Development of a Novel ELISA Kit for Screening Potential JAK3 Inhibitors

DESCRIPTION provided by applicant Janus kinase JAK is an intracellular kinase involved in cytokine and antibody receptor signaling it is frequently over activated during the course of inflammatory autoimmune diseases Therefore suitable JAK inhibitors that modulate the overexpression of JAK may be potential agents for treating autoimmune diseases Logically it is of significant importance to develop a sensitive precise and rugged screening tool to select agents that modulate the over activation of JAK Currently available screening ELISA tools use only the active site of JAK Generally the assay employs the quantitative sandwich enzyme immunoassay technique Briefly antibody specific for JAK is pre coated onto a microplate Standards and samples are transferred into the wells and any JAK present is bound by the immobilized antibody After removing any unbound substances a biotin conjugated antibody specific for JAK is added to the wells Following a wash to remove any unbound avidin enzyme reagent a substrate solution is added to the wells and color develops in proportion to the amount of JAK bound in the initial step Another technology utilizes an andquot induced fitandquot concept different from the classical andapos lock and key modelandapos of enzyme activity in thatit was believed that the D structures of the enzyme active sites are continuously reshaped by interactions with their substrates inhibitors Overall all currently available screening tools hav several common deficiencies including low sensitivity requiring an external substrate and requiring a radioactive detection As part of our continued efforts in the research in immune mediated disorders we have been working on improving the screening technology for finding better JAK inhibitors we herein present the novel screening kit under development Briefly the proposed JAK screening tool comprises the use of full length JAK different from currently available in that only the active site of JAK is used The advantages of our novel screening kit include significantly lower background noise higher sensitivity no need for the external substrate no need to use the radioactive method and capable of measuring auto tyrosine phosphorylation The ultimate goal of this project is to manufacture and sell a significantly improved ELISA kit to new drug discovery companies for screening JAK inhibitors The goal of this Phase I SBIR study is to show the feasibility of developing this novel kit as a superior commercial product The specific aims for this SBIR Phase I study are To further prove the concept of using the full length JAK antibody in the screening kit To test the kit o a number of compounds positive control and potential compounds We have published positive control as shown in Preliminary Studies section In this SBIR Phase I study we will synthesize at least potential JAK inhibitors and purchase a series of compounds from purchased library to test the novel ELISA kit To validate that our screening method is accurate precise reproducible and rugged We will choose two suitable JAK inhibitors for the validation of our screening kit To compare and contrast between already existing technology and our new technology under development PUBLIC HEALTH RELEVANCE Suitable inhibitors that modulate the overexpression of Janus Kinase JAK may be potential agents for treating autoimmune diseases The ultimate goal of this project is to manufacture and sell a significantly improved ELISA kit to new drug discovery companies for screening JAK inhibitors as drug candidates