Gold conjugated nanobodies in a signal-enhanced lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples

Abstract

Despite the transfer of COVID-19 from the pandemic to control, we are still in a state of uncertainty about long-term success. Therefore, there is a great need for rapid and sensitive diagnostics to sustain the control status. After several optimization trials, we developed lateral flow test (LFT) strips for rapid detection of SARS-CoV-2 spike 1 (S1) antigen in saliva samples. For signal enhancement of our developed strips, we applied dual gold conjugates. Gold-labeled anti-S1 nanobodies (Nbs) were employed as S1 detector conjugate, while gold-labeled angiotensin-converting enzyme 2 (ACE2) was used as S1 capturing conjugate. In a parallel strip design, we used an anti-S1 monoclonal antibody (mAb) as an antigen detector instead of anti-S1 Nbs. Saliva samples were collected from 320 symptomatic subjects (180 RT-PCR confirmed positive cases and 140 confirmed negative cases) and were tested with the developed strips. In early detection for positive samples with cycle threshold (Ct ≤ 30), Nbs-based LFT strips showed higher sensitivity (97.14%) and specificity (98.57%) than mAb-based strips which gave 90.04% sensitivity and 97.86% specificity. Moreover, the limit of detection (LoD) for virus particles was lower for Nbs-based LFT (0.4 × 104 copies/ml) than for the mAb-based test (1.6 × 104 copies/ml). Our results are in favor of the use of dual gold Nbs and ACE2 conjugates in LFT strips. These signal-enhanced strips offer a sensitive diagnostic tool for rapid screening of SARS-CoV-2 S1 antigen in the easily collected saliva samples.

Modified streptavidin–biotin based lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples

“…Due to their lower sensitivity, developing highly sensitive, rapid, and reliable techniques for COVID-19 diagnosis is a significant step towards early diagnosis and prevention of further infections, especially among healthcare workers (HCWs) 13 . In our previous work, we developed an enhanced signal strip for the detection of SARS-CoV-2 SI antigen in saliva from patients confirmed by RT-PCR, using gold-conjugated Nbs and ACE-2 as a matched pair of antigen probes 6 . This strip resulted in a sensitivity of 91.66% and specificity of 98.57% with a limit of viral detection (LVD) of 0.2 × 104 copies/ml.…”

“…Although sandwich ELISA is well-known and widely applied 25 , the assay approach utilizing the streptavidin–biotin interaction exhibited a tenfold higher sensitivity and reduced costs by 20-fold compared to RT-PCR 26 . We introduced several modifications for our previously developed enhanced signal strip, to enhance the sensitivity and specificity, while also reducing the cost of SARS-CoV-2 S1 antigen detection in saliva 6 . These modifications included bypassing several fabrication steps such as dispersing the conjugate pad, optimizing the strip, and employing a small sample volume (5 µl).…”

“…Due to their small size, simple structure, high antigen binding affinity, and remarkable stability in extreme conditions, using nanobodies overcame several of the limitations of conventional monoclonal antibodies and gave promising results in ELISA with overall sensitivity and specificity of 88.7% and 100%, respectively 5 . Subsequently, we developed an enhanced LFTS using dual gold conjugation of ACE-2 and Nbs that resulted in 91.66% sensitivity and 97.57% specificity 6 .…”

A High-Avidity, Thermostable, and Low-Cost Synthetic Capture for Ultrasensitive Detection and Quantification of Viral Antigens and Aerosols

“…Previously, many groups have reported the use of aptamers (nucleic acid affinity probes) and miniproteins (synthetic protein affinity probes) as captures for LFAs. − Compared to antibodies, both captures are thermally stable, economical, and comparably easier to synthesize in vitro using systematic evolution of ligands by exponential enrichment for aptamers and phage display for miniproteins. , However, recent advancements in protein engineering have made it possible to design miniproteins completely denovo, albeit with certain limitations in analyte size and complexity. , Thus, we wanted to explore the potential of denovo protein engineering for rapid deployment of thermally stable LFAs by developing a robust synthetic capture based on affibody (<100 amino-acid-long, highly thermostable 3 or 4-fold α helix bundles) technology with the purpose of detecting the SARS-CoV-2 spike protein.…”