Hybrid ELISA: Simple and specific one-tier assay for Lyme disease

Abstract Lyme disease, caused by infection with the spirochete Borrelia burgdorferi or closely related species, is the most common vector-borne disease in the United States, accounting for nearly 500,000 infections per year according to recent public health estimates. It is also one of the few infectious diseases that requires a two-step laboratory testing protocol, comprising a screening assay, typically ELISA, followed by a confirmatory immunoblot. This protocol is followed where the disease has progressed beyond the hallmark erythema migrans rash, or the rash cannot be identified, and symptoms or history suggest Lyme disease. The two-step protocol is a result of the poor specificity of most Lyme screening tests, which lead to a high false positive rate. This in turn is due to the cross-reactive nature of many bacterial antigens, combined with the limitations of conventional ELISA immunochemistry. In principle, a confirmatory result must be obtained prior to initiating treatment, but in practice, the multi-day delay that is often incurred in this process frequently leads physicians to prescribe antibiotic treatment in the absence of definitive lab results. As the vast majority of the more than three million annual Lyme tests in the U.S. are carried out on individuals who are ultimately found not to have Lyme disease, this practice leads to the unnecessary prescription and use of antibiotics, contributing to the growth of antibiotic resistance which has become a significant threat to public health. A first step test with significantly higher specificity would avoid the need for a second step test and enable clinicians to make informed treatment decisions in a timely manner, based on credible test results. This project is aimed at bringing about a significant change in medical practice by reducing Lyme testing from the current two-step process to a one-step process. To achieve this objective, we have developed a novel ELISA methodology for Lyme antibody detection that enables exceptionally high assay specificity. The test is based on well-established, highly specific and sensitive Borrelia antigens in a unique immunochemical format. The novel ELISA immunochemistry eliminates almost all non-specific reactivity, yielding results comparable in specificity but higher in sensitivity than those obtained with the conventional two-tier testing protocol. Consequently, this assay promises to deliver a one-step testing solution for Lyme disease, at a time when alternatives to the original two-step method are gaining legitimacy at the scientific as well as regulatory level. In Phase I, we developed a prototype ELISA assay, proving feasibility by demonstrating higher sensitivity and equivalent specificity on retrospective samples comprising Lyme patients and controls, in comparison with two- tier testing results. In Phase II, we will complete development of the assay into a commercial product, carry out prospective and retrospective clinical studies and submit a 510(k) application to FDA for use of the ELISA as a one-step test for Lyme disease, enabling commercial launch upon approval.