Microneme antigens of Eimeria bovis recognized by two monoclonal antibodies.
Two IgG1 monoclonal antibodies (mAbs 8-23F9 and 9-21G9) were developed after immunization of mice with homogenates of Eimeria bovis first-generation merozoites. Both mAbs reacted with antigens in the apical two-thirds of the parasites and immune electron microscopy determined the micronemes as targets. When tested by immunoblotting, mAb 8-23F9 failed to react with antigens separated under reducing conditions; under nonreducing conditions it recognized two components of >200 kDa. mAb 9-21G9 bound to antigens of 135 and 180 kDa after electrophoresis under reducing conditions and to a series of components when separated without reduction. The epitope of mAb 8-23F9 was destroyed 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 antigen was treated with phospholipase C the number of mAb 9-21G9-reactive constituents 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 Vero cells in vitro, epitope-bearing components were released onto the host cell surface and became part of the early parasitophorous vacuole wall. At day 5 the binding of the mAb was again confined to the intracellular parasite. 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 vitro. When testing was done against a variety of other Apicomplexa in various assays, the only cross-reaction observed occurred with mAb 8-23F9, which bound to a conformationally determined 180-kDa component of Toxoplasma gondii cystozoites.
Heise
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Institut für Parasitologie, Justus-Liebig-Universität Giessen, Germany.
Peters
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Zahner
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D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan / Q000276:immunology*; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000276:immunology* / Q000302:isolation & purification; D000954:Antigens, Surface / Q000276:immunology* / Q000302:isolation & purification; D002417:Cattle; D002522:Chlorocebus aethiops; D003429:Cross Reactions; D004539:Eimeria / Q000276:immunology* / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000276:immunology; D051379:Mice; D015388:Organelles / Q000276:immunology* / Q000648:ultrastructure; D010767:Phosphorylcholine / Q000276:immunology; D014122:Toxoplasma / Q000276:immunology; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D000939:Epitopes; D006023:Glycoproteins; D010767:Phosphorylcholine