A Method Of Detecting an Analyte and Related Systems
20230375531 · 2023-11-23
Inventors
Cpc classification
International classification
G01N33/52
PHYSICS
Abstract
There is provided a method of detecting an analyte in a sample, the method comprising: incubating said sample with a reporter agent to allow said reporter agent to bind to said analyte, if present, in said sample; applying the incubated sample to a cellulose substrate to allow said analyte that is bound to said reporter agent to be captured onto said cellulose substrate by a capture agent comprising a cellulose binding domain (CBD), wherein said capture agent is: (i) incubated with said sample prior to the applying step; and/or (ii) immobilised on said cellulose substrate prior to the applying step; and detecting a signal effected by the reporter agent to determine the presence or absence of said analyte. There is also provided related systems and methods of identifying an infection and detecting an antibody against an infection in a subject, in particular SARS-Cov-2 via lateral flow or vertical flow assays.
Claims
1. A method of detecting an analyte in a sample, the method comprising: applying the sample to a cellulose substrate to allow said analyte, if present in said sample, to be captured onto said cellulose substrate by a capture agent comprising a cellulose binding domain (CBD), wherein said capture agent is: (i) incubated with said sample prior to the applying step; and/or (ii) immobilised on said cellulose substrate prior to the applying step; and determining a presence or absence of said analyte captured on said cellulose substrate by said capture agent.
2. The method of claim 1, wherein determining a presence or absence of said analyte captured on the cellulose substrate by said capture agent comprises detecting a signal effected by a reporter agent on the cellulose substrate, wherein said reporter agent: (i) comprises an analyte binder and said reporter agent is contacted with the sample to allow said reporter agent to bind to said analyte, if present, in said sample; or (ii) comprises a competing binder having affinity for said capture agent and said reporter agent is contacted with said capture agent to allow said reporter agent to bind to said capture agent.
3. The method of claim 1, wherein the amount of said capture agent is not more than 1000-fold molar excess, optionally not more than 100-fold molar excess, further optionally not more than 60-fold molar excess of said analyte.
4. The method of claim 2, wherein where said reporter agent comprises an analyte binder, the method comprises incubating the reporter agent and the capture agent with said sample prior to the applying step and said analyte, when present, is part of a reporter agent-analyte-capture agent complex prior to the applying step.
5. The method of claim 2, wherein where said reporter agent comprises an analyte binder, the method comprises incubating the reporter agent with said sample and immobilizing said capture agent on the cellulose substrate prior to the applying step and said analyte when present, is bound to the reporter agent to form a reporter agent-analyte complex prior to the applying step.
6. The method of claim 2, wherein where said reporter agent comprises a competing binder, the method comprises incubating said capture agent with said reporter agent and/or said sample prior to the applying step to form a reporter agent-capture agent complex and/or an analyte-capture agent complex prior to the applying step.
7. The method of claim 2, wherein where said reporter agent comprises a competing binder, the method comprises incubating said reporter agent with said sample and immobilizing said capture agent on said cellulose substrate prior to the applying step and dispensing the reporter agent incubated with said sample on said cellulose substrate after the applying step.
8. The method of claim 1, wherein a plurality of capture agents is immobilised on said cellulose substrate in a substantially uniform orientation.
9. The method of claim 1, wherein in said capture agent, said CBD is coupled to a C-terminus of a binding protein for said analyte.
10. The method of claim 1, wherein the presence or absence of said analyte in said sample is determined within 15 minutes from the start of the incubating step.
11. The method of claim 1, wherein said method is a method of detecting coronavirus in a sample.
12. The method of claim 1, wherein said method is a lateral flow assay method.
13. The method of claim 1, wherein said method is a vertical flow assay method and the applying step comprises allowing the sample to flow through a plurality of cellulose substrate layers.
14. The method of claim 11, wherein the analyte is an antibody against SARS-CoV-2, and wherein the method comprises: applying a sample from the subject to a cellulose substrate to allow said antibody, if present in said sample, to be captured onto said cellulose substrate by a capture agent comprising a cellulose binding domain (CBD), wherein said capture agent is: (i) incubated with said sample prior to the applying step; and/or (ii) immobilised on said cellulose substrate prior to the applying step; contacting the capture agent with a reporter agent to allow said reporter agent to bind to said capture agent, wherein the reporter agent comprises a competing binder having affinity for said capture agent; and detecting a signal effected by the reporter agent to determine a presence or absence of said antibody captured on said cellulose substrate by said capture agent.
15. The method of claim 14, wherein the sample is selected from the group consisting of: a plasma sample, a serum sample and a whole blood sample.
16. The method of claim 14, wherein said capture agent is incubated with said sample before said capture agent is contacted with said reporter agent.
17. The method of claim 11, wherein the analyte is a SARS-CoV-2 protein, and wherein the method comprises: incubating a sample obtained from a subject with a reporter agent to allow said reporter agent to bind to a SARS-CoV-2 protein, if present, in said sample; applying the incubated sample to a cellulose substrate to allow the SARS-CoV-2 protein that is bound to said reporter agent to be captured onto said cellulose substrate by a capture agent comprising a cellulose binding domain (CBD), wherein said capture agent is: (i) incubated with said sample prior to the applying step; and/or (ii) immobilised on said cellulose substrate prior to the applying step; and detecting a signal effected by the reporter agent to determine a presence or absence of said SARS-CoV-2 protein captured on said cellulose substrate by said capture agent.
