Non-Neutralizing Monoclonal Antibodies Against RAS GTPase-Activating Protein: Production, Characterization and Use in an Enzyme Immunometric Assay

We studied several monoclonal antibodies (mAbs) raised against the 100 kD Ras GTPase activating protein (p100–GAP), which was purified from human placenta. These antibodies recognized p120–GAP and p100–GAP in native and in denatured forms. The most reactive, GP15 and GP200, both recognized distinct epitopes and did not neutralize GTPase stimulatory activity. These two mAbs were selected for a two–site enzyme immunoassay, using covalent conjugates of the antibodies coupled to the tetrameric form of acetylcholinesterase as tracer. This assay was used to quantify Ras–GAP in both normal and tumor tissues and cell extracts.