Microneme antigens of Eimeria bovis recognized by two monoclonal antibodies

Two IgG1 monoclonal antibodies (mAbs 8-23F9 and 9-21G9) were developed afte r immunization of mice with homogenates of Eimeria bovis first-generation m erozoites. Both mAbs reacted with antigens in the apical two-thirds of the parasites and immune electron microscopy determined the micronemes as targe ts. When tested by immunoblotting, mAb 8-23F9 failed to react with antigens separated under reducing conditions; under nonreducing conditions it recog nized two components of >200 kDa. mAb 9-21G9 bound to antigens of 135 and 1 80 kDa after electrophoresis under reducing conditions and to a series of c omponents when separated without reduction. The epitope of mAb 8-23F9 was d estroyed by treatment of the antigen with endoglycosidase H and removal of phosphocholine (PC) by phospholipase C. Since mAb 8-23F9 does not recognize cytidine-linked PC, the data suggest that PC in combination with N-linked sugars and/or N-glycans is part of its epitope. In the case of mAb 9-21G9, endoglycosidase H did not alter the epitope. When E. bovis merozoite antige n was treated with phospholipase C the number of mAb 9-21G9-reactive consti tuents increased, suggesting that PC may otherwise mask the epitope. mAb 8- 23F9 also bound to the apical area and the surface of E. bovis sporozoites and recognized a >200-kDa sporozoite component. When sporozoites invaded Ve ro cells in vitro, epitope-bearing components were released onto the host c ell surface and became part of the early parasitophorous vacuole wall. At d ay 5 the binding of the mAb was again confined to the intracellular parasit e. mAb 9-21G9 did not react with sporozoites but recognized the apical area of intra-cellular trophozoites on day 5 after invasion of host cells in vi tro. When testing was done against a variety of other Apicomplexa in variou s assays, the only cross-reaction observed occurred with mAb 8-23F9, which bound to a conformationally determined 180-kDa component of Toxoplasma gond ii cystozoites.