Antibody response to low-molecular-weight antigens of Aspergillus fumigatus in allergic bronchopulmonary aspergillosis.

Sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma and normal sera were analyzed for specific antibodies by Western (immuno-) blotting with Aspergillus fumigatus antigens transferred electrophoretically onto polyvinylidene difluoride membranes. Western blot analysis demonstrated consistent reactivity of low-molecular-weight A. fumigatus antigens against ABPA sera but not against uncomplicated aspergilloma or normal sera. None of these low-molecular-weight components had any lectin-binding activity. Sera from patients with aspergilloma, however, frequently reacted with high-molecular-weight components of A. fumigatus. The majority of these high-molecular-weight antigenic components demonstrated concanavalin A-binding activity. The low-molecular-weight bands were discernible in Western blots with sera from all ABPA patients irrespective of disease activities, such as relapse, flare, or treatment. Antibodies detected by methods such as immunodiffusion or enzyme-linked immunosorbent assays demonstrated total antibody responses to most or all antigenic components, while Western blots demonstrated the reactivities of the individual components with the specific antibodies. Western blot analysis thus provided more information for immunodiagnosis of ABPA than other methods, especially when only crude antigens were available.     

Serum antibodies and their role in antibody-dependent cell-mediated cytotoxicity in aspergillosis

A total of 22 sera from patients with aspergilloma and allergic bronchopulmonary aspergillosis (ABPA) were examined concomitantly for specific antibody against Aspergillus fumigatus antigen and for their activity in antibody-dependent cell-mediated cytotoxicity (ADCC) against Aspergillus antigen-coated target cells. These sera demonstrated significant precipitin bands in agar gel double diffusion test (78% of ABPA and 75% of aspergilloma sera), while in indirect immunofluorescence studies all sera showed positive reactivity with a titer distribution of 1:40 to 1:160 and 1:40 to 1:320, respectively, for ABPA and aspergilloma sera. In enzyme-linked immunosorbent assay all sera demonstrated titers varying from 1:200 to 1:6400. Several sera also displayed marked cytotoxic reactions against A. fumigatus antigen-coated SB target cells in ADCC assays using normal lymphocytes as effector cells (35% of aspergilloma and 25% of ABPA sera). These findings suggest a role for ADCC activity in patients with Aspergillus infections.     

The 18-kilodalton antigen secreted by Aspergillus fumigatus.

One of the major antigens secreted in vitro by Aspergillus fumigatus is an 18-kDa basic protein which has been purified by cation-exchange chromatography. It is recognized by sera from aspergilloma patients. It is also the major circulating antigen found in urine of patients with invasive aspergillosis. Our results indicated that this antigen has potential for the diagnosis of both aspergilloma and invasive aspergillosis.     

Isolation and characterization of a relevant Aspergillus fumigatus antigen with IgG- and IgE-binding activity

An immunologically relevant antigen fraction was isolated from a cytoplasmic extract of Aspergillus fumigatus mycelium. The fraction was obtained by concanavalin-A affinity chromatography and hydrophobic interaction chromatography. Two protein bands were discernible in polyacrylamide gel electrophoresis while two precipitin peaks were detected in crossed immunoelectrophoresis. When tested against sera from patients with Aspergillus-induced diseases, the fraction showed both IgG and IgE antibody-binding activity. All of the sera studied from patients with allergic bronchopulmonary aspergillosis (ABPA) showed high IgG and IgE antibody titers, while sera from patients with aspergilloma and cystic fibrosis with ABPA demonstrated high IgG titers and only a moderate increase in IgE titers. None of the other groups showed any significant antibody titers against this antigen in their sera. Because of its binding to both IgG and IgE antibodies this fraction was found to be useful in the immunodiagnosis of Aspergillus-induced diseases.     

Specific antibody detection in invasive aspergillosis by analytical isoelectrofocusing and immunoblotting methods.

