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.     

Identification, quantitation, and purification of cockroach allergens using monoclonal antibodies

A panel of murine IgG monoclonal antibodies (MAbs) was raised against German cockroach (CR) (Blattella germanica) extract and selectively screened to identify MAb directed against allergen(s) recognized by IgE antibodies. Sera from 28 CR-allergic patients were used as sources of IgE antibodies to detect allergens "presented" by the MAb. Four clones (10A6, 3G12, 8F4, and 1D4) produced MAb to allergen(s) that bound IgE antibodies. Quantitative radioimmunoassays were used to compare levels of the MAb-defined allergens in CR extracts. MAb 10A6 reacted with a cross-reacting allergen that was detected in 9/14 CR species, including Blattella, Periplaneta, Blatta, Leucophea, and Supella spp, at concentrations of 100 to 10,000 U/ml. In contrast, MAb 3G12, 8F4, and 1D4 were Blattella specific. The allergen defined by MAb 8F4 was purified by MAb affinity chromatography and size-exclusion by high-performance liquid chromatography. It is a 36 kd heat-sensitive protein, isoelectric point, 5.2 to 5.4. Allergen 10A6 was partially purified by isoelectric focusing and high-performance liquid chromatography. It is a heat-stable, acidic protein (isoelectric point 3.15). Based on comparison of their properties with properties of previously described CR allergens, the allergens defined by MAb 10A6 and 8F4 have been provisionally designated Blattella germanica allergen I (Bla g I) and Blattella germanica allergen II (Bla g II), respectively. Assays of six commercial CR skin test extracts demonstrated a 200-fold difference in Bla g I levels (4.7 to 1085 U/ml) and only two extracts that contained detectable Bla g II (248 and 324 U/ml). The results demonstrate that MAb can be used to identify and define CR allergens and that the strategy of the use of MAb as a first step in allergen analysis and purification can be very effective, especially for poorly characterized allergen extracts.     

Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.     

Production and characterization of murine monoclonal antibodies to Histoplasma capsulatum yeast cell antigens

Four monoclonal antibodies (MAbs) were produced by immunizing mice with a disrupted yeast cell homogenate of Histoplasma capsulatum. MAbs 1 and 2 reacted only with the yeast cell antigens of H. capsulatum and Blastomyces dermatitidis, whereas MAbs 3 and 4 showed broader cross-reactivity. MAb 3 cross-reacted with B. dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii, and Candida albicans, and MAb 4 cross-reacted with B. dermatitidis, C. albicans, Coccidioides immitis, Aspergillus fumigatus, and Mycobacterium tuberculosis. All four MAbs exhibited unique specificity when reacted with three different strains of H. capsulatum (G217B, A811, and P-IN). MAb 1 belonged to the IgG2b subclass, MAb 3 belonged to the IgG1 subclass, and MAbs 2 and 4 belonged to the IgG3 subclass. MAbs 1, 2, and 3 formed bands in the Western immunoblot assay; the two dominant distinct bands had apparent molecular masses of 72 and 62 kilodaltons.     

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.     

Contemporaneous occurrence of allergic bronchopulmonary aspergillosis, allergic Aspergillus sinusitis, and aspergilloma.

BACKGROUND: The clinical categories of Aspergillus-related respiratory disorders usually remain mutually exclusive. The coexistence of allergic bronchopulmonary aspergillosis (ABPA) with aspergilloma is uncommon, whereas concurrent ABPA and allergic Aspergillus sinusitis (AAS) is rare. The association of these 3 clinical entities has previously been documented only once in a patient who had earlier been operated on for an aspergilloma before the diagnoses of ABPA and AAS were established. OBJECTIVE: To describe an adult in whom ABPA, AAS, and aspergilloma were diagnosed simultaneously. METHODS: Spirometry, radiography, computed tomography, skin allergy testing with Aspergillus antigens, serum precipitins against Aspergillus, total and specific IgE, functional endoscopic sinus surgery, and fungal culture were performed. RESULTS: A 26-year-old man who had asthma and rhinitis since childhood presented with hemoptysis. Serial chest radiographs revealed transient pulmonary infiltrates and an aspergilloma. Computed tomography of the thorax confirmed the aspergilloma and showed bilateral central bronchiectasis along with patchy infiltrates. Strong bands of precipitins were detected against Aspergillus fumigatus, and intradermal testing with Aspergillus antigens elicited strong type I and III hypersensitivity reactions. Specific IgE and IgG antibodies against A fumigatus were positive, and total IgE levels were significantly elevated. Peripheral blood eosinophilia was also detected. Sinus involvement was confirmed on computed tomography, and pathologic material obtained by functional endoscopic sinus surgery demonstrated allergic mucin that contained fungal elements. In addition, A fumigatus was cultured. CONCLUSIONS: ABPA, AAS, and aspergilloma can occur simultaneously in the same patient.     

