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

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 × 104 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.

A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a newly emerging human infectious disease. Because no specific antiviral drugs or vaccines are available to treat COVID-19, early diagnostics, isolation, and prevention are crucial for containing the outbreak. Molecular diagnostics using reverse transcription polymerase chain reaction (RT-PCR) are the current gold standard for detection. However, viral RNAs are much less stable during transport and storage than proteins such as antigens and antibodies. Consequently, false-negative RT-PCR results can occur due to inadequate collection of clinical specimens or poor handling of a specimen during testing. Although antigen immunoassays are stable diagnostics for detection of past infection, infection progress, and transmission dynamics, no matched antibody pair for immunoassay of SARS-CoV-2 antigens has yet been reported. In this study, we designed and developed a novel rapid detection method for SARS-CoV-2 spike 1 (S1) protein using the SARS-CoV-2 receptor ACE2, which can form matched pairs with commercially available antibodies. ACE2 and S1-mAb were paired with each other for capture and detection in a lateral flow immunoassay (LFIA) that did not cross-react with SARS-CoV Spike 1 or MERS-CoV Spike 1 protein. The SARS-CoV-2 S1 (<5 ng of recombinant proteins/reaction) was detected by the ACE2-based LFIA. The limit of detection of our ACE2-LFIA was 1.86 × 105 copies/mL in the clinical specimen of COVID-19 Patients without no cross-reactivity for nasal swabs from healthy subjects. This is the first study to detect SARS-CoV-2 S1 antigen using an LFIA with matched pair consisting of ACE2 and antibody. Our findings will be helpful to detect the S1 antigen of SARS-CoV-2 from COVID-19 patients.

Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor.

Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV). Based on the rapid increase in the rate of human infection, the World Health Organization (WHO) has classified the COVID-19 outbreak as a pandemic. Because no specific drugs or vaccines for COVID-19 are yet available, early diagnosis and management are crucial for containing the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clinical samples. The sensor was produced by coating graphene sheets of the FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was determined using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Our FET device could detect the SARS-CoV-2 spike protein at concentrations of 1 fg/mL in phosphate-buffered saline and 100 fg/mL clinical transport medium. In addition, the FET sensor successfully detected SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 × 101 pfu/mL) and clinical samples (LOD: 2.42 × 102 copies/mL). Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a highly sensitive immunological diagnostic method for COVID-19 that requires no sample pretreatment or labeling.