SBIR-STTR Award

The proposed project is aimed at the development and implementation of a highly sensitive colorimetric detection technology for use in rapid medical diagnostic tests, and its application to a peptide-based Lyme disease rapid test as a first example. At the core of the new technology is a novel multi-color chromogenic substrate system for the widely used enzyme label horseradish peroxidase utilizing immobilized forms of color-generation reagents. This technology opens new opportunities for the application of enzyme -linked immunoassays to development of highly sensitive rapid diagnostic tests. The new detection technology will provide a level of sensitivity which is currently unattainable with conventional technologies, but preserving the simplicity, low cost, manufacturability and stability of conventional POC tests. Implementation of the new technology in POC tests will make it possible to develop rapid tests with very short assay time but with analytical sensitivity exceeding that of tests utilizing conventional colorimetric labels, such as colloidal gold or dyed latex particles, as well as microplate ELISA's. Moreover, the sensitivity is expected to compare favorably with the sensitivities of new sophisticated instrument-dependant technologies currently in development for rapid tests. The technology will be applicable to all major formats of current rapid diagnostic, lateral flow, flow through or dip-strip as well as bead filtration assays. The new stable substrate system with its unique multiplexing capabilities will produce bright, high-contrast, colored zones on diagnostic membranes ideally suited for either visual reading or densitometry. To validate applicability of the new detection technology to in vitro diagnostic tests, a new serological lateral flow test for Lyme disease will be developed. At present, there is no point-of-care test for Lyme disease on the market with clinically useful sensitivity and specificity. The new rapid test will utilize two peptide antigens, the C6 peptide of VLsE and the C10 peptide of OspC, and will provide a significant improvement in the diagnostic sensitivity for early phases of the disease over current methods including microplate ELISA's. Turnaround time for the rapid test will be 3-5 minutes, and whole blood as sample type will allow point-of-care use. Performance characteristics of the new rapid test will be evaluated using well-characterized serum samples from Lyme patients with culture-confirmed or physician documented early stage Lyme disease, and on control populations.

Public Health Relevance:
This project is aimed at development of a new colorimetric detection technology suitable for point-of-care diagnostic tests, which offers significantly greater sensitivity than current methods. This technology will provide a rapid turnaround, cost-effective solution for point-of-care testing using multicolor visual labels. As a first application, a rapid test for Lyme disease will be developed based on peptide antigens, which will be the first to enable accurate point-of-care serodiagnosis for early Lyme disease.

Thesaurus Terms:
Atgn; Antibodies; Antigens; Assay; Bedside Testings; Bioassay; Biologic Assays; Biological Assay; Blood Plasma; Blood Serum; C10; Characteristics; Chemistry; Chromogenic Substrates; Clinical Sensitivity; Colloidal Gold; Color; Coloring Agents; Densitometry; Detection; Development; Diagnostic; Diagnostic Sensitivity; Diagnostic Tests; Diffusion; Disease; Disorder; Dot Immunoblotting; Dyes; Elisa; Enzyme Immunoassay; Enzyme-Linked Immunosorbent Assay; Enzymes; Evaluation; Filtration; Fractionation, Filtration; Generations; Gold Colloid; Hrp; Horseradish Peroxidase; Immunoassay; Immunoblotting, Dot; Immunologic, Enzyme Linked Immunoassay; In Vitro; Label; Lateral; Latex Particles; Link; Lyme Borreliosis; Lyme Disease; Marketing; Medical; Membrane; Methods; Ospc; Ospc Protein; Outer Surface Protein C; Patients; Peptides; Performance; Peroxidases; Phase; Physicians; Plasma; Population Control; Publications; Reading; Reagent; Reticuloendothelial System, Serum, Plasma; Sampling; Science Of Chemistry; Scientific Publication; Sensitivity And Specificity; Serodiagnoses; Serologic; Serological; Serological Diagnosis; Serum; Serum, Plasma; Solutions; Specificity; Staging; Staining Method; Stainings; Stains; System; System, Loinc Axis 4; Technology; Testing; Testings, Bedside; Time; Visual; Whblood; Whole Blood; Base; Cost; Diagnostic Serology; Disease/Disorder; Dot Blotting; Immunogen; Instrument; Membrane Structure; New Technology; Novel; Ospc,; Point Of Care; Point Of Care Testing; Point-Of-Care Diagnostics; Public Health Relevance

