Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals

Abstract

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid (N) protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, a mAb-based bELISA was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition (PI) cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 6.95%, and 5.15%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with COVID-19-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals.

Digital Immunoassay for Rapid Detection of SARS-CoV-2 Infection in a Broad Spectrum of Animals

“…To evaluate the precision of the blocking biosensor assay for antibody detection, we tested a cohort of serum samples with known antibody statuses, which includes 86 known positive and 76 known negative samples from experimental cats. These samples were confirmed antibody statuses in our previous studies 29 . As shown in Figure 4A, receiver operating characteristic (ROC) analysis revealed that at PI value cut-off at 0.5877, the assay achieved a diagnostic sensitivity of 97.80% and a diagnostic specificity of 98.67%.…”

“…Purified protein was dialyzed using 1x phosphate-buffered saline (PBS) solution under 4℃ and then concentrated by polyethylene glycol 8000 (Thermo Fisher Scientific, 320 Waltham, MA). The SARS-CoV-2 N protein specific mAb #127-3 was generated in our previous study 6 .…”

“…Serological assays, especially enzyme linked immunosorbent assay (ELISA), are commonly utilized to detect specific host antibodies against SARS-CoV-2, providing insights into past virus exposure 5 . While indirect ELISA is highly sensitive and specific, its dependency on species-specific secondary antibodies limits its ability to detect antibodies across a diverse range of hosts 6 . The monoclonal antibody (mAb)-based blocking ELISA (bELISA) stands out for its increased specificity in detecting antibody responses from a broad spectrum of host species [6][7][8] .…”

Spatial and temporal clustering of anti-SARS-CoV-2 antibodies in Illinois household cats, 2021–2023

“…We suspected that there might be some possible transmissions between humans and cats during that period. Especially, the second seropositive peak could be related to human SARS-CoV-2 infections because about 14 days after being infected with SARS-CoV-2, the antibody level is detectable by our serological testing method [ 10 , 26 ], suggesting a possible human-to-cat transmission. However, we did not find any similarity between the positive case rates of human infections and cat seropositivity for the rest of the study period (between March 2022 and May 2023).…”

“…A blocking enzyme-linked immunosorbent assay (bELISA) for detecting SARS-CoV-2 specific antibody response was developed and validated in-house. The detailed method was described in our previous publication [ 26 ]. All the serum samples collected from the cats were initially screened by bELISA.…”