Aspergillus Allergens:
The chemical nature of the antigens of Aspergillus species may be protein, polysaccharides, and glycoproteins (Hearn, 1992; Kurup et al., 1977; Kurup and Kumar, 1991). Several studies indicate that synthetic media such as Czapek-Dox, glycine, asparagine or AOAC (Association of Analytical Chemists: Difco) broth supplemented with different nutrients are the suitable media for producing culture filtrate antigens by selectively removing the macromolecular components of the medium which may otherwise interfere with the immunoassays (Kim and Chaparas, 1978a; Kim and Chaparas, 1979; Kim et al., 1978b). The use of synthetic media and incubation of A. fumigatus culture at 37°C have been reported to yield more consistent antigen preparations (Kim and Chaparas, 1978a; Kurup et al., 1984). The available reports suggest that the most reliable cell-associated antigens for immunoassays can be obtained from a four day old aerated (shaken) culture while secreted antigens (culture filtrates) can be obtained from two to four weeks old stationary culture of the fungus (Hoehne et al., 1973; Kim and Chaparas, 1978a; Kurup et al., 1977; Kurup et al., 1989; van der Heide, et al., 1985a; van der Heide et al., 1985b). In spite of the difficulties in standardization of allergenic extracts, crude extracts are still in use for routine diagnosis (Borga, 1990). The results obtained using such antigens preparations must be interpreted with caution when assessing the antibody responses in patients due to the variability among the antigen preparation. Likewise, lack of comparability of the results obtained by different laboratories, which use different extracts, is also problematic.
A. fumigatus Enzymes Have Antigenicity
A. fumigatus antigens include a large number of enzymes such as chymotrypsin, proteinases, elastases, ribonucleases, catalases, and superoxide dismutases (Arruda et al., 1990; Biguet et al., 1967; Crameri, 1998a; Hamilton et al., 1995; Jaton-Ogay et al., 1992; Kolattukudy et al., 1993; Lopez-Medrano et al., 1995; Monod et al., 1991; Reichard et al., 1990; Rhodes et al., 1990; Schonheyder et al., 1988; Schonheyder et al., 1984c). The role of these enzymes in immunopathogenesis of ABPA and their usefulness in immunodiagnosis of the disease is yet to be elucidated. Eighteen enzymatically active components of Aspergillus have been detected, out of these, 13 reacted specifically with sera from patients with aspergillosis (Tran-van Ky et al., 1969). The antigenic fraction of A. fumigatus with chymotryptic activity reacts with sera from patients with aspergilloma, but not with ABPA. A high molecular weight antigen fraction of 250kDa, probably a tetramer of catalase has been characterized by Schonheyder et al (Schonheyder and Andersen, 1984a; Schonheyder et al., 1988). Cystic fibrosis patients with the complications of ABPA frequently showed IgE binding antibodies to catalase antigen from A. fumigatus (Hearn et al., 1990; Schonheyder and Andersen, 1984b). An immunodominant 90kDa A. fumigatus antigen was reported belonging to a catalase subclass which recognized over 90% of serum samples from patients with aspergilloma (Lopez-Medrano et al., 1995). Hamilton and associates (1995) purified a 19kDa superoxide dismutase (SOD) from A. fumigatus by isoelectric focusing, gel filtration, and fast protein liquid chromatography and this enzyme specifically reacted with the sera from patients with confirmed aspergillosis on Western blots (Hamilton et al., 1995).
The proteases from A. fumigatus were reported to be involved in the pathogenesis of aspergillosis. Two partially purified proteases of 25 and 50kDa reacted significantly with sera from aspergillosis patients (Piechura et al., 1990). In another study, two acid proteases of 73 and 43kDa purified from membrane vesicles of A. fumigatus showed reactivity with sera from ABPA patients in a biotin-avidin linked immunosorbent assay (Piechura et al., 1990). Recently, a number of A. fumigatus proteases have been isolated and characterized either by using conventional purification method or recombinant DNA technologies (Jaton-Ogay et al., 1992; Monod et al., 1991; Reichard et al., 1990; Rhodes et al., 1990; Sirakova et al., 1994). One of the recombinant alkaline proteases with elastinolytic activity exhibited weak antigenicity when studied in ABPA patients (Moser et al., 1994b). Although A. fumigatus proteases are reported to be involved in fungal penetration of the host barriers by catalyzing the hydrolysis of the major structural proteins of the basement membrane of lungs, their involvement in antibody and cellmediated immune responses in ABPA patients are yet to be explored.