18. The method of claim 17, wherein the SARS-CoV-2 protein comprises SARS-CoV-2 nucleocapsid (N) protein.
19. The method of claim 17, wherein said reporter agent and said capture agent are configured to bind to different epitopes of the SARS-CoV-2 protein.
20. The method of claim 17, wherein said sample is selected from the group consisting of: a saliva sample, a sputum sample, a nasal fluid sample, a pharyngeal fluid sample, a nasopharyngeal fluid sample and an oropharyngeal fluid sample.
Description
BRIEF DESCRIPTION OF FIGURES
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Limit of detections (LOD) were determined using mean+3SD formular and represented as the dotted lines. All data were represented as mean±SD. Each data points were performed in triplicates. 4 Parameters logistic model was used to draw the fitted curve with R.sup.2 value of 0.9534.
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[0186] Inhibitory percentages derived from cyan intensity signals obtained from different concentrations of mouse anti SARS-CoV-2 neutralizing antibodies and human anti S1 antibodies that did not possess neutralizing property. Limit of detections (LOD) were determined using mean+3SD formular and represented as a dotted line. All data points were represented as mean±SD. Each point was performed at least in triplicates.
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EXAMPLES
Example 1—Comparison Between a Conventional Rapid Diagnostic Test (RDT) Format and a Cellulose Pull Down Assay Format
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[0205] The conventional RDT format suffers from several shortcomings. In the conventional paper-based RDTs, the reporter agents have to be dry stored on the cellulose substrate or other types of substrate used as reporter agent pad 212 and the capture agents have to be immobilized to the test zone, commonly cellulose substrate, prior to performing the assay. Analytes are brought to the reporter agents and capture agents via the capillary force. Once they reach the reporter agent pad, the analytes have less than 10 seconds to form half sandwich complexes with the reporter agents. The half sandwich complexes are then brought to the test zone to form a full sandwich complex with the immobilized capture agents. Here, the capture agents only have a few seconds to capture the half sandwich complexes. With the limited interaction time, most half sandwich complexes flow past the test zone without being captured. The inefficient formation of full sandwich complexes results in low assay sensitivity and potentially false negative results.
[0206] Furthermore, the immobilization of capture agents onto the cellulose substrate occurs spontaneously through hydrophobic and electrostatic interactions. These interactions offer only moderate strength, and thus a substantial amount of capture agents is lost through this process. In addition, this process happens without control over the orientation of the capture agents. As a result, not all of the capture agent binding faces are oriented towards the sample solution, as shown in
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[0213] The low concentration of capture agents used in CP-H, CP-F and the hybrid method CP-H+CE is not expected to improve assay sensitivity. However, the results surprisingly indicate that the combination of (i) the reagents pre-incubation and (ii) the capture agents comprising CBD, allows the low concentration of the capture agents to effectively capture the full sandwich complexes and be efficiently pulled down to the cellulose substrate (in CPF). Embodiments of the method disclosed herein advantageously improve assay sensitivity and minimise resource waste i.e. use of capture agents and reporter agents are optimised.
[0214] In the comparative example, a high concentration of capture agent at 18 μM was required to be deposited on cellulose substrate to ensure efficient capture of analytes-reporter agents complex in sequential matter. Although analytes can typically be captured efficiently and rapidly using high molar concentration of capture agent, without being bound by theory, it is believed that the sequential application of reporter agents in the comparative example hinders the high sensitivity signal production because the reporter agents have short resident time to interact with the analytes on cellulose substrate. As a consequence, low sensitivity of the assay and false negative results are observed since the binding of the analytes and the reporter agents does not happen within seconds.
Example 2—Cellulose Pull Down of SARS-CoV-2 Nucleocapsid Protein (NP)
Engineering of Capture Agent (or Cbd Tagged Capture Reagent)
[0215] rcSso7d binder proteins that bind specifically to SARS-CoV-2 nucleocapsid protein (NP) were engineered using directed evolution approach. In brief, a library comprising theoretically 10.sup.9 variants of rcSso7d was generated. Each variant was cloned into a yeast surface display vector pCTcon2 and transformed into yeast S. cerevisiae. Recombinant SARS-CoV-2 N protein immobilized on magnetic particles were introduced to the rcSso7d yeast library. Yeast clones that bound to magnetic particles were sorted using magnetic stand and cultured to amplify the yeast cell number. His-tagged SARS-CoV-2 N protein was introduced to the yeast cells amplified from magnetic bead sorting. The SARS-CoV-2 N protein was stained using anti-His antibodies conjugated to fluorophore. Yeast cells that bound to SARS-CoV-2 and carried fluorescent antibody were sorted using FACS and cultured to amplify the yeast cell number. FACS sorting were repeated for 5 more rounds, each round with lower concentrations of SARS-CoV-2 N protein. Following 6 rounds of FACS sorting, rcSso7d sequences were sequenced and subcloned into bacterial expressing vector pET28b for protein expression in bacterial cells.
