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

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.

Introduction

COVID-19 affects > 200 million people and, to date, has killed > 4 million, worldwide. To prevent transmission of SARS-CoV-2—the virus that causes COVID-19—tight restrictions on movement and social interactions have been placed on populations across the globe. While this has had some effect on preventing the spread of the virus, they have plunged the global economy into a severe contraction. A phased relaxation of these social control measures is critical to allow business, and the world economy, to recover.

Achieving herd immunity against SARS-CoV-2, either naturally or through vaccination, is the ultimate long-term goal that will allow lifting of the widespread social control measures currently applied. Neutralizing antibodies (NAbs) are generated in response to either exposure to the virus, or to a vaccine. For effective prevention of viral infections, NAbs must be generated in sufficient quantity[1]. Screening populations for the presence of NAbs is essential to evaluate herd immunity against SARS-CoV-2, and to assess the effectiveness of vaccine immunization programmes, deployed in many countries since late 2020. To facilitate rapid screening of SARS-CoV-2 NAbs, NAbs detection tests that can be performed simply, rapidly and at low cost are highly desired.

Currently, NAbs are generally detected using virus neutralization tests (VNTs). Standard VNTs require handling of live virus (conventional VNT (cVNT)) or pseudovirus (pVNT), BSL3/BSL2 facility, skilled personnel, and 2–4 days processing time[2-5] making them unsuitable for mass testing the immune status of a population. SARS-CoV-2 initiates the process of host cell entry, by interacting with angiotensin-converting enzyme II (ACE2) receptors on the host cell via the receptor-binding domain (RBD) of the spike (S) protein[1,2,4,6-9]. Based on this observation, a rapid (1–2 h) plate-based ELISA, surrogate SARS-CoV-2 neutralization test (sVNT) has been developed using recombinant hACE2 receptor and viral RBD proteins[10]. NAbs are detected by their ability to bind RBD and block the formation of RBD/hACE2 complexes. Though much more rapid than the standard VNTs, the sVNT still require a laboratory setting and skilled personnel, presenting a barrier to large-scale screening.

Here we report a rapid cellulose pull-down viral neutralization test (cpVNT) that detects SARS-CoV-2 NAbs in plasma samples within 10 min and can be used at the point of care (POC). The test principle is based on the interaction of (i) RBD tagged cellulose-binding domain (CBD) (ii) ACE2 receptor tagged biotin (BA) and (iii) streptavidin conjugated horseradish peroxidase (SA-HRP), to detect NAbs binding to the RBD on cellulose paper. Despite the simplified and very rapid testing procedure, the cpVNT exhibits comparable performance to the lab-based tests in determining the level of NAbs in COVID-19 convalescent plasma samples with accuracy well above 80% and 90%, compared to pVNT and sVNT, respectively.

Methods

Materials

Materials were purchased from the following sources, mouse anti-SARS-CoV-2 NAb (cat# 40591-MM43-100), monoclonal mouse anti Influenza A H10 Hemagglutinin/HA NAb (cat# 40359-M001), monoclonal mouse anti Influenza A Nucleoprotein IgG (cat# 11675-MM03T) and polyclonal rabbit anti-SARS-CoV-2 nucleocapsid protein IgG (cat# 40588-T62) from Sino Biological, USA; monoclonal rabbit anti MERS Coronavirus Spike protein NAb (cat# MA5-29975) and polyclonal rabbit anti-Dengue Virus Type 2 NS3 protein IgG (cat# PA5-32199) from Invitrogen, USA; polyclonal rabbit anti-Zika virus NS5 protein IgG (cat# GTX133312), polyclonal rabbit anti-Zika virus NS3 protein IgG (cat# GTX133309), monoclonal mouse anti-Dengue virus envelope protein IgG (cat# GTX629117) and monoclonal mouse anti-SARS-CoV-2 spike protein IgG (cat# GTX632604) from GeneTex, USA. Other chemicals were of analytical grades from Merck, Singapore, otherwise stated.

Collection of clinical samples

The collection of COVID-19 convalescent samples were reviewed and approved under the DSRB reference # 2020/00120, National University of Singapore (NUS). Subjects with COVID-19 PCR positive results were recruited for this study. Informed consents including the use of clinical information were obtained from the participants for the COVID-19 convalescent samples. Peripheral blood was collected in EDTA blood tubes and subsequently diluted with an equal amount of sterile PBS. This was then gently layered on top of 13 mL Ficoll-Plaque density gradient media (GE Healthcare) in a 50 mL Falcon tube. The tube was centrifuged at 2400 rpm for 30 min with acceleration and deceleration set at 0. Plasma was harvested from the top layer and stored at −80 °C. Buffy coat layer was washed with sterile PBS at 2000 rpm for 6 min followed by another wash at 1500 rpm for 5 min. Peripheral blood mononuclear cells (PBMNCs) were harvested, resuspended in freezing media containing 90% FBS (Hyclone) + 10% dimethyl sulfoxide (Sigma Aldrich), and stored in liquid nitrogen.

