One-pot pre-coated interface proximity extension assay for ultrasensitive co-detection of anti-SARS-CoV-2 antibodies and viral RNA

In the field of in vitro diagnostics, detection of nucleic acids and proteins from biological samples is typically performed with independent platforms; however, co-detection remains a major technical challenge. Specifically, during the coronavirus disease 2019 (COVID-19) pandemic, the ability to simultaneously detect viral RNA and human antibodies would prove highly useful for efficient diagnosis and disease course management. Herein, we present a multiplex one-pot pre-coated interface proximity extension (OPIPE) assay that facilitates the simultaneous recognition of antibodies using a pre-coated antigen interface and a pair of anti-antibodies labeled with oligonucleotides. Following anti-antibody-bound nucleic acid chain extension to form templates in proximity, antibody signals can be amplified, together with that of targeted RNA, via a reverse transcription-polymerase chain reaction. Using four-color fluorescent TaqMan probes, we demonstrate the co-detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies and viral nucleic acids in a single bio-complex sample, including nucleocapsid protein-specific IgG and IgM, and the RNA fragments of RdRp and E genes. The serum detection limit for this platform is 100 fg/mL (0.67 fM) for the anti-SARS-CoV-2 antibody and 10 copies/μL for viral RNA. The OPIPE assay offers a practical and affordable solution for ultrasensitive co-detection of nucleic acids and antibodies from the same trace biological sample without the additional requirement of complicated equipment.