare IgG antibodies to A. fumigatus in sera from 25 patients with Aspergillus-related diseases. IgG antibodies were significantly higher in patients with allergic bronchopulmonary aspergillosis (geometric mean, 437 U/ml) than in patients with asthma (geometric mean, 14 U/ml; p less than 0.001), but undetectable (less than 5 U/ml) in 43/48 control subjects. A good correlation was found between levels of IgG antibodies to the 125I-labeled 0.15M fraction and the 125I-labeled 2M fraction in sera from 106 patients with cystic fibrosis (r = 0.77; p less than 0.001). Five murine IgG MAbs and two IgM MAbs were raised against the 2M fraction, and immunoprecipitation with the IgG MAb demonstrated two distinct antigens within the 2M fraction, Asp f I, and a 16 kd antigen. The results of a solid-phase RIA with IgG MAb 4A6 demonstrated that approximately 85% of A. fumigatus-allergic patients with allergic bonchopulmonary aspergillosis had IgE antibodies to Asp f I. The three protein antigens defined in these studies are useful probes for investigating the immunopathogenesis of diseases associated with colonization by A. fumigatus.     

Cytophilic antibodies in bronchopulmonary aspergilloma and cryptogenic pulmonary eosinophilia.

The immunoglobulin class and subclass of cytophilic antibodies have been studied using peripheral leucocytes from twenty-two patients with allergic bronchopulmonary aspergillosis, aspergilloma and cryptogenic pulmonary eosinophilia. In patients with allergic bronchopulmonary aspergillosis, significantly increased histamine liberation occurred following challenge of their leucocytes with antisera to IgE, IgG2, IgG3 and IgG4 as well as with Aspergillus fumigatus antigen. The results were considerably modified if the patient was receiving corticosteroids at the time of the test. The presence of IgG2-specific antibody to A. fumigatus in the serum of one patient, capable of sensitizing donor leucocytes, was demonstrated in passive sensitization experiments. In two patients with uncomplicated aspergillomas no evidence of cytophilic antibody to any class was found although large amounts of precipitating IgG antibody was present in the serum. Two patients with aspergilloma and systemic symptoms of weight loss and fatigue (which have been interpreted by others as 'hypersensitivity' responses) had increased amounts of cytophilic antibody similar to those with allergic bronchopulmonary aspergillosis. Six patients with cryptogenic pulmonary eosinophilia were also studied. No evidence of specific antibody to A. fumigatus was found but, as a group, significantly increased histamine liberation using antisera to IgG2 was demonstrated. Individual patients also showed evidence of other classes of cytophilic antibody, one having IgE, three IgG3 and two IgG4. The relationship between heat-stable short-term sensitizing antibody (IgG STS) inducing immediate skin responses and the pattern of cytophilic antibodies found in our patients with bronchopulmonary aspergillosis having dual (immediate and late reactions) is discussed. Clinically these tests are of diagnostic value and they may be helpful in assessing symptomatic patients with aspergillomas for corticosteroid treatment.     

Comparison of antibody measurements against Aspergillus fumigatus by means of double-diffusion and enzyme-linked immunosorbent assay (ELISA)

IgG antibody titers against Aspergillus fumigatus from different sera were measured by means of a standardized ELISA and compared with precipitates measured by double diffusion (D.D.). There was a significant correlation between the number of precipitates and the ELISA IgG titers in the 758 sera examined. However, in several individual sera within this group, large deviations between these two immunologic parameters were found. Further analysis indicated that ELISA detects antibodies against nonprecipitating antigenic components in addition to the antibodies detected by D.D. Furthermore, not all precipitating antigenic components appear to play a role in the detection of antibodies against A. fumigatus by ELISA. Patients with aspergillosis largely show increased titers of IgG antibody by ELISA even when the results of D.D. are negative, except those of patients with Aspergillus-provoked asthma which fall within a normal range.     

Immunoblot analysis of serological responses in invasive aspergillosis.