Collection of pre-COVID samples were reviewed and approved by the Institutional Review Board of Nanyang Technological University, Singapore (IRB 003/2010, IRB 11/08/03, IRB 13/09/01, IRB-2016-01-045 and IRB-2020-11-047). The whole blood was donated by healthy adult volunteers at the National University Hospital, Singapore. Informed consents were obtained from all donors in accordance with the approved protocols. Whole blood samples collected were centrifuged at 2000 rpm for 10 min. Plasma was collected and stored at −80 °C until used.

Isolation and cloning of SARS-CoV-2 Spike RBD-specific human antibodies

Memory B cells were isolated from PBMNC derived from blood samples drawn from COVID-19 convalescent patients using a Human Memory B cell isolation kit (Miltenyi Biotec, #130-093-546). Small pools of purified Memory B cells were seeded into 384-well plates on irradiated CD40L-expressing feeder cells for differentiation into plasma cells as described previously[11]. After 7 days of culture, supernatants from B cell pools were screened for binding activity on SARS-CoV-2 Spike by ELISA. Antibody Heavy and Light Chain variable regions were cloned from positive wells by PCR (Collibri™ Stranded RNA Library Prep Kit for Illumina™ Systems) and whole human IgG reconstructed as described previously[12]. Confirmation of binding specificity of cloned human monoclonal antibodies was confirmed by ELISA.

Protein expression and purification

The soluble extracellular fragment of human ACE2 (residues 19–615; GenBank: AB046569.1) was cloned into a modified pHLSec[13] mammalian expression vector following an N-terminal monoFc, hexahistidine tag and Tobacco Etch Virus (TEV) protease cleavages site. SARS-CoV2-Spike RBD[14] fused to CBD (residues 276-434 of Hungateiclostridium thermocellum CipA) was cloned into the pHLmMBP-10 vector[15] (a gift of Luca Jovine; Addgene plasmid 72348) which encodes an N-terminal octahistidine tag, codon-optimized maltose-binding protein (MBP) tag and a TEV site. The coding sequence for the single-chain variable fragment (scFv) of the anti-SARS-CoV CR3022[16], and was subcloned into pHLmMBP-10 to generate an MBP-scFv fusion construct. Verified plasmids were transfected into Expi293F cells by using the Expifectamine293 transfection kit (ThermoFisher Scientific, #A1435101) to express the secreted proteins following the supplier’s standard protocol. Cells were harvested by centrifugation after 6 days of transfection and the supernatants were collected for protein purification. The media were conditioned for Ni-NTA binding by adding 2.5 mL of conditioning buffer, 200 mM HEPES pH 7.5, 3 M NaCl and 10% glycerol; 10 µL mammalian protease inhibitor cocktail (Nacalai Tesque, #25955-11) per 50 mL media. Proteins were first purified by affinity chromatography using Ni-NTA cartridges (Qiagen, #1046323), followed by size exclusion chromatography by using HiLoad 16/60 Sephadex 200 (Cytiva, formerly GE Healthcare) in gel filtration buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol). To avoid protein crosslinking and aggregation, pooled fractions were supplemented with 0.5 mM TCEP before being concentrated by using Vivaspin centrifugal concentrators (Cytiva). The His-MBP tag of sRBD-CBD was cleaved off by using TEV protease (a gift of NTU Protein Production Platform, proteins.sbs.ntu.edu.sg) at 4 °C overnight with 1:40 mass ratio. Untagged sRBD-CBD was separated from His-tagged proteins by passing the reaction mixture through HisPur-Ni-NTA resin (Thermo Scientific, #88222) pre-equilibrated in 20 mM HEPES pH 7.5, 300 mM NaCl, 10 mM Imidazole. The purified sRBD-CBD sample was buffer exchanged and concentrated in 20 HEPES pH 7.5, 300 mM NaCl, 10% glycerol and 0.5 mM TCEP for storage.