Lyme disease is the most prevalent vector-borne disease in the U.S., with about 30,000 cases per year reported by CDC and a range now covering 24 states. Early diagnosis and treatment of Lyme disease is important to prevent the disease from progressing to a systemic infection characterized by chronic arthritis, carditis or neurological disorders. Where a patient does not truly have Lyme disease, an accurate negative test result would be valuable in promptly directing a physician to seek other causes of illness and hopefully minimize unnecessary antibiotic therapy. No Lyme rapid test suitable for physician's office use is commercially available, and the current laboratory-based two-tier Lyme testing algorithm (ELISA followed by Western blot) suffers from high cost, slow time to results (2-5 days), subjective interpretation and low sensitivity for early infections. Accordingly, there s a critical need for a rapid Lyme diagnostic test with improved performance. The overall objective of the proposed project is the development and clinical validation of a rapid, point-of-care test for Lyme disease that will for the first time make accurate Lyme testing accessible to any physician's office. In Phase I we have developed a prototype Lyme rapid test with 15 minute total turnaround time, which exhibited >99% specificity and sensitivity equal to or greater than that of FDA-cleared ELISA tests in both early and late stage infections. In particular, the Lyme C6/C10 rapid test provided high detection sensitivity for very early Lyme infections (<1 week post-onset of erythema migrans rash), significantly better than several FDA- cleared Lyme ELISA tests. The rapid Lyme test is based on three innovative components: 1) novel detection chemistry with ELISA-equivalent sensitivity, 2) a combination of C6 and C10 synthetic peptide antigens yielding high sensitivity for early infections, and 3) an immunoassay format that overcomes previous limitations of rapid tests for sensitive antibody detection. In Phase II we willoptimize and validate the rapid test for both whole blood and serum/plasma samples, to enable its use as a CLIA-waived test in a physician's office environment. Performance of the rapid test will be compared to that of the current two-tier Lyme testing algorithm in prospective and retrospective clinical studies to support a 510(k) submission for FDA clearance. Based on the high sensitivity and specificity which we expect to demonstrate in these studies, we will propose an FDA claim that the Lyme rapid test may substitute for one or both steps of the two-tier protocol. The Lyme C6/C10 rapid test will be the first commercial implementation of our novel rapid test technology, and will, for the first time, enable accurate, same-day Lyme disease diagnostic results which could lead to a basic paradigm shift in the standard protocol for Lyme testing.

Thesaurus Terms:
Algorithms;Antibiotic Therapy;Antibiotics;Antibodies;Antigens;Arthritis;Base;Bedside Testings;Biological Assay;Bite;Blood Tests;C10;Carditis;Centers For Disease Control And Prevention (U.S.);Chemistry;Chronic;Clinic;Clinical;Clinical Research;Consult;Cost;Design;Detection;Development;Diagnosis;Diagnostic;Diagnostic Tests;Disease;Documentation;Drug Formulations;Early Diagnosis;Early Treatment;Environment;Enzyme-Linked Immunosorbent Assay;Erythema Migrans;Evaluation;Exanthema;Exhibits;Fingers;Health;Heart Diseases;Housing;Immunoassay;Improved;In Vitro Testing;Infection;Innovation;Laboratories;Lateral;Lead;Lyme Disease;Lyme Infection;Meetings;Nerve;Nervous System Disorder;Novel;Patients;Performance;Performance Tests;Phase;Physicians;Physicians' Offices;Plasma;Point Of Care;Prevent;Procedures;Prospective;Prospective Studies;Protocols Documentation;Prototype;Public Health Relevance;Reagent;Reagent Testing;Relative (Related Person);Reporting;Research Study;Sampling;Scale Up;Sensitivity And Specificity;Serodiagnoses;Serum;Specimen;Staging;Symptoms;Synthetic Peptide;Systemic Infection;Technology;Test Result;Testing;Ticks;Time;Validation;Vector-Transmitted Infectious Disease;Western Blotting;Whole Blood;Work;Writing;