Aspergillus fumigatus antigens have been tested to determine their potential as aids in the diagnosis of invasive aspergillosis (IA). Immunoglobulin G (IgG) antibodies to these antigens were detected by analytical isoelectrofocusing in conjunction with immunoblotting. A total of 12 antigenic fractions, including culture filtrates and surface and mycelial extracts of A. fumigatus, were investigated. Eleven were reactive with serum specimens from patients with aspergilloma, which served as positive controls for the evaluation of a specific IgG response. Eight of 12 antigens showed good responses with serum specimens from patients with allergic bronchopulmonary aspergillosis, which were used to assess the sensitivity of IgG detection. No measurable reactivity was detected in 18 negative control serum specimens, while 11 of 13 patients with proven, highly probable, or probable cases of IA had anti-Aspergillus IgG to multiple antigenic preparations. Patients with IA who were capable of mounting a substantial humoral response to Aspergillus antigens gave an antibody profile with five antigenic preparations which seemed to be characteristic of the disease. Data show that this method is highly sensitive and may allow the selection of fractions which are both highly antigenic and specific for the detection of antibodies to Aspergillus antigens. They also indicate that the use of a spectrum of antigenic molecules is advisable, given the variability observed in the immune responses of individual patients.     

AFLMP1 encodes an antigenic cel wall protein in Aspergillus flavus

We have previously reported the cloning and characterization of the MP1 gene in Penicillium marneffei and the AFMP1 gene in Aspergillus fumigatus and their use for serodiagnosis of penicilliosis and aspergilloma and invasive aspergillosis, respectively. In this study, we describe the cloning of the AFLMP1 gene, which encodes the homologous antigenic cell wall protein in Aspergillus flavus, the most common Aspergillus species associated with human disease in our locality and in other Asian countries and the second most common Aspergillus species associated with human disease in Western countries. AFLMP1 codes for a protein, Aflmp1p, of 273 amino acid residues, with a few sequence features that are present in Mp1p and Afmp1p, the homologous antigenic cell wall proteins in P. marneffei and A. fumigatus, respectively, as well as several other cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Aflmp1p antibody was generated with recombinant Aflmp1p protein purified from Escherichia coli to allow further characterization of Aflmp1p. Indirect immunofluorescence analysis indicated that Aflmp1p is present on the surface of the hyphae of A. flavus. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. flavus develop a specific antibody response against Aflmp1p. This suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with aspergilloma or invasive aspergillosis, and the protein may represent a good cell surface target for host humoral immunity.     

Partial characterization of a rapidly released antigenic/allergenic component (Ag 5) of Aspergillus fumigatus

An antigenic component of Aspergillus fumigatus, which was responsible for inducing an initial early antibody response in rabbits immunized with A. fumigatus germlings, has been identified and partially characterized. By immunofluorescent antibody and ELISA techniques, this antigen was demonstrated to be present on the germling surface, although it was also detected in supernatants of conidia/germlings within 1 hour and was present in all shake and stationary culture-filtrate extracts. By incorporation of a monospecific antiserum to this component in an intermediate gel of the reference self-crossed radioimmunoelectrophoresis pattern for allergic bronchopulmonary aspergillosis sera, it was recognized as Ag 5 and was also demonstrated to bind specific IgE. Further immunochemical analyses have revealed that Ag 5 is relatively heat labile, does not bind to concanavalin A, and has a molecular weight of approximately 35 kd. This rapidly released antigenic/allergenic component may play an important role in the initiation of immunologic responses in patients with allergic bronchopulmonary aspergillosis.     

Immunochemical characterization of Aspergillus fumigatus antigens.

Culture filtrate antigens of Aspergillus fumigatus Ag 534 were purified by preparative isoelectric focusing and affinity chromatography. One of the pooled antigen fractions from the preparative isoelectric focusing step (pool 2) was passed through a concanavalin A column and yielded two components, designated antigens IIa and IIb. Antigen IIb reacted more strongly than antigen IIa with all of the aspergilloma and allergic bronchopulmonary aspergillosis sera tested by enzyme-linked immunosorbent assay. The glycoprotein nature of antigen IIb was shown by the concanavalin A binding properties and staining reactions of the components.     

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.     

Characterization of a monoclonal antibody against concanavalin A binding antigen of Aspergillus fumigatus

A monoclonal antibody was produced against a Concanavalin A (Con A) binding major epitope of Aspergillus fumigatus using a novel method of immunization. The antigen was purified using monoclonal antibody affinity chromatography reacted with specific antibodies present in human sera. Both allergic bronchopulmonary aspergillosis and aspergilloma showed high levels of antibody, against this purified antigen, when compared to normal controls. Similar results were obtained when the monoclonal antibody was used in a capture antigen assay. The antibody reacted with several A. fumigatus extracts in rocket electrophoresis demonstrating a single precipitin arc, which disappeared when Con A intermediate gel was used. This monoclonal antibody demonstrated reactivity only with cytoplasmic components of hyphae and spores of A. fumigatus, when a colloidal gold was used as a probe in immunoelectron microscopy.     

