The goal of this project is to develop nucleic acid aptamers that can discriminate the four human alkaline phosphatase isozymes for use in the development of enhanced blood tests. Alkaline phosphatases catalyze the removal of a phosphate group from proteins, nucleic acids, carbohydrates, and inorganic pyrophosphate. Humans express four distinct alkaline phosphatase isozymes; germ cell alkaline phosphatase, intestinal alkaline phosphatase, placental alkaline phosphatase, and tissue non-specific alkaline phosphatase. Due to the high structural similarity of these isozymes, it has not been possible to generate monoclonal antibodies that have a stringent isozyme specificity. Currently available alkaline phosphatase antibodies all display varying degrees of isozyme cross reactivity, which reduces their usefulness in diagnostic applications. To address this issue, we will generate nucleic acid aptamers that possess stringent isozyme specificity using systematic evolution of ligands by exponential enrichment (SELEX). The binding kinetics of the aptamers will be measured using surface plasmon resonance and purified alkaline phosphatase isozymes. The aptamer?s detection limit will be determined using an enzyme-linked aptamer assay.
A routine blood test typically includes total alkaline phosphatase level measurement. While an abnormally high alkaline phosphatase level in blood typically indicates a serious condition such as liver cancer, cirrhosis, hepatitis, diabetes mellitus, chronic alcoholism, and osteosarcoma, the disease affected organ cannot be identified from the total alkaline phosphatase level reading alone. We will develop aptamers that can distinguish the four human alkaline phosphatase isozymes in the serum, thus enabling development of an enhanced blood test that will report the level of each individual isozyme, which in turn will allow earlier and faster diagnosis.
