EXPERIMENTAL PARASITOLOGY 62, 40-50 (1986) Amblyomma americanum: Physiochemical Isolation of a Protein Derived from the Tick Salivary Gland That Is Capable of Inducing Immune Resistance in Guinea Pigs STEPHEN J. BROWN Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801, U.S.A. AND PHILIP W. ASKENASE Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, U.S.A. (Accepted for publication 20 January 1986) BROWN, S. J., AND ASKENASE, P. W. 1986. Amblyomma americanum: Physiochemical iso- lation of a protein derived from the tick salivary gland that is capable of inducing immune resistance in guinea pigs. Experimental Parasitology 62, 40-50. Crude salivary gland derived proteins from Amblyomma americanum ticks were analyzed by physiochemical (gel filtration and ion exchange chromatography) and immunochemical guinea pig IgG, (anti-tick immunoaffinity column) techniques for the presence of antigens responsible for the induc- tion of host immune resistance responses. Gel filtration (G-75 Sephadex) and ion exchange (diethyl aminoethyl cellulose) chromatography of crude salivary gland antigen yielded mul- tiple fractions, but only one fraction from each procedure induced significant cutaneous anaphylaxis bluing reactions when used for skin tests in tick sensitized animals treated intravenously with 0.05% Evans blue dye. Salivary gland antigen (200 ng) eluted from the immunoaffinity column by 0.2 M Na,CO,, pH 11.3, and emulsified with incomplete Freund’s adjuvant conferred a significant level of tick rejection (24%, P < 0.001) on naive guinea pigs compared with that seen in controls, but less than (P < 0.01) the level of immu- nity conferred by crude salivary gland antigen (380 kg). The immunizing dose of immunoaf- finity purified salivary gland antigen was 111900 the dose of the crude antigen preparation representing 99.9% purification. Furthermore, engorged ticks from animals immunized with salivary gland antigen exhibited a significant decrease (P < 0.001) in weight compared with ticks from naive animals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rz51 labeled proteins in the Na,CO, eluate and the skin reactive fraction from gel filtration and ion-exchange chromatography, after immunoprecipitation with a guinea pig IgG, antibody to the tick that transferred resistance, revealed the presence of a 20 kDa weight protein reported previously to be the antigen responsible for the induction of host resistance. These studies present physiochemical and immunochemical procedures for the purification of an important tick protein that (1) induces skin reactions in tick sensitized guinea pigs, (2) is recognized by antibody to the tick, and most importantly, (3) is capable of immunizing naive guinea pigs against tick challenge. 0 1986 Academic PIES, 1~. INDEX DESCRIPTORS AND ABBREVIATIONS: Tick; Amblyomma americanum; Gel filtration chromatography; Ion exchange (DEAE) chromatography; Immunoaffinity chromatography; Salivary gland antigen (SGA); Immunization; Resistance; Phosphate buffered saline (PBS); Sodium dodecyl sulfate (SDS); Polyacrylamide gel eletrophoresis (PAGE); Incomplete Freund’s adjuvant (IFA); Complete Freund’s adjuvant (CFA); Tris-EDTA-NaCl (TEN). 40 0014-4894186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.