SARS-CoV-2 N protein (residues 1–419; GenBank: YP_009724397.2) was synthesized by Genewiz (USA) and cloned into pET28b(+) bacterial expression vector following a hexahistidine tag and a thrombin cleavage site. Constructed plasmid was transformed into BL21 (DE3) competent cell for protein expression, briefly, culture in LB broth miller (1st Base, #BIO-4000-1kg) supplemented kanamycin (GOLDBIO, #K-120-25) was allowed to grow till OD600 of 0.8 prior it was induced using IPTG (isopropyl β-D-1-thiogalactopyranoside) at final concentration of 0.5 mM for overnight at 16 °C. Bacterial cell pellet was then lysed in lysis buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl) supplemented with protease inhibitor cocktail (Nacalai Tesque, #04080-11) by sonication. Soluble portion was collected and incubated with HisPur-Ni-NTA resin for metal affinity purification. Size exclusion chromatography with HiLoad 16/60 Superdex 75 was carried out for final purification of SARS-CoV-2 N protein with gel filtration buffer (1 × PBS pH7.9). Collected protein fractions were pooled and concentrated with Vivaspin centrifugal concentrators prior storage at −80 °C.

Biotinylation of monoFc-ACE2

Chemical biotinylation of monoFc-ACE2 was carried out by using EZ-link Sulfo-NHS-LC -Biotinylation kit (ThermoFisher, #21435). Protein was incubated with 20 molar excess of Sulfo-NHS-LC biotin at 4 °C for 2 h. The level of biotinylation was measured by HABA assay provided from the kit.

Antibody profiling by ELISA

SARS-CoV-2 Spike protein, MBP-RBD protein, or nucleocapsid protein was coated on 96-well flat-bottom maxi-binding immunoplate (SPL Life Sciences, #32296) at 7.5 nM, 27 nM, or 40 nM, respectively, 100 µL/well at 4 °C overnight. The plate was washed three times in PBS and blocked for 2 h with blocking buffer: 4% skim milk in PBS with 0.05% Tween 20 (PBST) at 350 µL/well. After three washes in PBST, 100 µL of 80 times diluted plasma samples were added to each well for 1 h incubation. The plate was then washed three times in PBST and 100 µL of 5000 times diluted goat anti-human IgG-HRP (Invitrogen, #31413), or 5000 times diluted F(ab’)2 anti-human IgA-HRP (Invitrogen, #A24458), or 7500 times diluted goat anti-human IgM-HRP (Invitrogen, #31415) was added to each well for 1 h incubation protected from light. After three times of plate wash in PBST, 100 µL of 1-Step Ultra TMB-ELISA (Thermo Scientific, #34029) was added to each well. After 3 min incubation in dark, the reaction was stopped with 100 µL of 1 M H2SO4 and OD450 was measured using a microplate reader (Tecan Sunrise). OD450 reported was calculated by subtracting the background signal from plasma binding to the blocking buffer.

Bio-layer interferometry (BLI)

The N-terminally biotinylated monoFc-ACE2 interaction with RBD–CBD was measured on an 8-channel Octet RED96e system (Forté Bio) with streptavidin biosensor tips (Sartorius). These tips were pre-incubated with assay buffer: PBS, 0.2% BSA and 0.05% Tween 20 for 10 min at 25 °C. Then, they were coated with biotinylated mFc-ACE2 to yield a loading thickness of 0.9 nm. After washing the tips with assay buffer, the binding with RBD–CBD was measured in real time by recording the increase in optical thickness of the tips during 600 s of the association phase. The tips were transferred back into assay buffer during the dissociation phase. A two-fold dilution series of RBD–CBD ranging from 6.25 to 100 nM was used. For negative control, the concentration of N-protein was kept at 100 nM for comparison with the highest concentration of RBD–CBD. The data were processed by Octet Data Analysis software then transferred into GraphPad Prism 9 for association-dissociation non-linear regression model curve-fitting.

SARS-CoV2 pseudotyped lentivirus production

This method was optimized from Poh et al.[17] A third-generation lentivirus system, was used to produce pseduotyped viral particles expressing SARS-CoV2 S proteins via reverse transfection. 36 × 106 HEK293T cells 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 transfection reagent (Invitrogen, #L3000-150) and cultured in a 37 °C, 5% CO2 incubator for 3 days. The viral supernatant was then, harvested and filtered through a 0.45 µm filter unit (Merck). The filtered pseudovirus supernatant was concentrated using 40% PEG 6000 by centrifugation at 1600g for 60 min at 4 °C. Lenti-X p24 rapid titer kit (Takara Bio, #632200) was used to quantify the viral titres, as per the manufacturer’s protocol.

