Development and preliminary clinical validation of A29L mAb-based ELISA and lateral flow immunoassays for monkeypox virus detection.

The monkeypox virus (MPXV) poses a significant and ongoing public health threat, highlighting the urgent need for accessible and accurate diagnostic tools. The viral envelope protein A29L is an immunodominant and highly conserved target for immunoassay development. This study aimed to generate a panel of novel monoclonal antibodies (mAbs) against A29L and to develop both a highly sensitive laboratory-based enzyme-linked immunosorbent assay (ELISA) and a rapid lateral flow immunoassay (LFIA) for specific antigen detection. Following immunization and hybridoma screening, six high-affinity mAbs against A29L were selected and fully characterized. Utilizing this antibody panel, a sandwich ELISA was developed, achieving a notably low detection limit of 31.25 pg/mL, with an excellent linear range. In clinical evaluations using samples from suspected MPXV cases (plasma, vesicular fluid, and swabs), the ELISA demonstrated 95% sensitivity and 100% specificity for plasma samples, performing comparably to gold-standard PCR, and outperforming available commercial kits. Furthermore, the developed LFIA demonstrated sensitivities of 58.3% (7/12) in plasma and 40.0% (4/10) in vesicular fluid samples, with perfect detection in high viral load cases, confirming its strong potential for point-of-care deployment. In conclusion, we present a practical, tiered diagnostic strategy that integrates a highly sensitive confirmatory ELISA with a rapid frontline LFIA. This complementary approach effectively bridges the critical gap between centralized confirmatory testing and decentralized field surveillance, significantly enhancing outbreak response capabilities for MPXV.IMPORTANCEMonkeypox virus has emerged as a global infectious disease threat, creating an urgent need for accurate and accessible diagnostic tools. Currently, the confirmation of an infection necessitates the execution of sophisticated laboratory tests, which can result in delayed results and impede a swift public health response, particularly in settings characterized by limited resources. This study addresses this critical gap by creating a complete diagnostic toolkit. We developed new, highly specific antibodies against the virus and used them to build two complementary tests: a very sensitive lab-based test for definitive confirmation and a simple, rapid paper-strip test that can be used at the point of care, such as in a clinic or field site. This dual approach provides a practical strategy for improving outbreak control, enabling both accurate laboratory diagnosis and immediate on-site screening to quickly identify infected individuals and help stop the chains of transmission.