Preparation of Aspergillus fumigatus antigens and their analysis by two-dimensional immunoelectrophoresis

The water-soluble components of Aspergillus fumigatus mycelium were partially separated by fractional precipitation with ammonium sulphate. The total protein and neutral-sugar content were determined for each of the four fractions prepared and their immunological activity was examined by double diffusion. Partial chemical characterisation of these isolates by polyacrylamide-gel electrophoresis was linked to their precipitability by allowing the separated components to diffuse from the gel into an agarose medium containing an appropriate antiserum. The distribution and reactivity of antigens was monitored by two-dimensional immunoelectrophoresis with rabbit sera raised against either mycelial or culture-filtrate antigens and human sera, obtained from patients with aspergilloma and from patients with allergic bronchopulmonary aspergillosis. This technique was also used to establish that considerable variation exists in the precipitation profile seen among patient specimens. Several antigens were found to possess sugar residues that interacted with concanavalin A, when this lectin was used in an intermediate gel in two-dimensional immunoelectrophoresis.     

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.     

A case report of pulmonary infiltration with eosinophilia syndrome induced by Candida albicans]

A sixty six-year-old female who had been treated for bronchial asthma for about 25 years was admitted to the hospital with complaints of episodes of dyspnea, eosinophilia and infiltrative shadows in the chest X-ray film. An infiltrative shadow appeared to move from the left to the right lung field and finally formed a shadow of atelectasis in the middle field of the right lung. A sputum culture showed only Candida albicans. Allergic and immunologic examination revealed high IgE serum levels with specific IgE against Candida albicans in high titer, and Aspergillus fumigatus in low titer. The precipitating antibody was shown only against Candida antigen. Additionally, the blastogenic response to Candida antigen was high in comparison with other fungal antigens including Aspergillus fumigatus. The clinical features and laboratory findings of this patient were found to satisfy Rosenberg's criteria for allergic bronchopulmonary aspergillosis (ABPA), except for the existence of Candida albicans in place of Aspergillus species as the causative antigen. The pathogenesis of PIE syndrome has been studied and various allergic mechanisms against many antigens reported. In this patient Candida albicans could be playing the crucial role in the formation of PIE syndrome, which might be best described as allergic bronchopulmonary candidiasis (ABPC).     

Diagnostic specificity of a sandwich ELISA for Aspergillus-related diseases

A sandwich ELISA with specificity for a major antigen (Ag 7) of Aspergillus fumigatus has been compared with indirect ELISAs with use of crude antigenic (culture filtrate) extracts and found to have a sensitivity of 100% and specificity of 92.3% for antibody detection in sera of patients with allergic bronchopulmonary aspergillosis (ABPA) and aspergilloma. In the Ag 7 ELISA, all sera from groups with ABPA (21) and aspergilloma (15) had positive titers, and mean values for both these groups were significantly higher (p less than 0.001) than control levels. By comparison, in the indirect ELISAs, most sera of patients with ABPA were positive; the sensitivity was 81% to 90%, and the specificity was 87%. Sera from other disease groups including sera from 13 patients with farmer's lung, 16 with tuberculosis, and 10 individuals with positive prick test to either A. fumigatus or Alternaria alternata were also tested by both types of assay. Up to five of these sera elicited significant positive results in the indirect ELISA, and although two sera were also positive in the Ag 7-specific ELISA, these latter titers were both below the range of values for the sera from patients with ABPA. With the use of an IgG calibration curve, the sensitivity of the assay was determined as within the range of 1 to 10 microgram of specific IgG antibody per milliliter. The Ag 7-specific ELISA is therefore a highly specific, sensitive assay for antibody detection in Aspergillus-related diseases.     

A polymerase chain reaction enzyme immunoassay for diagnosing infection caused by Aspergillus fumigatus

AIM: To develop a polymerase chain reaction enzyme immunoassay (PCR-EIA) to measure levels of circulating aspergillus DNA in invasive aspergillosis caused by Aspergillus fumigatus. METHODS: The PCR reaction was based on primers from the 18s rRNA gene. Binding of the product to a streptavidin coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR and this was the tag to which antibody was bound in the subsequent EIA. RESULTS: The optical density (OD) endpoint was < 0.1 in 10 sera from neutropenic patients with no evidence of invasive aspergillosis, and in 10 sera from nonneutropenic patients with bacterial pneumonia (group 1). The OD from five of 12 patients with allergic bronchopulmonary aspergillosis (ABPA) (group 2), three with an aspergilloma (group 3), and five with possible invasive aspergillosis (group 4) was > or = 0.1. In 63 sera from 33 cases of proven invasive aspergillosis (group 5) an OD > or = 0.1 was achieved in 48 sera from 30 patients. The maximum OD was 0.510. The level fell in survivors and gradually rose in fatal cases. CONCLUSIONS: This assay validated the concept of diagnosing invasive aspergillosis by measuring levels of circulating fungal DNA in serum.     

