Detection of Antibody to Theileria Parva Schizonts and Cell Surf Ace Membrane Antigens of Infected L Ymphoblastoid Cells by Immunoperoxidase Techniques

Abstract

Peroxidase-labeled antibody procedures were described for detecting bovine antibodies reactive with intracellular Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells. Indirect tests were performed where the reacting bovine antibodies were localized with affinity purified rabbit-anti-bovine IgG coupled to horseradish peroxidase. A 4- to 8-fold increase in sensitivity for detecting bovine antibodies was obtained with unlabeled rabbit-anti-bovine IgG which in turn was detected with peroxidase labeled goat-anti-rabbit IgG. The T. parva infected cells used as antigen were attached to poly-I-lysine treated glass slides and all reaction steps were performed on the slides. The intracellular schizonts and cell surface staining reactions were dependent upon the status of the cells; acetone-fixed cells were required for schizont reactions and viable unfixed cells for cell surface membrane reactions. Sera from cattle stimulated in various ways with T. parva were examined by the techniques. Cattle infected by stabilate inoculation or inoculated with infected autologous lymphoblastoid cells developed relatively high levels of antibody to schizonts, but no detectable antibody to cell . surface membrane antigens. This would indicate that parasite antigens do not occur on the surface of infected lymphoblasts. Cattle inoculated with infected allogeneic lymphoblasts developed low-levels of antibody to schizonts and readily demonstrable antibody to cell surface antigens. The immunoperoxidase procedures have certain advantages over immunofluorescence in that light microscopy is used; therefore, the reactions do not fade which permits a more detailed examination and provides a relatively permanent record, the preparations can be counterstained, and the reagents may be used for immunoelectron- microscopy. The procedures could provide suitable alternatives to immunofluorescence methods for East Coast fever investigations and other systems having intracellular and/or cell surface membrane antigens.