Crude salivary gland derived
proteins from Amblyomma americanum ticks were analyzed by physiochemical (gel filtration and ion exchange chromatography) and immunochemical guinea pig
IgG1 (anti-tick immunoaffinity column) techniques for the presence of
antigens responsible for the induction of host immune resistance responses. Gel filtration (G-75 Sephadex) and ion exchange (diethyl
aminoethyl cellulose) chromatography of crude salivary gland
antigen yielded multiple 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 0.5%
Evans blue dye. Salivary gland
antigen (200 ng) eluted from the immunoaffinity column by 0.2 M Na2CO3, pH 11.3, and emulsified with
incomplete Freund's adjuvant conferred a significant level of tick rejection (24%, P less than 0.001) on naive guinea pigs compared with that seen in controls, but less than (P less than 0.01) the level of immunity conferred by crude salivary gland
antigen (380 micrograms). The immunizing dose of immunoaffinity purified salivary gland
antigen was 1/1900 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 less than 0.001) in weight compared with ticks from naive animals.
Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis of 125I labeled
proteins in the Na2CO3 eluate and the skin reactive fraction from gel filtration and ion-exchange chromatography, after immunoprecipitation with a guinea pig
IgG1 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 induces skin reactions in tick sensitized guinea pigs, is recognized by antibody to the tick, and most importantly, is capable of immunizing naive guinea pigs against tick challenge.