[0216] The pair of engineered rcSso7d proteins that bind to different epitopes of SARS-CoV-2 N protein were used as capture and reporter reagents for all proof-of-concept studies in this disclosure. The DNA sequence encoding CBD (Table 1) was tagged to the C-terminus of the capture reagent using standard restriction enzyme and ligation methods. In brief, the DNA sequence encoding CBD that contains (i) BamHI restriction enzyme sequence and a 3×GS spacer sequence (GGA GGT GGA GGT TCT GGT GGA GGA GGA TCT GGA GGT GGT GGT TCT) at its N-terminus and (ii) XhoI restriction enzyme sequence at its C-terminus was synthesized by a commercialized service provider and cloned into His-pET28b vector using indicated restriction enzyme sites and T4 ligase (New England BioLabs®, Inc., USA). Cloning of CBD into His-pET28b vector created a His-CBD-pET28b vector template for the subsequent cloning of capture reagent. To tag CBD to capture reagent, the DNA sequence encoding capture reagent that contains (i) NdeI restriction enzyme sequence at its Nterminus and (ii) BamHI at its C-terminus, was amplified (using Phusion high-fidelity DNA polymerases, ThermoFisher Scientific, USA) and cloned into His-CBD-pET28b vector using indicated restriction enzyme sties and T4 ligase. This process created His-rcSso7d.sub.SARS-CoV-2-NPcapture-pET28b vector that will be used to generate the ‘capture reagent’. All restriction enzymes and T4 ligase were obtained from New England BioLabs®, Inc., USA. The procedure for restriction enzyme digestion and ligation were performed as per manufacturer recommendation.
TABLE-US-00001 TABLE 1 CBD DNA and protein sequences CBD CCGGTATCAGGCAATTTGAAGGTTGAATTCTACAACAGC DNA AATCCTTCAGATACTACTAACTCAATCAATCCTCAGTTC sequence AAGGTTACTAATACCGGAAGCAGTGCAATTGATTTGTCC AAACTCACATTGAGATATTATTATACAGTAGACGGACAG AAAGATCAGACCTTCTGGTGTGACCATGCTGCAATAATC GGCAGTAACGGCAGCTACAACGGAATTACTTCAAATGTA AAAGGAACATTTGTAAAAATGAGTTCCTCAACAAATAAC GCAGACACCTACCTTGAAATAAGCTTTACAGGCGGAACT CTTGAACCGGGTGCACATGTTCAGATACAAGGTAGATTT GCAAAGAATGACTGGAGTAACTATACACAGTCAAATGAC TACTCATTCAAGTCTGCTTCACAGTTTGTTGAATGGGAT CAGGTAACAGCATACTTGAACGGTGTTCTTGTATGGGGT AAAGAACCC CBD PVSGNLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLS protein KLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNV sequence KGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRF AKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWG KEP
Engineering of Reporter Reagent (or Analyte Binder or Binding Protein)
[0217] To allow the reporter reagent to generate colorimetric signal, the reporter reagent was tagged to biotin acceptor (BA) sequence (GGC CTG AAC GAT ATT TTT GAA GCG CAG AAA ATT GAA TGG CAT GAA). In brief, DNA sequence encoding NdeI-BA3×GS_linker-EcoRI was synthesized by a commercialized service provider and cloned into HispET28b vector via NdeI and EcoRI restriction enzyme sites using T4 ligase. This process generated His-BA-pET28b vector. DNA sequence encoding maltose binding protein (MBP) that contains EcoRI sequence at its N-terminus and BamHI at its C-terminus was amplified and cloned into His-BA-pET28b at the indicate restriction enzyme sites using T4 ligase. This process created His-BA-MBP-pET28b vector. To prepare ready-to-used vector template with a spacer protein after MBP, DNA sequence encoding 3×GS_linker with BamHI and SpeI restriction enzyme sequences at its N- and C-terminus was synthesized and cloned into His-BA-MPB-pET28b vector using T4 ligase. Finally, the rcSso7d reporter reagent sequence containing SpeI and XhoI at its N and C terminus was amplified and cloned into the His-BA-MBP-pET28b vector using T4 ligase. This process created His-BA-MBP-rcSso7d.sub.SARS-CoV-2-NP-reporter-pET28b vector which will be used to generate the ‘reporter agent’.
Expression and Purification of Capture and Reporter Agents
[0218] In brief, the pET28b plasmids containing capture reagent tagged with CBD or reporter reagent tagged with BA were transformed to BL21-DE3 cells, plated on LB-agar plate containing 50 μg/mL of kanamycin and incubated for 12-18 hr at 37° C. A single colony was selected from each plate and inoculated in 10 mL LB media containing 50 μg/mL of kanamycin at 37° C. on a shaker for 12-18 hr. Cells were passage into a larger volume of 0.5-1 L of LB media containing kanamycin and cultured at 37° C. on a shaker until they reach log phase growth (˜OD of 0.6 at 600 nm). Protein expression was induced using 0.5 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG). For the reporter reagent 0.3 mM biotin was also supplemented to the culture. The cells were continued to be cultured at 20° C. on a shaker for 16-20 hr.
