IMMUNOHISTOCHEMICAL DIAGNOSIS OF SYSTEMIC BOVINE ZYGOMYCOSIS BY MURINE MONOCLONAL-ANTIBODIES

Murine monoclonal antibodies (Mabs) against water-soluble somatic anti gens (WSSA) and the wall fraction (WF) from Rhizopus arrhizus (Rhizopu s oryzae) were produced in vitro by fusion of splenocytes from immuniz ed BALB/c mice with mouse myeloma X63-Ag 8.653 cells. Supernatants rea cting only with homologous antigens in an enzyme-linked immunosorbent assay were subsequently screened for reactivity with homologous fungi in immunohistochemical techniques. All four Mabs raised against the WF of A. arrhizus failed to react on tissues. However, four of the Mabs raised against the WSSA of R. arrhizus (Mab-WSSA-RA-1 through Mab-WSSA -RA-4) revealed a high homologous reactivity on tissues and the cross- reactivity of these were subsequently evaluated on tissues containing other members of the family Mucoraceae and other unrelated fungi. On t issues and on immunoblots all four Mabs reacted identically and specif ically with members of the family Mucoraceae, i.e., Absidia corymbifer a, R. arrhizus, and Rhizomucor pusillus. The Mabs were all isotyped as IgM antibodies, were nonprecipitating, and reacted with homologous an tigens with molecular masses from 14 to 110 kDa. With WSSA from A. cor ymbifera and R. pusillus the four Mabs were bound to antigens from 14 to 52 kDa and from 20 to 28 kDa, respectively. The diagnosis of 145 bo vine lesions obtained by one of the specific Mabs (Mab-WSSA-RA-1) were compared to results obtained by heterologously absorbed polyclonal an tibodies. In most lesions (n = 140 [similar to 97%]) the Mab and the p olyclonal antibodies reacted in a similar pattern, i.e., positively fo r zygomycosis in 89 lesions, negatively in 41 aspergillosis lesions, a nd negatively in 10 undiagnosed lesions. Hyphae within two of four les ions in lymph nodes, which were not stained by the polyclonal antibodi es, reacted with the specific Mab. However, in another three lesions o f lymph nodes stained by the polyclonal antibodies no reactivity was s een with the Mab-WSSA-RA-1. The immunoreactivity of the Mabs (Mab-WSSA -RA-1 through Mab-WSSA-RA-4) raised against WSSA of R. arrhizus justif y their application for the in situ diagnosis of systemic bovine zygom ycosis.