A solid-phase radioimmunoassay for detection of human antibodies. I. Measurement of IgG antibody to bee venom antigens.

A solid-phase radioimmunoassay (SPRA) has been developed to measure IgG antibodies to bee venom (BV) and phospholipiase A2 (PLA) in human sera. The principle of the test is similar to that of the radioallergosorbent test (RAST) measuring IgE antibody. Cyanogen-bromide-activated paper discs coupled with BV or PLA followed by supplementary coupling with human serum albumin were incubated with standard or test sera, washed, and incubated with 125I-labeled anti IgG. The serum levels of the IgG antibody have been temporarily expressed in arbitrary units. the reaction between the antigen and antibody was specific and the results were reproducible. Sera from 19 beekeepers, 42 beesting-sensitive patients and 20 blood donors (controls) were assayed by the SPRA. IgG antibodies to BV and PLA could not be detected (less than 4 U/ml) in all control sera, in 25 of the 42 patients and in one beekeeper. The IgG antibodies in 17 patients ranged between 5 to 58 U/ml (mean 7.6 U/ml), and in the 18 beekeepers ranged between 8 to 160 U/ml (mean 59 U/ml).     

Identification of two distinct allergenic sites on ryegrass-pollen allergen, Lol p IV

Lol p IV is an important allergen of ryegrass pollen. For the immunochemical identification of antigenic and/or allergenic site(s), murine monoclonal antibodies (MAbs) were prepared against Lol p IV. The hybridoma cell-culture supernatants were screened for anti-Lol p IV antibodies by a combination of ELISA and Western immunoblot analyses. The MAbs were finally purified from ascites on a Mono Q ion-exchange column. In a competitive radioimmunoassay with Lol p IV as the solid phase and 125I-labeled MAbs, it was established that MAbs 90, 91, 92, 93, and 94, although they differed in their relative affinities, recognized in common with one another an epitope designated as antigenic site A, whereas MAb 12 recognized a different epitope referred to as site B. Sites A and B were also demonstrated to constitute allergenic determinants of Lol p IV. Differences in the repertoire of specificities of the human IgE antibodies directed to Lol p IV were also demonstrated. Interestingly, it was found that sera from both allergic as well as from nonatopic individuals had IgG antibodies to sites A and/or B.     

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.     

Use of a synthetic peptide epitope of Asp f 1, a major allergen or antigen of Aspergillus fumigatus, for improved immunodiagnosis of allergic bronchopulmonary aspergillosis

Allergic bronchopulmonary aspergillosis (ABPA) is an immunologically complex allergic disorder caused by the fungal pathogen Aspergillus fumigatus. Elevated levels of total immunoglobulin E (IgE), specific IgE, and IgG antibodies in sera are important immunodiagnostic criteria for ABPA. International reference standards or standardized immunodiagnostic assays are not available due to a lack of well-defined diagnostic antigens. The present study was carried out to identify and evaluate the immunodiagnostic relevance of synthetic epitopic peptides of Asp f 1, a major allergen, antigen, or cytotoxin of A. fumigatus. Five overlapping peptides were synthesized from the N terminus of Asp f 1, one of the potential immunodominant regions predicted by algorithmic programs. The 11-amino-acid synthetic peptide (P1) significantly inhibited both IgG binding (89.10% +/- 4.45%) and IgE binding (77.32% +/- 3.38%) of the standardized diagnostic antigen (SDA) (a well-defined pool of diagnostically relevant allergens and antigens of A. fumigatus). With a panel of sera of ABPA patients, allergic patients with skin test negativity to A. fumigatus, and healthy individuals, P1 showed a higher diagnostic efficiency than SDA (specific IgG, 100%; specific IgE, 98.3%). The diagnostic efficiency of P1 could be attributed to the presence of homologous epitopes in various immunodominant allergens or antigens of A. fumigatus. The ability of P1 to induce histamine release from sensitized mast cells and a Th2 type of cytokine profile in peripheral blood mononuclear cells of ABPA patients suggests its potential for use in intradermal testing. P1 could be further explored for development of a standardized, specific, and sensitive immunodiagnostic test for aspergillosis.     

