Abstract
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.
Keywords: Antibody; Co-detection; Immunoassay; Pre-coated solid interface; SARS-CoV-2; Viral RNA.
【저자키워드】 SARS-CoV-2, antibody, immunoassay, Viral RNA, Co-detection, Pre-coated solid interface, 【초록키워드】 COVID-19, coronavirus disease, Coronavirus disease 2019, coronavirus, pandemic, antibody, equipment, Diagnosis, in vitro, severe acute respiratory syndrome Coronavirus, diagnostics, nucleocapsid protein, Antigen, RNA, Protein, immunoassay, polymerase chain reaction, nucleic acid, serum, Viral, anti-SARS-CoV-2 antibody, nucleic acids, nucleocapsid, management, RdRP, Viral RNA, respiratory, multiplex, platform, specific antibodies, Reverse transcription-polymerase chain reaction, Detection limit, biological samples, IgG and IgM, Chain Reaction, acute respiratory syndrome, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, disease course, viral nucleic acid, probes, oligonucleotides, fluorescent, pair, extension, TaqMan, viral nucleic acids, offer, independent, e genes, amplified, performed, detect, facilitate, RNA fragment, biological sample, 【제목키워드】 proximity extension assay,