The serological response to Aspergillus fumigatus was investigated in patients with pulmonary aspergillosis using the immunoblot technique. Antibody was detected against nine components of the fungus, ranging in molecular weight from 88 000 to 33 000 daltons. Antibody to a 40 000 dalton component was present in most patients (13 of 16). In surviving patients it was high (two with aspergilloma) or low but persistent (two with invasive aspergillosis). It was absent or fell in the 12 patients who died of invasive aspergillosis. Antibody to this 40 000 dalton antigen was not present in 10 sera from healthy controls. This antigen may form the basis of a more sensitive and specific serodiagnostic test for systemic aspergillosis.     

Class-specific antibody determination against Aspergillus fumigatus by means of the enzyme-linked immunosorbent assay. III. Comparative study: IgG, IgA, IgM ELISA titers, precipitating antibodies and IgE binding after fractionation of the antigen

IgG, IgA and IgM ELISA antibody titers against Aspergillus fumigatus were elevated in sera of patients with aspergilloma and allergic bronchopulmonary aspergillosis (ABPA), showing higher titers for the IgG antibodies compared with the IgA and IgM antibodies. No differences were found between titers of identical antibody classes in the two groups of sera. IgG and IgA ELISA titers were highly specific whereas IgM ELISA showed more unspecific binding of IgM antibodies. Antibodies, as measured by ELISA, studied after fractionation of the antigen into fractions of decreasing molecular weight, showed a preferential binding by the high molecular weight fractions. Precipitating antibodies studied in patient sera did not always correspond with the IgG ELISA titers. IgE antibody binding was observed in all fractions from Sephadex G-100 fractionated components; maximum binding was found with fractions of 28,000-60,000 daltons. The low molecular weight fractions (18,000-less than 5,000 daltons) showed less IgE binding but the quantity of this fraction was higher. The discrepancies noted between the IgG and IgA ELISA titers and the binding of IgM or IgE antibodies indicate that antigenic components may in part differ in the binding of antibody classes.     

Identification and partial characterization of diagnostically relevant antigens of Aspergillus fumigatus

Metabolic antigens of Aspergillus fumigatus, soil strain 2605 and sputum isolate, were evaluated for their diagnostic applicability using hyperimmune sera and sera of adults and pediatric patients of allergic bronchopulmonary aspergillosis. An indirect ELISA was standardised by using 2-10 micrograms/ml of coating antigen for detection of specific IgG and IgE antibodies in the sera of patients. The ratios of absorbance for specific IgE and IgG antibodies by ELISA (normal to patients) were observed to be in the range of 1:2 to 1:3 to 1:8 respectively. These antigenic preparations were further analyzed to identify and characterize the individual components by immunoblotting. This analysis indicated the presence of allergenic and antigenic determinants in the antigens of molecular weights 70, 34, and 28 Kd. The utility of the antigens of soil strain for diagnostic purpose is suggested.     

A partially purified glycoprotein antigen from Aspergillus fumigatus

Culture filtrate antigens of Aspergillus fumigatus were fractionated by isoelectric focusing using a pH gradient of 4-6.5. Three fractions, namely, 18, 19 and 20 were pooled and subjected to immunochemical analysis. It contained a concanavalin A binding glycoprotein. Antibody raised against this component was used to prepare an affinity column of IgG-Sepharose and was used to purify crude culture filtrate antigens. This component produced three precipitin arcs in crossed immunoelectrophoresis using anti-A. fumigatus rabbit serum. This fraction has an isoelectric point of 6.5 and showed three components in two-dimensional electrophoresis with approximate molecular weights of 20, 40 and 80 kilo daltons. This antigen reacted with patient sera in the biotin-avidin linked immunosorbent assay and showed high levels of anti-A. fumigatus IgG and IgE antibodies in allergic bronchopulmonary aspergillosis and IgG antibodies in aspergilloma. Both controls and Aspergillus skin test positive asthmatics showed only low levels of specific antibodies. Because of the purity of this antigen, its potential use as a standardized antigen in the detection of antibody is discussed.     