Characterization of AFMP1: a Novel Target for Serodiagnosis of Aspergillosis

We cloned the AFMP1 gene, which encodes the first antigenic cell wall galactomannoprotein in Aspergillus fumigatus. AFMP1 codes for a protein, Afmp1p, of 284 amino acid residues, with a few sequence features that are present in Mp1p, the antigenic cell wall mannoprotein in Penicillium marneffei that we described previously, as well as several other cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidyl inositol attachment signal sequence. Specific anti-Afmp1p antibody was generated with recombinant Afmp1p protein purified from Escherichia coli to allow further characterization of Afmp1p. Afmp1p has a high affinity for Galanthus nivalis agglutinin, a characteristic indicative of a mannoprotein. Furthermore, it was recognized by a rat monoclonal antibody against the galactofuran side chain of galactomannan, indicating that it is a galactomannoprotein. Ultrastructural analysis by immunogold staining indicated that Afmp1p is present in the cell walls of the hyphae and conidia of A. fumigatus. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. fumigatus develop a specific antibody response against Afmp1p. This suggested that the recombinant protein and its antibody may be useful for serodiagnosis in patients with aspergilloma or invasive aspergillosis, and the protein may represent a good cell surface target for host humoral immunity.     

Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis

Aspergilloma and invasive aspergillosis are important opportunistic infections caused by Aspergillus species, among which Aspergillus fumigatus is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of A. fumigatus. Evaluation of the test with guinea pig sera against A. fumigatus and other pathogenic fungi indicated that this assay was specific for A. fumigatus. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis.     

Ultrastructural demonstration of specific IgG and IgE antibodies binding to Aspergillus fumigatus from patients with aspergillosis.

Aspergillus-induced diseases usually demonstrate elevated circulating antibodies belonging to different isotypes. The antigens currently used to detect antibodies are crude culture filtrate and mycelial extracts of A. fumigatus (Af). Most Af-associated diseases result from the inhalation of the spores of the organisms present in the environment. However, it is not known whether specific circulating antibodies directed only against spore or mycelia of Af exist in the sera of patients with Af-induced diseases. With colloidal gold we have investigated thin sections of spores and hyphae of Af for their reactivity with Af-specific IgG and IgE antibodies. The results indicate that both spores and hyphae reacted identically with IgG and IgE antibodies from patients. None of the sera from normal control subjects reacted in this system, although low levels of antibodies were detected in the sera by ELISA. Sera from both patients with allergic bronchopulmonary aspergillosis or aspergilloma reacted with cell envelope antigens, whereas sera from patients with invasive aspergillosis also bound to cell sap. This method therefore demonstrates localization of antigens binding to different isotypes in the sera from different clinical forms of aspergillosis and may be useful in purifying specific antigens for immunodiagnosis.     

Antigens/allergens of Aspergillus fumigatus. Identification of antigenic components reacting with both IgG and IgE antibodies of patients with allergic bronchopulmonary aspergillosis

Evidence that both IgE and IgG antibodies present in the sera of allergic bronchopulmonary aspergillosis patients (ABPA) combine with several of the antigenic components of Aspergillus fumigatus has been obtained using a self-crossed radioimmunoelectrophoresis test. In this test, patient's serum was used to develop its own crossed immunoprecipitation pattern to a reference antigen before proceeding to autoradiography with 125I-anti-human IgE in the usual way. Comparison of autoradiograph with immunoprecipitate showed a varied pattern of reactivity, i.e. precipitin peaks with and without associated radioactivity, as well as some peaks appearing only in the autoradiograph. Control sera, including sera from A. fumigatus skin test positive patients and a skin test negative aspergilloma patient with multiple precipitins showed no such combined activity. The specificity of the test was further demonstrated using an ABPA serum pool, both by absorbing the IgE from the serum before incorporation in the gel and also by heat inactivation (56 degrees C, 4 h) of the serum to destroy the heat labile determinants on IgE molecules, thus preventing the uptake of the anti-IgE specific for these determinants.     

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.