Immunoprint pattern in patients with allergic bronchopulmonary aspergillosis in different stages

Individual immunoprint patterns of sodium dodecylsulfate-polyacrylamide gel electrophoresis-separated Aspergillus fumigatus (Af) allergens/antigens were evaluated in 28 patients with allergic bronchopulmonary aspergillosis in stages II to V. It could be demonstrated that active disease (stage III) is characterized by a very strong IgE response against a variety of Af components, whereas the IgE antibody pattern in corticosteroid-treated patients who have demonstrated improvement is much weaker. In patients in remission (stage II), it is minimal. In contrast, many patients in stage V without corticosteroid treatment demonstrated a strong reactivity of IgE antibodies, indicating persisting active disease. The pattern of IgG antibodies with individual Af components resembles, in general, that of IgE antibodies; however, discrimination between different stages and between treated patients is much weaker. Our results indicate that a certain relationship between the different stages of allergic bronchopulmonary aspergillosis and Af immunoprint patterns exists.     

Rat monoclonal antibodies against Aspergillus galactomannan.

Monoclonal antibodies (MAbs) against Aspergillus fumigatus galactomannan were produced in rats. Seven of them, EB-A1 through EB-A7, were characterized in more detail. They were all immunoglobulin M antibodies, reacting in an indirect enzyme-linked immunosorbent assay with purified A. fumigatus galactomannan, with avidity constants of between 2 x 10(9) and 5 x 10(9)/M. Enzyme-linked immunosorbent assay inhibition experiments with modified galactomannan and synthetic oligomers of beta (1----5)galactofuranose demonstrated that the MAbs bound to an epitope located on the beta(1----5)galactofuranose-containing side chains of the galactomannan molecule. An identical or similar epitope also seemed to be present in other fungi. Immunofluorescence and immunoelectron microscopy experiments with EB-A2 revealed the presence of the antigen in the fungal wall and inside the cell. Immunoblotting experiments demonstrated that the epitope recognized by the MAbs was a common oligosaccharide moiety of a wide range of intracellular and extracellular glycoproteins in A. fumigatus. The characteristics of the MAbs justify their use in the diagnosis of invasive aspergillosis by antigen detection.     

Monoclonal antibodies to 125 kd and 95 kd proteins on human melanoma cells: comparison with other monoclonal-defined melanoma antigens

Two monoclonal antibodies produced by hybridomas were identified by an indirect 125I-protein A binding assay that define cell surface antigens expressed on cultured human melanoma cells but not on autologous lymphoblastoid cells. The first antibody, 705F6 (an IgG2b immunoglobulin), bound to 14/14 melanoma lines, 6/9 carcinomas and sarcomas, 7/7 gliomas and neuroblastomas, 2/2 fetal cell lines, 0/8 lymphoblastoid cell lines, and weakly to 2/4 leukemia lines. The second monoclonal antibody, 436G10 (IgG1), reacted with 10/14 melanomas 5/13 carcinomas and sarcomas, 2/7 gliomas and neuroblastomas, and weakly with the fetal cells, but not with the leukemic or lymphoblastoid cell lines. Comparison of 705F6 and 436G10 with 28 other monoclonal antibodies from different laboratories identified several with similar binding patterns to a panel of tumor and nontumor cell lines. Crossblocking of 125I-labeled 436G10 was not observed by R23, I12 or L10 antibodies. However, 705F6 was completely blocked by monoclonal 376.96S, showing that these two antibodies bind to the same antigenic determinant. The 705F6 antibody immunoprecipitated a 95 kd (kilodalton) membrane protein and the 436G10 antibody bound a 125 kd protein from 125I-labeled melanoma cells. The broad distribution of these two proteins on melanomas and other solid tumors suggests that they define common oncodevelopmental antigens expressed on proliferating cells.     

Aspergillus fumigatus specific IgE and IgG antibodies for diagnosis of Aspergillus-related lung diseases

IgE and IgG antibodies against Aspergillus fumigatus were detected by crossed radio immunoelectrophoresis (CRIE) on the sera of seven patients with aspergilloma, six patients with allergic broncho-pulmonary aspergillosis (ABPA) and 25 patients with extrinsic asthma with Aspergillus allergy. IgE-CRIE analysis indicated the presence of A. fumigatus-specific IgE in sera of patients with ABPA and Aspergillus asthma but not of aspergilloma patients. IgG-CRIE showed that both aspergilloma and ABPA patient sera contained high levels of circulating specific IgG antibodies in contrast to sera of Aspergillus asthma patients, which did not show detectable amounts of Aspergillus-specific IgG antibodies. Specific IgE binding could be demonstrated for the major allergens Ag-10 and AG-40 in all ABPA patients, in 80% of Aspergillus asthma patients but not in sera from aspergilloma patients. Specific IgG antibodies directed towards the major allergens could be detected in most of the aspergilloma patients, between 30-70% of the ABPA patients but not in sera from patients with Aspergillus asthma.     

Monoclonal antibodies against a 97-kilodalton antigen from Aspergillus flavus.