Allergic bronchopulmonary aspergillosis does exist in Brazil

A case of allergic bronchopulmonary aspergillosis is presented. The patient had corticosteroid-dependent asthma, recurrent pulmonary infiltrates clearing with oral prednisone bursts, positive dual (immediate and late) skin test (prick and intradermal) reactivity to Aspergillus fumigatus, 11% blood eosinophilia, elevated total serum IgE, positive precipitating antibody against Aspergillus fumigatus, and elevated specific serum antibodies to Aspergillus fumigatus (positive IgE and IgG antibody indices, and elevated IgE levels by both RAST and FAST). To our knowledge this is the first immunologically documented case of allergic bronchopulmonary aspergillosis in Brazil. Future survey studies are required.     

Clinical analysis of chronic pulmonary aspergillosis and discovery of an elastase inhibitor]

We studied the clinical features of 59 chronic pulmonary aspergillosis cases (aspergilloma, chronic necrotizing pulmonary aspergillosis) which we experienced in our hospital. To diagnose this disease, X-rays, sputum culture and serologic tests were mainly examined, X-ray findings were a fungus ball type in 47% of cases and thickened wall of a cavity type in 32%. Positive sputum culture found was A. fumigatus 78%, A. niger 13% and A. flavus 2%. Positive rates of serologic tests showed precipitating antibody 81% and antigen 11%; 39% of beta-D glucan exceeded the reference value. As clinical symptoms, bloody sputum and hemoptysis were found at high frequency. Antifungal agents were administered intravenously or topically for treatment, primarily AMPH-B, ITCZ and MCFG. As adjuvant therapy, we administered Ulinastatin which is an elastase inhibitor for use against hemoptysis, and we performed steroid combination for cases considered to be associated with allergy. In all of 6 cases of chronic necrotizing pulmonary aspergillosis which were administered MCFG, X-ray findings improved. A pathogenic factor, elastase was isolated from Aspergillus spp., and we also found the elastase inhibitor from this series. Five of 12 strains of A. fumigatus, and one of 2 strains of A. flavus expressed elastase inhibitory activity when we screened for the culture supernatant of various Aspergillus spp. of a clinical isolate. Elastase inhibitory activity from A. niger was very weak. Culture supernatants from 5 strains of A. fumigatus and one strain of A. flavus were stable for a fever, and human leucocyte elastase was inhibited, but these did not inhibit porcine pancreas elastase. We are aiming at clinical application and plan to continue further study.     

Aspergillus fumigatus: Identification of 16, 18, and 45 kd antigens recognized by human IgG and IgE antibodies and murine monoclonal antibodies

The immunochemical properties of antigens produced by Aspergillus fumigatus were investigated with biochemical purification techniques in conjunction with the production of murine monoclonal antibodies (MAbs) and binding studies with human IgG and IgE antibodies. A. fumigatus antigens were partially purified by gel filtration and hydrophobic interaction chromatography on phenyl-Sepharose. Two fractions that eluted with either 2 mol/L or 0.15 mol/L of NaCl demonstrated strong binding to human IgG and IgE antibodies. Immunoprecipitation analysis with IgG antibodies from six patients with different Aspergillus-related diseases demonstrated that the 2M and 0.15M fractions contained major antigens of molecular weight 18 kd (Asp f I) and 45 kd, respectively. The ¹²⁵I-labeled 2M fraction was used to compare IgG antibodies to A. fumigatus in sera from 25 patients with Aspergillus-related diseases. IgG antibodies were significantly higher in patients with allergic bronchopulmonary aspergillosis (geometric mean, 437 U/ml) than in patients with asthma (geometric mean, 14 U/ml; p < 0.001), but undetectable (<5 U/ml) in 43148 control subjects. A good correlation was found between levels of IgG antibodies to the ¹²⁵I-labeled 0.15M fraction and the ¹²⁵I-labeled 2M fraction in sera from 106 patients with cystic fibrosis (r = 0.77; p < 0.001). Five murine IgG MAbs and two IgM MAbs were raised against the 2M fraction, and immunoprecipitation with the IgG MAb demonstrated two distinct antigens within the 2M fraction, Asp f I, and a 16 kd antigen. The results of a solid phase RIA with IgG MAb 4A6 demonstrated that ≈85% of A. fumigatus-allergic patients with allergic bonchopulmonary aspergillosis had IgE antibodies to Asp f 1. The three protein antigens defined in these studies are useful probes for investigating the immunopathogenesis of diseases associated with colonization by A. fumigatus.     

Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.