# Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses

## TL;DR

Scientists developed a new test to detect a group of bat viruses that can jump to humans, offering a fast and adaptable tool for monitoring and pandemic preparedness.

## Contribution

A novel sandwich ELISA targeting pan-merbecoviruses with high specificity and sensitivity is established.

## Key findings

- Monoclonal antibodies 1A8/10H6 effectively detect N proteins from six merbecoviruses with LODs below 7.81 ng/mL.
- The ELISA shows no cross-reactivity with SARS-CoV-2 or endemic human coronaviruses.
- The test can detect infectious EjCoV-3 at 1.3 × 10³ PFU/mL and is suitable for high-throughput and point-of-care use.

## Abstract

Merbecovirus, a subgenus of Betacoronavirus, includes MERS-CoV and multiple bat-derived viruses with zoonotic potential. Given the unpredictable emergence of these viruses and their genetic diversity, development of broad-spectrum diagnostic tools is expected. In this study, we established a sandwich ELISA targeting the nucleocapsid (N) protein of merbecoviruses. We generated monoclonal antibodies (mAbs) using recombinant N protein of a bat merbecovirus, VsCoV-1, and selected cross-reactive clones for other merbecoviruses. Three mAbs showed strong reactivities with multiple merbecoviruses but not with SARS-CoV-2 or endemic human coronaviruses. Pairwise ELISA screening identified 1A8/10H6 mAbs as the optimal combination for detection of N protein from six merbecoviruses—VsCoV-1, EjCoV-3, MERS-CoV, NeoCoV, HKU4, and HKU5—with limits of detection (LODs) below 7.81 ng/mL, including 1.25 ng/mL for VsCoV-1. Infectious bat merbecovirus EjCoV-3 was detected at 1.3 × 103 PFU/mL. No cross-reactivity was observed with non-merbecoviruses, indicating its high specificity. This sandwich ELISA offers a rapid, reproducible, and cost-effective diagnostic platform with potential for high-throughput screening and automation. Moreover, its design is amenable to adaptation into point-of-care formats such as lateral flow assays, highlighting its value for field-based surveillance and pandemic preparedness.