Monoclonal antibodies to TAT-1 were raised in mice using a subcutaneous injection of 150 μg of the purified recombinant TAT-1 protein (amino acid 369-734, designated as rTat-S2 protein and expressed and purified from Escherichia coli) emulsified in Freund's complete adjuvant, followed by an intravenous booster injection of 50 μg of rTat-S2 two weeks later. The resulting hybridomas were screened for secretion of monoclonal antibodies specific to rTat-S2 using an indirect enzyme-linked immunosorbent assay. Monoclonal antibodies were prepared by injecting hybridoma cultures into the peritoneal cavities of pristane-primed BALB/c mice. The antibodies were purified from the collected ascites using ammonium sulfate precipitation followed by Mabselect-Xtral affinity chromatography (Amersham GE Health). Immunoglobulin concentrations were determined on a DU800 spectrophotometer (Beckman Coulters). One monoclonal antibody, designated 7G1, was used here.
tat-1