[0219] Cells were harvested by centrifugation at 4,000 g for 10 min. The cell pellets were re-suspended in binding buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 7.6) at a ratio of 1 g/10 mL and lysed by sonicating at 50% amplitude with 5 s on 10 s off cycles for 5 min. The lysed cells were centrifuged at 20,000 rpm for 30 min to remove cell debris. To purify the proteins (capture/reporter reagents), supernatants were collected and incubated with nickel resin (Nuvia IMAC Resin, BIORAD, USA) at a ratio of 1 mL resin/1 mL culture for 2-3 hr at 4° C. This step allows His-tagged proteins to be captured to the nickel resin, isolating the desired protein from other endogenous background proteins. The supernatant containing nickel resin bound to His tagged proteins was flown through a column where the nickel resin would be trapped in the column and excess liquid would flow pass the column. With this column, the resin was washed twice, each with 10 mL of binding buffer. The proteins were eluted using binding buffer containing 500 mM of imidazole. The eluted proteins were buffer exchanged to phosphate buffer saline (PBS) using Amicon® Ultra Centrifugal Filters (Merck, Singapore).
Generation and Preparation of the Cellulose Paper-Based Matrix
[0220] Cellulose paper (Whatman No. 1) was printed with wax ink (Xerox ColorCube, Xerox, USA) to create a 2.5 mm hydrophilic test zone and baked at 150° C. for 1 min. An 11.4 cm×21.6 cm Kimwipes paper (Kimberly-Clark Professional™, Singapore) was folded in half for 4 times and placed underneath the printed cellulose test zone to form an absorbent pad. The cellulose test zone and the absorbent pad were kept in tight contact using 2 binder clips. For all experiments, the test zones were blocked with 10 μL of 5% bovine albumin serum (BSA) (Sigma Aldrich, Singapore) in PBS. All other reagents were prepared in 10% human serum in PBS. CBD tagged rcSso7d SARS-CoV-2 N protein capture reagent and BA tagged rcSso7d SARS-CoV-2 N protein reporter reagent were used in all experiments.
Paper-Based Colorimetric Assays (FIG. 7)
[0221] For very short incubation time (SI) test format, 10 μL of each of the following reagents was applied to the test zone sequentially (i) 500 nM capture reagent, (ii) 0 or 50 nM of SARS-CoV-2 N protein, (iii) 500 nM of reporter reagent, (iv) 250 pM of streptavidin horse radish peroxidase (SA-HRP) (Biolegend, USA) and (v) ready-to-use 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma Aldrich, Singapore). All reagents were applied soon after the previous reagent was fully absorbed away from the test zone.
[0222] For the limited incubation time (LI) test format, 10 μL of 500 nM capture reagent was added to the test zone. Subsequently, 10 μL of reagent containing mixture of (i) 0 or 50 nM SARS-CoV-2 N protein, (ii) 500 nM reporter reagent and (iii) 250 pM SA-HRP was incubated for 10 sec at room temperature. Following the incubation, the mixture reagent was applied to the test zone. Finally, 10 μL of TMB was applied to generate colorimetric signal.
[0223] For half complex cellulose pull-down (CP-H) test format, 10 μL of 500 nM capture reagent was added to the test zone. For the subsequent steps, similar protocol to LI test format was carried out. However longer, 1 min, incubation time was allowed to incubate the mixture reagent before the reagent was added to the test zone. Finally, 10 μL of TMB was added to generate colorimetric signal.
[0224] For full complex cellulose pull-down (CP-F) test format, 10 μL of mixture reagent containing (i) 500 nM capture reagent, (ii) 0 or 50 nM SARS-CoV-2 N protein, (iii) 500 nM reporter reagent and (iv) 250 pM SA-HRP was incubated at room temperature for 1 min before applying to the test zone. Subsequently, 10 μL of TMB was applied to the test zone to generate visible signals. For all test formats, the cellulose test strips were allowed to develop colorimetric signals for 3 min before images were captured.
Comparison of the Current Cellulose-Pull Down Assay Performance to a Comparative Example (FIG. 8)
[0225] To perform the assay in the comparative example (CE), 2 μL of 20 μM capture reagent (40 pmol) was added to the test zone to immobilize high molar concentration of capture reagent. Afterward, sequential application of the following reagents was carried out (i) 10 μL of 0 or 50 nM SARS-CoV-2 NP, (ii) 10 μL of 500 nM reporter reagent, (iii) 10 μL of 250 pM SA-HRP and (iv) 10 μL of TMB. The CP-H and CP-F assays were carried out as described earlier. The combination of CE and CP-H was done by immobilizing the cellulose test zone with 2 μL of 20 μM capture reagent. Subsequently, a mixture of the following reagents was prepared in a 10 μL solution: (i) 0 or 50 nM SARS-CoV-2 N protein, (ii) 500 nM reporter reagent and (iii) 250 pM SAHRP. The mixture was allowed to incubate at room temperature for 1 min before it was applied to the cellulose test zone. Finally, 10 μL of TMB was applied to the test zone to generate colorimetric signal. All tests were allowed to develop colorimetric signals for 3 min before images were captured.
Image Analysis
[0226] Images were taken by phone camera or scanner and stored in .jpg format. The color intensities were analyzed using ImageJ software by measuring cyan values in CYMK color format.