Pseudovirus neutralization assay (pVNT)

This method was modified from Poh et al.[17] The ACE2 stably expressed CHO cells were seeded at a density of 5 × 104 cells in 100 µL of complete medium [DMEM/high glucose with sodium pyruvate (Gibco, #10569010), supplemented with 10% FBS (Hyclone, # SV301160.03),10% MEM Non-essential amino acids (Gibco, #1110050), 10% geneticin (Gibco, #10131035) and 10% penicillin/streptomycin (Gibco, #15400054)] in 96-well white flat-clear bottom plates (Corning, #353377). Cells were cultured at 37 °C with the humidified atmosphere at 5% CO2 for one day. Patient plasma samples were diluted to a final dilution factor of 80 with PBS. The pseudovirus is diluted to a final concentration of 2 × 106 PFU/ ml. In 25 µl there will be 50,000 lentiviral particles. The diluted samples were incubated with an equal volume of pseudovirus to achieve a total volume of 50 µL, at 37 °C for 1 h. The pseudovirus-plasma mixture was added to the CHO-ACE2 monolayer cells and left incubated for 1 h to allow pseudotyped viral infection. Subsequently, 150 µL of complete medium was added to each well for further incubation of 48 h. The cells were washed twice with sterile PBS. 100 µL of ONE-gloTM EX luciferase assay reagent (Promega, #E8130) was added to each well and the luminescence values were read on the Tecan Spark 100 M. The percentage neutralization was calculated as follows:

Modified ELISA-based sVNT

ACE2-Fc was conjugated to peroxidase using Peroxidase Labeling Kit- NH2 (Abnova, #KA0014) according to the manufacturer’s protocol. Each well of 96-well flat-bottom maxi-binding immunoplate was coated with 100 µL of 13 nM MBP-RBD at 4 °C overnight. The plate was washed and blocked as described above. The plate was washed three times in PBST and incubated for 1 h with 100 µL/well of plasma samples diluted ten times in blocking buffer. No inhibitor control wells were incubated with blocking buffer. Positive and negative control wells were established by incubating with functionally characterized recombinant monoclonal antibodies targeting SARS-CoV-2 RBD. A characterized neutralizer was included as the positive control and a non-neutralizing binder was included as the negative control. Both monoclonal antibodies were tested at concentrations from 64 nM to 0.5 nM, prepared via 2× serial dilution in the blocking buffer. Subsequently, the plate was washed three times and incubated for 1 h with 0.4 nM ACE2-Fc-peroxidase, 100 µL/well, protected from light. The following steps of color development and absorbance measurement were performed as described above. Inhibition% was calculated as

cpVNT assay

Cellulose test strips were prepared using Whatman No. 1 chromatography paper (GE healthcare, #3001-861). The papers were printed with wax-ink printer (Xerox ColorQube 8570, Xerox, USA) to define liquid flow path and testing region. The non-testing regions were printed with the wax ink whereas the testing region were left unprinted. Circular testing region with diameters of 5 mm and 6 mm were prepared. The printed papers were baked at 150 °C for 1 min to allow the wax ink to diffuse through the paper forming hydrophobic boundary throughout the paper thickness. The wax-free testing regions were blocked with 10 µL of 5% BSA in PBS. After air-drying, the test strips were stacking into three layers with the 5 mm strips on the topmost layers and 6 mm strips on the second and third layers. The three layered wax printed paper allows consistent flow of liquid at ~10 s when 40 µL of liquid are applied. One piece of Kimwipes paper (11.4 cm × 21.6 cm, Kimberly-Clark Professional, # 34155) folded in half for 6 times was used as absorbent pad. The three-layered test strips were stacked on top of the folded Kimwipes. All layers were secured together using two paper binders.

10 nM RBD–CBD in 1% BSA in PBS was prepared and assigned as reagent “A”. 10 nM biotinylated monoFc-ACE2 with 6 nM SA-HRP (Biolegend, #405210) in 1% BSA in PBS was prepared and assigned as reagent “B”. To perform the test, one part of reagent A and one part of reagent B were mixed with two parts of plasma samples, i.e. for one reaction, the mixture contains 10 µL of A, 10 µL of B and 20 µL of sample. The mixture was incubated for 5 min at room temperature. 40 µL of the mixture was applied to the testing region. Once sample was fully absorbed the test was washed once with 40 µL of PBS, followed by 40 µL of TMB/H2O2 solution (Merck, #T0440). Signals were allowed to develop for 3 min. Images were taken using Xiaomi Redmi A9 phone in a light box equipped with LED lights and save as.jpg format. Images were transferred to a PC. and analyzed using the opened source ImageJ software from NIH. Images were converted from RGB color space to CMYK. Cyan intensity in the testing regions were analyzed. Inhibition% was calculated using the following formula:

Pearson’s correlation

Pearson’s correlation coefficiency was calculated using Microsoft Excel function PEARSON.

Calculation of test performance

Disease prevalence was calculated from the sample size. It may not represent the true prevalence. Calculations of each parameter of test performance were done using the following formula:

Accuracy = (Sensitivity ∗ Prevalence) ∗ (Specificity ∗ (1 − Prevalence))

Statistics and reproducibility

All data points were performed at least in triplicates. Each data point represented a mean value with an error bar that represented standard deviation (SD). Some data points from clinical samples that were grouped together may not represent error bar. These data points represent mean value from at least three separate run.