Purification of a major membrane protein of Toxoplasma gondii by immunoabsorption with a monoclonal antibody.
The principal iodinatable surface protein (P30) of our cloned RH strain of Toxoplasma gondii has an apparent molecular weight of 30,000, as measured by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Monoclonal antibody B specifically immunoprecipitated protein P30 from a detergent extract of surface radioiodinated T. gondii. Monoclonal antibody B in the presence of complement was also parasiticidal for T. gondii, and this parasiticidal effect could be blocked by protein P30. Monoclonal antibody B was purified from mouse ascitic fluid and linked to cyanogen bromide-activated Sepharose. The resulting immunoabsorbent was used to purify 1.7 mg of protein P30 from a large number of parasites. The efficiency of recovery of protein P30 was measured by assays of radioactivity and of parasiticidal blocking activity. Protein P30 represented 3 to 5% of the total protein. It is also present in a recently isolated strain of T. gondii. A convalescent human antitoxoplasma serum immunoprecipitated radiolabeled protein P30. Three convalescent antisera when quantitated by an ELISA test had a high anti-protein P30 titer. Charge shift electrophoresis showed that protein P30 has an extensive hydrophobic region and thus is probably an integral membrane protein. Electrophoresis under nonreducing conditions showed no evidence that protein P30 exists as a disulfide linked homo- or heterodimer, although it probably has intramolecular disulfide bonds.
D000042:Absorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000502:physiology; D000917:Antibody Formation; D001667:Binding, Competitive; D003289:Convalescence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008970:Molecular Weight; D011817:Rabbits; D014122:Toxoplasma / Q000276:immunology / Q000378:metabolism; D014123:Toxoplasmosis / Q000276:immunology* / Q000469:parasitology
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D008565:Membrane Proteins