[0227] For all applications described in the examples that follow, cellulose-based vertical flow device was used to host chemical reactions and capture the desire target analytes. The cellulose test strips were constructed by placing one or several layers of the cellulose paper above absorbent pads. Absorbent pads can be any porous materials that possess liquid absorbing properties. The cellulose test paper(s) and the absorbent pads were secured together using pressure applied by various means e.g., paper binders, chamber, cassettes, manifold, etc.
Example 3—a Rapid Simple Point-of-Care Assay for the Detection of SARS-CoV-2 Neutralizing Antibodies
[0228] Neutralizing antibodies (NAbs) prevent pathogens/virus from infecting host cells. Determination of SARS-CoV-2 NAbs are 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. A 10 minute cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma is described herein. 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. This 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. Importantly, this approach can be easily extended to test RBD variants or to evaluate NAbs against other pathogens.
[0229] COVID-19 is the biggest pandemic of the modern era. It 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.
[0230] 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 in place. 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. Screening populations for the presence of NAbs is a critical step 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.
[0231] 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, thus 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 present on the host cell via the receptor binding domain (RBD) of the spike (S) protein. 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. 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.
[0232] Here, a rapid cellulose pull-down viral neutralization test (cpVNT) that detects SARS-CoV-2 NAbs in plasma samples within 10 minutes and that can be used at the point of care (POC) is described. The test principle relies on the interaction between RBD and ACE2 to determine the presence of NAB. To allow the test to be compatible with cellulose matrix, RBD is fused to the cellulose binding domain (CBD), generating RBD-CBD which is used as a capture reagent. ACE2 is conjugated to biotin (BA), creating ACE2-BA which is capable of interacting with various kinds of reporting molecule via BA—streptavidin (SA) interaction. In this application, SA-horse radish peroxidase (SA-HRP) bound to ACE2-BA (ACE2-BA/SA-HRP) is used as a reporting reagent. Thus, components of the test include: (i) RBD, tagged with cellulose binding domain (CBD), (ii) ACE2 receptor, tagged with 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.
[0233] Biological fluids including non-diluted or diluted plasma or serum can be used. Briefly, samples may be mixed with capture (RBD-CBD) and reporter (ACE2-BA/SA-HPR) reagents and incubated for at least 5 minutes prior to applying it onto the cellulose test strip. A washing step can be applied to wash away non-specific molecules on the cellulose surface. 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution may then be applied to generate colorimetric signal. The enzymatic colorimetric reaction can be left developed on the test strip for ˜3 minute before colorimetric signal can be measured.
[0234] In the absence or low concentrations of NAb, RBD-CBD and ACE2-BA/SA-HRP are able to form complex effectively, therefore high colorimetric signals may be observed on the cellulose test strip. In the presence of high concentration of NAb, the antibodies may compete with ACE2-BA/SA-HRP complex to bind to RBD-CBD, thereby inhibiting colorimetric signals on the test strip.
[0235] Using this developed assay workflow and construction, the test is able produce well above 80% and 90% test accuracies as compared to the lab-based pseudovirus virus neutralization test (pVNT) and surrogate virus neutralization test (sVNT) (see Results section below for more details).
Results
[0236] Optimization of rapid paper-based cpVNT. The assay times for the currently established lab-based and commercialized NAb tests range from 1.5 hours to 4 days. A shortening of the overall assay time and a simplified workflow are primary requirements for POC NAb tests suitable for large-scale surveillance applications. Vertical flow assays are a type of assay formats that allows reagents to flow in a top-to-bottom fashion for detection of biomolecules. Vertical flow assays offer short liquid flow paths that enable rapid and controllable flow speed for handling of liquid reagent. With an aim for POC NAb test, the vertical flow assay format was selected as a test format for this study. Cellulose is a cost-effective material that can be easily manufactured at scale. It has been demonstrated that cellulose can be used as a test matrix for vertical flow assays, whereby the assay reaction is allowed on the cellulose matrix using high affinity interaction between cellulose binding domain (CBD) and the cellulose matrix. The common construction of the assay is to fuse CBD to the capture reagent. Cellulose was adopted as the test materials to bypass the surge of high nitrocellulose demand from global ramp up of rapid COVID-19 tests which presses massive risk onto the supply chain shortage. The test principle relies on a complex formation between RBD/ACE2 receptor whereby presence of NAb interferes with the RBD/ACE2 receptor complex formation, thereby reducing the reporting signal intensity. To enable the test to be compatible to cellulose paper, CBD was tagged to RBD (RBD-CBD), allowing the RBD protein to be captured rapidly and at high affinity onto cellulose surface. ACE2 receptor was engineered to be a reporting molecule. This was done by tagging biotin (BA) on to ACE2, creating ACE2-BA. Horse radish peroxidase (HRP) conjugated streptavidin (SA), SA-HRP, was used as a colorimetric signal generator. A complex of ACE2-BA/SA-HRP was used to generate colorimetric signal via application of 3,3′,5,5′-tetramethylbenzidine (TMB)/H.sub.2O.sub.2 which hydrolyzes HRP, producing vivid blue color signals.
