Establishment of a Sandwich ELISA for Detection of Pan-Merbecoviruses

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.

Keywords: antigen detection; bat merbecoviruses; monoclonal antibody; sandwich ELISA.

Figure 1

Phylogenetic tree of merbecoviruses. Phylogenetic trees were generated using the N gene sequences of coronaviruses via maximum-likelihood analysis combined with 1000 bootstrap replicates. Bootstrap values are indicated in the major nodes. Scale bars indicate nucleotide substitutions per site. Viruses colored blue were tested in sandwich ELISA.

Figure 2

Expression and purification of recombinant merbecovirus proteins. GST-tagged merbecovirus N proteins were expressed in E. coli. The proteins were purified using glutathione affinity column and the GST-tags were removed by digestion of PreScission proteases. The purities of recombinant proteins were subjected to SDS-PAGE.

Figure 3

Characterization of monoclonal antibodies (mAbs). (A) Purified mAbs obtained using Protein L affinity chromatography were analyzed by SDS-PAGE. (B) Amino acid sequences of the variable regions of the mAbs were determined by Sanger sequencing. Asterisks (*) indicate positions with identity among all three aligned sequences, while dots (·) indicate identity between two of the three.

Figure 4

Optimization of the sandwich ELISA antibody pair combinations. The bar graph represents the sandwich ELISA absorbance values (OD 450) obtained from different mAb pair combinations. The capture mAbs (13E8, 1A8, 10H6) and HRP-conjugated detection mAbs (13E8-HRP, 1A8-HRP, 10H6-HRP) were tested using rVsCoV-1-N (500 ng/mL) (A) and EjCoV-3-infected cell lysate (1:100 dilution) (B). Bars indicate the mean absorbance values (OD 450) (n = 3). The signal-to-background (S/B) ratio for each mAb pair is annotated above the respective bars. (C) A contour plot illustrates the signal-to-background (S/B) ratio across various combinations of 1A8 and 10H6-HRP concentrations. Higher S/B ratios (shown in white arrow) indicate better assay performance.

Figure 5

Evaluation of sandwich ELISA for merbecovirus detection. (A) ELISA absorbance values (OD 450) for lysates from various coronavirus-expressing cells. Bars indicate mean ± SD (n = 3). Lysates were tested at dilutions of 1, 1/10, 1/100, and 1/1000. (B) ELISA absorbance values for purified recombinant N proteins of various coronaviruses diluted from 500 to 7.8125 ng/mL (2-fold). Bars represent mean values (n = 3). PBS-T served as the negative control. The dashed line shows the cut-off value, defined as the mean of PBS-T plus 3 SD. (C) Detection limit determination for rVsCoV-1-N. The x-axis shows antigen concentration (ng/mL), and the y-axis indicates absorbance (OD 450). The dashed line represents the cut-off value (PBS-T mean + 3 SD). (D) Detection sensitivity for infectious EjCoV-3 virus lysate. The x-axis shows dilution factors, and the y-axis shows absorbance (OD 450). The dashed line indicates the cut-off value (mock-infected control mean + 3 SD).