DEVELOPMENT OF MURINE MONOCLONAL-ANTIBODIES FOR THE IMMUNOHISTOCHEMICAL DIAGNOSIS OF SYSTEMIC BOVINE ASPERGILLOSIS

Murine monoclonal antibodies (MAbs) against water-soluble somatic anti gens (WSSA) and the wall fraction (WF) from Aspergillus fumigatus were produced by fusion of splenocytes from immunized BALB/c mice with mou se myeloma X63-Ag 8.653 cells. The supernatants of in vitro cultured h ybridomas were initially screened for reactivity with the WSSA and the WF from A. fumigatus and WSSA of other fungi in an enzyme-linked immu nosorbent assay (ELISA). Supernatants reacting only with A. fumigatus antigens were subsequently screened for homologous and heterologous re activity with immunohistochemical techniques using formalin-fixed, par affin-embedded tissues from experimentally infected mice. Because of a high immunohistochemical reactivity with homologous fungi, 4 MAbs rai sed against A. fumigatus WSSA and WF were selected for a further evalu ation of cross-reactivity (diagnostic specificity) in immunohistochemi cal and immunoblotting assays. In immunohistochemical assays, all MAbs raised against WSSA cross-reacted heavily with a number of other fung al species. All 4 MAbs (MAb-WF-AF-1-4) raised against the WF reacted s trongly with hyphae of Aspergillus spp.; hyphae of Scedosporium apiosp ermum were also strongly labeled by MAb-WF-AF-3 and-4. The 2 specifica lly reacting MAbs (MAb-WF-AF-1 and-2) were of the IgM biotype and were precipitating, and in immunoblotting experiments both bound to a 106- kD antigen of the WF, whereas they did not bind to WSSA of A. fumigatu s. One of the 2 aspergillosis-specific MAbs (MAb-WF-AF-1) was used to screen 145 mycotic lesions of cattle. The diagnoses on bovine lesions obtained by MAb-WF-AF-1 were compared with results based on reactivity with heterologously absorbed polyclonal antibodies and, for some lesi ons, to culture results. In the vast majority of lesions (n = 133), th e MAb-WF-AF-1 and the polyclonal anti-Aspergillus antibodies reacted i n a similar pattern, i.e., positively in 41 aspergillosis lesions and negatively in 92 zygomycotic lesions. Hyphae in 3 of 12 lesions that w ere not stained by the polyclonal antibodies reacted with the specific MAb-WF-AF-1; i.e., aspergillosis was diagnosed. The characteristics o f the 2 MAbs (MAb-WF-AF-1 and-2) raised against the WF of A. fumigatus in ELISA and immunoblotting and immunohistochemical assays justify th eir application for the in situ diagnosis of systemic aspergillosis of cattle.