[0237] To construct the test, recombinant (i) RBD-CBD and (ii) biotin (BA) tagged monoFc-ACE2 receptor proteins were expressed, purified (
[0238] To engineer the vertical flow assay (
[0239] To capture the colorimetric signal from the cellulose vertical flow assay, Xiaomi Redmi A9 phone was used to capture the image and save the image in a .jpg format. To fix the camera distance and angle as well as to prevent interference from the surrounding light, a ‘light box’ was created. The box has a W×L×H dimension of 150×230×90 mm (
[0240] Different cellulose vertical flow assay formats were performed to determine the most effective assay workflows (
[0241] Based on these optimizations, the assay can be performed by mixing the plasma sample with the reagents and incubating for 5 min to allow efficient formation of RBD-CBD/NAbs or RBD-CBD/ACE2 complexes in aqueous phase before applying it onto the cellulose paper (
[0242] Concentrations of RBD-CBD and ACE2-BA/SA-HRP were further optimized to obtain the highest signal difference between presence and absence of NAb (
[0243] The rapid paper-based cpVNT performance. To validate the cpVNT test, different concentrations of mouse anti hSARS-CoV-2 neutralizing antibodies were spiked into non-diluted human plasma and evaluated for their ability to inhibit RBD-CBD/ACE2 complex formation (
[0244] Assessments of SARS-CoV-2 immunological profile from COVID-19 convalescent plasma. Prior to validating the cpVNT using clinical samples, general assessments of the clinical samples were performed. Plasma samples from 24 confirmed COVID-19 patients were collected between 29 days and 73 days (median of 49 days) post positive PCR test (follow up visit 1 (FV1)). Subgroups of patient plasma samples were collected on two subsequent occasions (FV2 (n=13); and FV3 (n=10)) (Table 2).
TABLE-US-00002 TABLE 2 No. days post admission No. of days FV1 of symptoms (Follow before Sample Severity up visit) FV2 FV3 admission Remark P01 Mild 38 7 P02 Moderate 50 6 P04 Mild 29 114 196 14 P06 Mild 30 99 183 8 P07 Severe 36 5 P08 Severe 36 7 P09 Mild 36 3 P10 Mild 35 4 P11 Mild 33 NA Asymptomatic P13 Mild 51 82 194 −9 Symptom started on Day 9 post admission P14 Mild 48 NA Asymptomatic P15 Mild 38 129 192 12 P19 Mild 53 92 192 −3 Symptom started on Day 3 post admission P20 Mild 44 93 191 0 P21 Severe 59 121 213 0 P22 Mild 54 100 199 0 P23 Mild 67 107 190 −7 Symptom started on Day 7 post admission P24 Mild 73 0 P25 Severe 44 0 P30 Mild 51 98 30 P37 Mild 60 101 0 P42 Mild 64 103 196 NA Asymptomatic P45 Mild 50 7 P48 Mild 55 108 −8 Symptom started on Day 8 post admission
[0245] General assessments were performed, for each convalescent plasma sample, to obtain immunological profiles against SARS-CoV-2 and to determine reference points for cpVNT evaluation. Antibody subtypes IgA, IgM and IgG against SARS-CoV-2's S, RBD, and nucleocapsid (N) proteins were evaluated using ELISA (
[0246] To determine the levels of NAbs present in different plasma samples, two lab-based VNTs were used (i) chemiluminescent-based pseudovirus VNT (pVNT) and (ii) a modified format of the published ELISA-based surrogate VNT (sVNT) (Tan, C. W. et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat. Biotechnol. 38, 1073-1078 (2020)). The established pVNT protocols (Jahrsdörfer, B. et al. Characterization of the SARS-CoV-2 Neutralization Potential of COVID-19-Convalescent Donors. J. Immunol. 206, 2614-2622 (2021) and Nie, J. et al. Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay. Nat. Protoc. 15, 3699-3715 (2020)) were used as references for pVNT performed in this study. pVNT determined NAb status by measuring chemiluminescent signals from cells infected with pseudovirus. Presence of NAbs prevent virus from infecting the cells thereby reducing chemiluminescent signals. Based on the reference studies, a wide range of sample dilution factors from 1-10.sup.6 were used to determine the effective cut-off between presence and absence of NAb. A 50% signal inhibition was indicated as an effective value to distinguish between presence and absence of NAb. During the test optimization, it was found that, a dilution factor of at least 1:80 is necessary to minimize false positives produced by healthy control samples, therefore a fix 1:80 dilution factor is used in this study. The chemiluminescent signals measured were normalized with the signals from non-infected and pseudovirus infected cells. The status of NAbs was expressed as neutralization percentage.
[0247] For sVNT, while it retains the same test principles as the published method, the modified sVNT configuration used recombinant RBD protein as a capture reagent and ACE2 receptor conjugated with HRP as a reporter reagent. In this test format, the presence of NAbs causes signal reduction that can be expressed as inhibitory percentages. The higher percentages represent higher level of NAbs and vice versa. 10×dilution were chosen as the lowest dilution factors that give reliable results without showing false positives from healthy control samples. The cut-off value for sVNT was determined by analyzing the test sensitivity and specificity using known concentrations of NAbs and non-NAbs spiked in plasma samples (
[0248] Data obtained from pVNT and sVNT are shown in
[0249] pVNT presents a test format that is closely related to events that occur in a physiological condition. Therefore, pVNT will be used as a baseline to determine the accuracy of sVNT. Using the defined 50% cut-off for pVNT and 20% cut-off for sVNT, the sVNT provides test sensitivity and specificity of 90.0% and 86.5%, respectively with an overall accuracy of 87.2% (with 95% CI of 74.3% to 95.2%) (Table 3).