We prepared a panel of five monoclonal antibodies (MAbs) directed against Aspergillus flavus that all reacted against one 97-kDa antigen by western blot (immunoblot). Flow cytometry demonstrated that these antibodies bound (in increasing degrees) to all morphologic stages of A. flavus growth: conidia, swollen conidia, and hyphae. Cross-reactivity among species was examined by enzyme-linked immunosorbent assay of fungal culture filtrates. Four MAbs reacted with 10 of 11 A. flavus isolates, and the fifth one reacted with 9 of them. One MAb also reacted with A. fumigatus, two reacted with A. niger, A. wentii, and A. nidulans, and all five reacted with A. ochraceus. None reacted with A. terreus, A. glaucus, A. versicolor, or a Penicillium species. Each MAb bound to A. flavus hyphae in formalin-fixed paraffin sections of a muscle biopsy from a confirmed human case of invasive aspergillosis. In summary, these MAbs identified a 97-kDa antigen found on A. flavus that is both surface bound and an exoantigen. Either the same or a cross-reacting antigen is present in A. fumigatus and other Aspergillus species.     

Generation of monoclonal antibodies against surface antigens of Moraxella bovis.

Six hybridoma lines producing monoclonal antibodies (MAbs) against Moraxella bovis were established from fusions between the SP2/0 myeloma cells and BALB/c mice splenocytes. Three antibodies were of the IgG1 isotype, two were IgG2a, and one was IgG2b. The specificity of the antibodies was determined by indirect enzyme-linked immunosorbent assay (ELISA) using whole cells of M. bovis and of other Gram-negative bacteria, and lipopolysaccharide (LPS) from M. bovis JUR2 and E. coli as antigens. Ascitic fluid produced by the six hybridoma lines inhibited hemagglutination by M. bovis GF9. One MAb (35F) reacted specifically with purified M. bovis LPS in the ELISA test. The MAb panel detected heterogeneity among the isolates recovered from different geographical regions.     

IgG antibodies to purified Aspergillus fumigatus antigens determined by enzyme-linked immunosorbent assay

IgG antibodies to three purified Aspergillus fumigatus antigens were determined by enzyme-linked immunosorbent assay (ELISA) in sera from 26 patients with definite pulmonary aspergillosis (group 1), 23 patients with suspected pulmonary aspergillosis (group 2), 8 patients with precipitating antibodies to A. fumigatus of undetermined clinical significance (group 3), and 113 healthy blood donors (group 4). With a 470,000-dalton antigen fraction ELISA values exceeded the upper range of group 4 (0.72) in 92, 91 and 13% of cases in groups 1, 2 and 3, respectively. With a 250,000-dalton antigen fraction the figures were 96, 78 and 13%, respectively (upper range of group 4 = 0.25). With an antigen fraction of 25,000-50,000 daltons the figures were 96, 65 and 13%, respectively (upper range of group 4 = 0.18). In 48 of 49 cases of definite or suspected aspergillosis (groups 1 + 2) ELISA values with at least one antigen exceeded the upper range of controls (group 4). It appears that antibody determination with these three antigen fractions may have a high diagnostic sensitivity.     

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.     

Monoclonal immunoglobulin G1 directed against Aspergillus fumigatus cell wall glycoprotein protects against experimental murine aspergillosis

Most of the biological functions related to pathogenicity and virulence reside in the fungal cell wall, which, being the outermost part of the cell, mediates the host-fungus interplay. For these reasons much effort has focused on the discovery of useful inhibitors of cell wall glucan, chitin, and mannoprotein biosynthesis. In the absence of a wide-spectrum, safe, and potent antifungal agent, a new strategy for antifungal therapy is directed towards the development of monoclonal antibodies (MAbs). In the present study the MAb A9 (immunoglobulin G1 [IgG1]) was identified from hybridomas raised in BALB/c mice immunized with cell wall antigen of Aspergillus fumigatus. The immunoreactive epitopes for this IgG1 MAb appeared to be associated with a peptide moiety, and indirect immunofluorescence microscopy revealed its binding to the cell wall surface of hyphae as well as with swollen conidia. MAb A9 inhibited hyphal development as observed by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (25.76%), reduced the duration of spore germination, and exerted an in vitro cidal effect against Aspergillus fumigatus. The in vivo protective efficacy of MAb A9 was also evaluated in a murine model of invasive aspergillosis, where a reduction in CFU (>4 log(10) units) was observed in kidney tissue of BALB/c mice challenged with A. fumigatus (2 x 10(5) CFU/ml) and where enhanced mean survival times (19.5 days) compared to the control (7.1 days) and an irrelevant MAb (6.1 days) were also observed.     

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.