Protein Antigens:
Among the four antigens purified by Longbottom et al. Ag3 and Ag5 are thermolabile ConA nonbinding antigens of 18kDa and 35kDa, respectively (Longbottom, 1983; Longbottom, 1986a; Longbottom et al., 1989; Taylor and Longbottom, 1988). These two antigens are also found to be useful in detecting antibodies in sera of ABPA patients. The low molecular weight (>14kDa) ConA nonbinding antigens of A. fumigatus were reported to exhibit specific antibody binding with sera from patients with ABPA (Kurup et al., 1977).
Purification of a 18kDa ConA nonbinding protein from different sources and strains of A. fumigatus using diverse purification methods has been reported by researchers (Latge et al.,1991). These methods include gel filtration and phenyl Sepharose chromatography, ion exchange chromatography, gel permeation chromatography, and Sephacryl S 200 column chromatography followed by phenyl Sepharose chromatography (Kurup and Kumar, 1991; Latge et al., 1991). The preparations of the purified antigen retained immunological reactivities. In another study, a 20kDa ConA nonbinding antigen was purified to apparent homogeneity using a combination of size-exclusion chromatography on a Sephacryl S 200 column and preparative isoelectric focusing in a pI 2.5-6.5 gradient. This antigen showed IgE binding reactivity by radio allergosorbent test (RAST) (Samuelsen et al., 1991).
A diagnostically relevant protein of molecular weight of 18kDa was purified to homogeneity from a third week culture filtrate of a clinical isolate of A. fumigatus and from a soil strain using phenyl Sepharose chromatography followed by preparative HPLC (Arruda et al., 1990). The sequence data of the purified 18kDa protein was found to be homologous to the deduced sequence of Asp f 1 (Arruda et al., 1990; Arruda et al., 1992). Immunoreactivity of the fractionated and purified 18kDa antigen with the monoclonal antibody (MAb) and sera of patients with ABPA and aspergilloma demonstrate the presence of 18kDa antigen in A. fumigatus and suggests the usefulness of this protein as a marker in the diagnosis of aspergillosis (Arruda et al., 1992; Moser et al., 1992; Moser et al., 1994a; Samuelsen et al., 1991).
Raising monoclonal antibodies against diagnostically relevant antigens of A. fumigatus was attempted in order to obtain a permanent source of pure, standardized antigens for immunodiagnosis of A. fumigatus- sensitization. Monoclonal antibodies were raised against culture filtrate and mycelial extracts and against recognized diagnostically relevant antigens of A. fumigatus (Fratamico and Buckley, 1991a; Fratamico et al., 1991b; Kumar et al., 1993a; Kurup, 1998a; Ste. Marie et al., 1990). A ConA nonbinding antigen purified using monoclonal antibody affinity chromatography reacted preferentially with the sera from ABPA patients compared to those sera from aspergilloma patients and normal controls (Kumar et al., 1993a). A monoclonal antibody was also produced against Asp f 1, a protein with ribonuclease activity and strong reactivity with sera from ABPA patients (Arruda et al., 1990). A monoclonal antibody immunoaffinity column was used to purify this major allergen associated with ABPA. The purified Asp f 1 reacted with IgE antibody in the sera of 85% of patients with ABPA. A 37kDa ConA nonbinding allergen was purified by ConA fractionation followed by monoclonal antibody based immunoaffinity chromatography (Banerjee et al., 1996). It reacted strongly with IgE antibody in Western blots against pooled ABPA sera. The mean ELISA value for IgE antibody against this purified allergen was 10-fold higher in patients with ABPA compared with values in normal controls. The high specificity of these allergens facilitates rapid and accurate diagnosis of the disease as well as the direct comparison of results among different laboratories. However, such antigens are still not available commercially for diagnostic use.
