A rapid simple point-of-care assay for the detection of SARS-CoV-2 neutralizing antibodies

Abstract

Background Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive. Method We develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor—RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper. Results Here we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma. Conclusions A rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens.

Defining Factors that Influence vaccine-induced, cross-variant neutralizing antibodies for SARS-CoV-2 in Asians

“…4a-d ). Using 50% pseudovirus neutralization as the threshold to distinguish neutralizers versus non-neutralizers, the ACE2 inhibition ELISA has a sensitivity of 87.8% and specificity of 81.3% at 40% inhibition, which is employed as a threshold to define neutralizing response 30 ( Supplementary Fig. 4e , f ).…”

Finger stick blood test to assess postvaccination SARS‐CoV‐2 neutralizing antibody response against variants

“…The expression and purification of soluble extracellular fragment of human ACE2 (residues 19–615; GenBank: AB046569.1) and WT SARS‐CoV2‐Spike (EMBL: QHD43416.1 with silent mutations c.A1452>G and c.T1470>C) RBD fused to CBD followed the same protocol as described in Kongsuphol et al 10 Similarly, alpha c.A1501>T (p.N501Y), beta c.A1501>T, c.G1251>C, c.G1450>A (p.N501Y K417N E484K), gamma c.A1501>T, c.A1250>C, c.G1450>A (p.N501Y K417T E484K), delta c.T1355>G, c.C1433>A (p.L452R T478K), kappa c.T1355>G, c.G1450>C (p.L452R E484Q), epsilon c.T1355>G (p.L452R), delta plus c.T1355>G, c.C1433>A, c.G1450>A (p.L452R T478K K417N), eta c.G1450>A (p.E484K), lambda c.T1355>A c. T1469>C (p.L452Q, F490S), and AD c.A1501>T, c.C1433>A (p.N501Y T478K) RBD‐CBD variants were expressed in Expi293F cells (Thermo Fisher Scientific; A1435101) according to the supplier's protocol. The purification protocol followed that of WT RBD‐CBD.…”

Finger stick blood test to assess post vaccination SARS-CoV-2 neutralizing antibody response against variants

“…The expression and purification of soluble extracellular fragment of human ACE2 (residues 19–615; GenBank: AB046569.1) and wildtype (WT) SARS-CoV2-Spike (EMBL: QHD43416.1 with silent mutations c.A1452>G and c.T1470>C) RBD fused to CBD followed the same protocol as described in Kongsuphol et al 10 . Similarly, alpha c.A1501>T (p.N501Y), beta c.A1501>T, c.G1251>C, c.G1450>A (p.N501Y K417N E484K), gamma c.A1501>T, c.A1250>C, c.G1450>A (p.N501Y K417T E484K), delta c.T1355>G, c.C1433>A (p.L452R T478K), kappa c.T1355>G, c.G1450>C (p.L452R E484Q), epsilon c.T1355>G (p.L452R), delta plus c.T1355>G, c.C1433>A, c.G1450>A (p.L452R T478K K417N), eta c.G1450>A (p.E484K), lambda c.T1355>A c. T1469>C (p.L452Q, F490S) and AD c.A1501>T, c.C1433>A (p.N501Y T478K) RBD-CBD variants were expressed in Expi293F cells (Thermo Fisher Scientific, A1435101) according to the supplier’s protocol.…”

“…We applied the same protocol for production of SARS-CoV-2 pseudotyped lentiviral particles and pseudovirus neutralization assay as previously reported 10 . Briefly, to produce SARS-CoV-2 pseudovirus, HEK293T cells at 36 ×10 6 cell density were transfected with 27 µg pMDLg/pRRE (Addgene, #12251), 13.5 µg pRSV-Rev (Addgene, #12253), 27 µg pTT5LnX-WHCoV-St19 (SARS-CoV2 Spike) and 54 µg pHIV-Luc-ZsGreen (Addgene, #39196) using Lipofectamine 3000 (Invitrogen, #L3000-150).…”

“…We previously developed a rapid paper-based SARS-CoV-2 neutralization assay known as cellulose pulled-down virus neutralization test (cpVNT) that detects SARS-CoV-2 neutralizing antibody (nAb) in plasma or serum within 10 minutes 10 . The principle of cpVNT is based on the complex formation between the receptor binding domain (RBD) of the SARS-CoV-2 and the angiotensin converting enzyme II receptor (ACE2) of the host cell.…”