TABLE-US-00003 TABLE 3 Statistic Value 95% CI Sensitivity 90.0% 55.5% to 99.8% Specificity 86.5% 71.2% to 95.5% Positive Predictive Value* 64.3% 43.7% to 80.7% Negative Predictive Value* 97.0% 83.2% to 99.5% Accuracy* 87.2% 74.3% to 95.2%
[0250] It is also observed that most samples showed negative NAb status (
[0251] Evaluating of cpVNT performance using COVID-19 convalescent plasma. To evaluate the ability of the cpVNT in determining the level of NAbs, results obtaining from cpVNT using COVID-19 convalescent plasma, from different visits, were plotted against the pre-COVID plasma samples collected from earlier studies. Based on these data, the inhibitory signal cut-off which distinguished positive from negative NAbs levels was determined at 20% (
[0252] Data obtained from cpVNT show a high correlation with pVNT and sVNT with Pearson correlation coefficients of 0.70 and 0.87, respectively, (
TABLE-US-00004 TABLE 4 Statistic Value 95% CI Sensitivity 80.0% 44.4% to 97.5% Specificity 84.4% 67.2% to 94.7% Positive Predictive Value* 61.5% 40.31% to 79.1% Negative Predictive Value* 93.1% 79.5% to 97.9% Accuracy* 83.3% 68.6% to 93.0%
[0253] As compared to sVNT, cpVNT exhibits a sensitivity and specificity of 85.71% and 96.55%, respectively, with an overall test accuracy of 93.02% (with 95% CI of 74.37% to 96.02%, Table 5).
TABLE-US-00005 TABLE 5 Statistic Value 95% CI Sensitivity 85.7% 57.2% to 98.2% Specificity 96.6% 82.2% to 99.9% Positive Predictive Value* 92.3% 63.4% to 98.8% Negative Predictive Value* 93.3% 79.5% to 98.1% Accuracy* 93.0% 80.9% to 98.5% *These values depend on the prevalence of the disease. The prevalence is calculated from the sample size. It may not reflect the real disease prevalence.
[0254] Cross reactivity tests were performed to ensure the test specificity. High concentrations (100 nM) of IgG against different viruses were spiked in plasma from healthy controls and tested on cpVNT. Data show minimal cross reactivity to antibodies against other viruses, or to SARS-CoV-2 S and N proteins (
[0255] Altogether, the cpVNT offers a rapid test, that can be performed in ten minutes, for reliable detection of NAbs against SARS-CoV-2. Accuracy of cpVNT, compared to the lab-based methods are well above 80% and 90%, for pVNT and sVNT. The test can be performed both in plasma and serum samples without dilution, therefore facilitating simple workflow at POC settings.
[0256] Unlike other report which predicted that high level of NAb could be detected from convalescent samples, most of the convalescent samples collected for this study showed relatively low NAb level from all three VNT formats tested. The plasma samples collected for this study begun at ˜1 month post admission. Samples containing NAb came mostly from FV1. pVNT, sVNT and cpVNT detected 10, 9 and 9 NAb positive samples, which accounted for 41.7%, 37.5% and 47.4%, of the sample size, respectively. pVNT showed 1 and 0 positive samples in FV2 and 3, whereas sVNT and cpVNT, each, showed 2 positive NAbs samples in FV2 and 2 in FV3. Only 3 patients which showed positive NAb status in FV1 completed all 3 visits. Although a trend of reduction in NAbs is observed in these 3 samples, the sample sizes are too small to observe statistical differences in all test formats. Aligning with the findings, a study reported that a substantial number of convalescent samples did not produce sufficient NAb to be detected using sVNT. In addition, a large subgroup of convalescent population showed rapid waning of NAb at 2-month post symptom onset, a timeline in which majority of samples were collected for this study.
Discussion
[0257] Global efforts are underway to improve SARS-CoV-2 surveillance and manage long term prevention of COVID-19. Assessment of SARS-CoV-2 NAbs is one of the key surveillance criteria required to evaluate herd immunity and the impact of SARS-CoV-2 on a larger population scale. Existing technologies for NAb detection (cVNT, pVNT and sVNT) all require laboratory facilities, skilled personnel and long execution times (1 hr-4 days) that are not favorable for large-scale surveillance outside a laboratory setting. cpVNT provides a robust NAb surveillance detection test, that is simple, rapid and can be easily conducted both inside and outside of laboratory settings in as little as 10 minutes. In addition to the cpVNT performance evaluated in this study, it is now feasible to compare its performance to the commercialized sVNT, c-Pass™ from GenScript®.
