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

> **NIH NIH R43** · KEPHERA DIAGNOSTICS, LLC · 2020 · $299,829

## Abstract

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 over 300,000 infections per year
according to 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 results in 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 for this purpose leads to physicians
frequently prescribing antibiotic treatment in the absence of definitive lab results. As the vast majority of the
roughly three million annual Lyme tests in the U.S. are carried out on individuals who do not 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
both sensitivity and specificity to 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 this Phase I project, we will develop a prototype ELISA assay, proving feasibility by demonstrating sensitivity
and specificity on retrospective samples comprising Lyme patients and controls, in comparison with two-tier
testing results. In Phase II, we will complete development of a commercial product, carry out prospective and
retrospective clinical studies and submit an application to FDA for in vitro diagnostic cla...

## Key facts

- **NIH application ID:** 9886194
- **Project number:** 5R43AI142903-02
- **Recipient organization:** KEPHERA DIAGNOSTICS, LLC
- **Principal Investigator:** Andrew E. Levin
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $299,829
- **Award type:** 5
- **Project period:** 2019-03-06 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9886194

## Citation

> US National Institutes of Health, RePORTER application 9886194, Hybrid ELISA: Simple and specific one-tier assay for Lyme disease (5R43AI142903-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9886194. Licensed CC0.

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