A double-direct sandwich enzyme-linked immunosorbent assay that uses a rat anti-galactomannan monoclonal antibody as the acceptor and detector antibody was designed. This immunoassay, which detects less than 1 ng of galactomannan per ml, was assessed in a retrospective study with samples from patients with invasive aspergillosis. Serum is more appropriate than urine for use in the search for circulating galactomannan. Antigenemia does not have a transient character. Galactomannan can be detected at least 39 days before the death of the patients.

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.

Two monoclonal antibodies (MAbs) of the immunoglobulin G2A isotype, reacting with a Nocardia-specific 54-kDa antigen, were generated. As determined by Western blot (immunoblot), both MAbs reacted only with the 54-kDa band. As determined by indirect immunofluorescence or enzyme immunoassay with whole microorganisms, the MAbs did not react with Nocardia cells. One of the MAbs showed weak cross-reactivity with mycobacterial antigens, while the other showed no cross-reactivity.

Aspergillus species and dermatophytes are medically important fungi (Bodey and Vartivarian, 1989; Grappel et al., 1974). Both fungal groups produce exocellular antigens containing galactomannan molecules. Chemical characterization of these polysaccharidic antigens remain incomplete. However, the presence of non-reducing galactofuranose end units have been demonstrated unequivocally in both Aspergillus and dermatophyte species (Latgé et al., chapter 11; de Haan et al.,chapter 28). Polyclonal antibodies have been produced against Aspergillus galactomannans for the detection of antigens either in serum of patients with invasive aspergillosis or in foodstuffs, contaminated by fungi (Bennett et al., 1985; Notermans et al.,1988; Banks et al., 1990). These studies have shown that the galactofuran side chains of the galactomannan are immunodominant. Cross-reaction has been observed among these antisera and other fungi, including dermatophytes. Such cross-reaction may be due to structural relatedness of the polysaccharides of different fungal species but also to the polyclonal character of these antisera. Therefore, monoclonal antibodies (MAbs), directed against a single epitope, would be more usçful. A recent paper by Ste-Marie et al. (1990) describes two murine MAbs which react to Aspergillus galactomannan epitopes, which cross-reacted with fungi from other genera. In this paper we describe the production of rat MAbs against Aspergillus galactomannan and the initial characterization of rat MAbs against exocellular antigens of the dermatophyte Trichophyton rubrum.