Carbohydrate Antigen:
Polysaccharide fractions from either cell wall or cytoplasm also showed reactivity with antibodies in the sera of patients. However, these antigens frequently demonstrate crossreactivity with antigens from other fungi limiting the specificity of diagnostic applications (Azuma et al., 1967; Azuma et al., 1968; Azuma et al., 1969; Azuma et al., 1971; Bennett et al., 1985). Galactomannan present in the cell wall of A. fumigatus has structural features common to galactomannan of A. flavus and A. niger. Galactomannan antigen was isolated from mycelial extract using sequential chromatography on anion exchange and ConA affinity columns and the purity of the antigen was established by gas liquid chromatography (Bennett et al., 1985). The detection limit for the estimation of galactomannan in serum of patients with invasive aspergillosis was established between 10-40 ng/ml. A galactomannan of approximate size of 50kDa was purified by Azuma and co-workers and showed skin test reactivity and precipitin reaction in patients (Azuma et al., 1969; Dupont et al., 1987). Some of these carbohydrates are useful in the diagnosis of invasive aspergillosis (Dupont et al., 1987).
Glycoprotein Antigens:
Partial purification and characterization of glycoprotein antigens of A. fumigatus have been reported from different laboratories (Calvanico et al., 1981; Piechura et al., 1983; Piechura et al., 1990). Several glycoproteins isolated from both culture filtrate and mycelial extracts exhibited binding to lectin (ConA). The high molecular weight glycoprotein antigens from mycelial or culture filtrate preparations purified using conventional purification technique have been used to detect specific antibodies in the serum of patients suffering from ABPA. The cell sap protein isolated by Calvanico and associates consisted of four polypeptides of 45kDa, linked through disulfide bonds (Calvanico et al., 1981). The isoelectric point (5.2-5.6) and carbohydrate content (~12.5 % neutral hexose) indicated that this protein is an acidic glycoprotein. It showed antibody binding with 75% of sera from ABPA patients and was not reactive with sera from normal individuals. Four antigens (Ag3, Ag5, Ag7 and Ag13) were purified by Longbottom using gel filtration, isoelectric focusing and affinity chromatography (Longbottom, 1978; Longbottom and Austwick, 1986b; Longbottom et al., 1989; Taylor and Longbottom, 1988). Ag7 of 150-200 kDa and Ag13 with a molecular mass of 70kDa bound to ConA and reacted in an ELISA with sera from patients with ABPA and aspergilloma. We have demonstrated that A. fumigatus antigens transferred to polyvinyldifluoride (PVDF) membrane resolved 18 bands, and 12-14 of these were ConA-binding proteins indicating their glycoprotein nature (Kurup et al., 1977). Another fraction isolated from A. fumigatus showed significant antibody binding with sera from ABPA patients, focused at pH6.5 and showed 20, 40, and 80kDa size components in two-dimensional electrophoresis (Kurup et al., 1989). Two other fractions identified and characterized by using preparative isoelectric focusing and affinity chromatography also demonstrated reactivity against patient sera (Kurup et al., 1983). On the other hand, using hydrophobic interaction chromatography, Schonheyder and Andersen (1984a) obtained A. fumigatus components spanning a molecular mass from 25 to 50kDa. Out of 14 components detected on polyacrylamide gel electrophoresis three showed ConA binding property. None of these components were purified or characterized further (Schonheyder and Andersen, 1983). An antigenic fraction with two components of 35 and 65kDa were purified and analyzed for IgE and IgG binding with sera from ABPA patients (Kurup et al., 1985).
A major allergenic component (gp55) of A. fumigatus has been purified from water soluble extract of A. fumigatus cultures by gel filtration and SDS gel electrophoresis. This 55kDa protein has N-glycosidically linked oligosaccharides and demonstrated specific IgE binding with the sera from ABPA patients in Western blot analysis (Teshima et al., 1993). An immunodominant glycoprotein allergen of 45kDa (gp45) with specific antibody binding to ABPA patient sera was also purified and characterized (Banerjee et al., 1995). It was observed that deglycosylation destroys the IgE binding while IgG reactivity was retained. The allergen gp66 represents a glycoprotein of A. fumigatus obtained by ammonium sulfate precipitation followed by immuno affinity column purification (Little and Warner, 1996). This allergen showed IgE reactivity on crossed-radioimmunoelectrophoresis (CRIE) and ELISA with sera from cystic fibrosis patients complicated by ABPA. In a recent report, a glycoprotein with an apparent size of 93kDa was purified from water soluble extract of A. fumigatus by a single step affinity chromatography using mannose specific lectin coupled to agarose (Haynes, 1996). This antigen also exhibited IgE binding with sera from ABPA patients (Haynes, 1996). Thus, glycoproteins are significant allergenic components of A. fumigatus, possessing reactivity with both IgE and IgG antibodies of the sera from patients with aspergillosis.