[0258] SARS-CoV-2 vaccination programmes have been rolled out in many countries, since late 2020, initially providing vaccines to healthcare frontline workers and members of high-risk communities. A serological neutralizing antibody test is a valuable companion test to evaluate the effectiveness of available vaccines. Data from different VNT formats obtained from this study suggested that ˜37-48% of the samples showed positive NAb status in the first 1-2 months (FV1), post admission. The number declined to ˜7-15% in the subsequent visits. However, due to the small sample size of follow up visits, no statistical difference was observed from different visits. Nonetheless, findings from other studies demonstrated that NAbs declined gradually over 3-month period post symptom onsets, therefore suggesting that vaccine boosters may be required to maintain the immunity status at a desirable level. The rapid neutralization test described here would be a suitable tool to regularly assess the immune status of individuals, particularly in the vulnerable population. The simple nature and speed of this test provides an accessible POC tool, which can be used at community clinics or in low resource settings, to prioritize vaccine administration. In addition, the test can be rapidly adapted to evaluate the efficiency of NAbs to new virus variants and thereby guide the decision-making process in relation to the need of new booster vaccines.
[0259] Despite a number of lateral flow assay (LFA) tests available for detection of antibodies against SARS-CoV-2, to knowledge, only one pre-print report is found for rapid NAbs test. In most LFA antibody detection tests (rapid serology tests), specific antigens are either immobilized on the testing matrix or used as reporting molecules whereas the counter reporting/capturing part are anti-IgA/IgM/IgG antibodies. It was reported that, when RBD and anti-IgA/IgM/IgG antibodies are used for detection of NAbs in the plate-based ELISA format, non-NAbs are often detected along with NAbs (due to antibodies that bind to RBD but do not possess neutralizing ability). This method is thus unable to predict the level of NAbs accurately. Adapting a NAbs test to a LFA format seems feasible due to the well-established LFA technology. However, with the test format employed for the cpVNT and sVNT reported in this study, it would be challenging for LFA to report a loss of a colorimetric signal as a positive result, particularly when anti-IgA/IgM/IgG were to use as reporting molecules. ˜15-20 incubation time is required for LFA, thus allowing substantial amount of time for non-specific binding to occur at the test line on the LFA test strip. To overcome this issue, a suitable control system would have to be designed to ensure that a positive colorimetric signal generated (i.e., lack or low level of NAb) is not due to non-specific binding.
[0260] The rapid cpVNT neutralization test developed in this study identifies and measures the very specific interaction between RBD and ACE2 receptor. Non-NAbs will not interfere with the RBD/ACE2 receptor complex formation and the signal detected is specific to neutralizing antibodies (
[0261] The sudden high demand for LFA COVID-19 rapid diagnostic tests has created a worldwide shortage of materials required for LFAs, particularly the nitrocellulose membrane, leading to supply chain issues. The cpVNT presented in this study utilizes cellulose membrane which is more economical to produce and supply chains are unimpeded. In addition, cellulose paper can be easily manufactured, enabling large-scale production of the test strips in a very short period, thereby facilitating mass manufacture of the test with low production cost.
[0262] Plasma/serum is currently optimized for cpVNT. As such, a device capable of separating plasma from whole blood is needed for the POCT applications. Different aspects of test stability are currently being investigated. Based on the preliminary data, with the right preservatives and additives the cellulose test paper can last up to 6 months when kept at ambient temperature (25° C.) without controlling of humidity. The test papers last up to 3 years in a desiccator. RBD-CBD and ACE2-BA retain their activities for at least 3 months when kept in optimized conditions. In the POC settings, a test strip may comprise one ‘Test’ spot and one ‘Control’ spot, whereby the control spot hosts a chemical reaction that indicates active function of the reagents. It is critical for the test to have a control spot because the positive result is derived from loss of colorimetric signal. The control spot ensures that the loss of signal at the test is due to binding of NAb to RBD-CBD and not the malfunction of the chemistry reaction. The optimization of the control spot was started and it was found that immobilizing of RBD-CBD at high concentrations on the cellulose paper could potentially serve as a control spot reaction to capture ACE2 tagged to reporting molecules from the assay mixture. The preliminary data showed that this design of control spot produced high cyan intensity signals regardless of presence or absence of NAb in the samples. For signal analysis of cpVNT, a pre-set cyan intensity value could be pre-determined from pre-COVID or non-infected samples during the assay optimization stage. This value can be implemented for the POC applications in which the pre-determined cyan intensity value defines a reference signal for analysis of the cpVNT test results.
[0263] In conclusion, a rapid, paper-based cpVNT that can be used at POC for effective identification of neutralizing antibodies against COVID19 is developed. Comparison of cpVNT against the existing VNTs, including, the pVNT and the sVNT (
Materials and Methods
[0264] 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-MMO3T) 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.
[0265] Collection of clinical samples. Collection of COVID-19 convalescent samples were reviewed and approved under the DSRB reference #2020/00120, National University of Singapore (NUS). 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 (PBMNC) were harvested, resuspended in freezing media containing 90% FBS (Hyclone)+10% dimethyl sulfoxide (Sigma Aldrich), and stored in liquid nitrogen.
[0266] Collection of pre-COVID samples were reviewed and approved by 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.
[0267] 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 (Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-vornhagen, A. & Bergwelt-baildon, M. S. Von. Generation of Human CD40-activated B cells. J. Vis. Exp. 1373 (2009). doi:10.3791/1373). 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 (Gu, Y. et al. Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A*11:01. