Development of a SARS-CoV-2-specific biosensor for antigen detection using scFv-Fc fusion proteins

Abstract

Coronavirus disease 2019 (COVID-19) is a newly emerged human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a global pandemic, development of a cheap, rapid, accurate, and easy-to-use diagnostic test is necessary if we are to mount an immediate response to this emerging threat. Here, we report the development of a specific lateral flow immunoassay (LFIA)-based biosensor for COVID-19. We used phage display technology to generate four SARS-CoV-2 nucleocapsid protein (NP)-specific single-chain variable fragment-crystallizable fragment (scFv-Fc) fusion antibodies. The scFv-Fc antibodies bind specifically and with high affinity to the SARS-CoV-2 NP antigen, but not to NPs of other coronaviruses. Using these scFv-Fc antibodies, we screened three diagnostic antibody pairs for use on a cellulose nanobead (CNB)-based LFIA platform. The detection limits of the best scFv-Fc antibody pair, 12H1 as the capture probe and 12H8 as the CNB-conjugated detection probe, were 2 ng antigen protein and 2.5 × 10 4 pfu cultured virus. This LFIA platform detected only SARS-CoV-2 NP, not NPs from MERS-CoV, SARS-CoV, or influenza H1N1. Thus, we have successfully developed a SARS-CoV-2 NP-specific rapid diagnostic test, which is expected to be a simple and rapid diagnostic test for COVID-19.

Novel approaches for rapid detection of COVID-19 during the pandemic: A review

“…A research work focused on LFIA-based biosensor for COVID-19 detection was conducted by Kim et al [ 83 ]. They utilized phage technology to produce four SARS-CoV-2 nucleocapsid protein (NP)-specific single-chain variable fragment-crystallizable fragment (scFv-Fc) fusion antibodies.…”

“…In addition, the results indicated that the LFIA biosensor could successfully distinguish between SARS-CoV-2 positive and negative samples. The detection limit of LFIA biosensor would meet the conditions for the clinical use through optimization [ 83 ]. The effect of applying nanoparticles on sensitivity and selectivity of LFIA detection is investigated by Huang et al [ 26 ].…”

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy

“…Phage display-derived antibodies are among the multiple approaches quickly deployed to generate monoclonal antibodies directed to SARS-CoV-2 ( Jiang et al., 2020 ). In this emergence scenario, anti-nucleocapsid protein antibodies have been generated through phage display for diagnostic purposes ( Kim et al., 2021 ). However, the most efforts have been focused on spike protein, and specifically to the receptor binding domain (RBD), to directly block the interaction with the host receptor, aiming to select neutralizing antibodies for novel therapeutics development.…”

State-of-the-art colloidal particles and unique interfaces-based SARS-CoV-2 detection methods and COVID-19 diagnosis

“…ScFv-Fc antibodies are specific to SARS-CoV-2-NP antigen with high affinity, and three specific antibody pairs are used on the cellulose nanosphere (CNB)-based LFIA kit. The detection limits for the best pair are found as 2 ng of antigen protein and 2.5 x 104 pfu of the cultured virus ( 66 ).…”