Development of a Laboratory Test for TTP

DESCRIPTION (provided by applicant): This is a Phase I STTR application to develop a laboratory test for the diagnosis and monitoring of patients with thrombotic thrombocytopenic purpura (TTP). As there is no reliable, controlled laboratory test, TTP must be diagnosed on clinical grounds alone. Thus, some cases go undiagnosed, while others are misdiagnosed and subjected to costly therapy including daily therapeutic plasma exchange (about $1500 per treatment, 4 hrs per treatment x 7-14 days), that utilizes fresh frozen plasma as the plasma exchange replacement fluid (associated with allergic reactions and transfusion transmitted diseases including HIV, HCV and HBV). Recently, a metalloprotease (MP [ADAMTS13]) has been "identified" that cleaves large von Willebrand factor (vWF) protein multimers. Inhibition of the activity of this MP is thought to be the primary cause of acquired TTP. Only research laboratory based tests are available to determine the presence and/or quantify activity of this MP. These tests are cumbersome, slow, time and labor-intensive, and expensive. Additionally, there are no standards or controls for which the lab-based MP assay against which can be normalized or made widely available. Therefore, after considerable experience with our own modified, lab-based MP assay, we have developed a concept that would allow for an inexpensive, rapid, and specific, ELISA based test that could be made commercially available. It is this concept for which we seek Phase I funding. Specifically, we have/will design(ed) an amino acid target protein analogous to the A2 domain of vWF containing the specific cleavage site for the MP; when present, MP activity would cleave the target protein into 2 unequal protein fragments, which, initially, will be identified by HPLC. We will then design an ELISA wherein the target protein will contain a quenched fluorochrome at the cleavage site, which will fluoresce when the breakdown products, and thus MP activity, are present. Finally, we will design a target protein which will contain an enzyme that will become activated after its cleavage and convert a color substrate allowing automated detection. These 3 assay formats (HPLC, fluorochrome and colorimetric) will allow both qualitative and quantitative determination of MP activity. After feasibility is demonstrated, Phase II funding will be sought to optimize and commercialize the above prototype tests into a rapid, inexpensive, sensitive and specific, ELISA-format, clinical laboratory, automated, diagnostic and monitoring test for TTP, and to test it against reference samples from patients with clinically diagnosed TTP as well as others with related hematologic disorders.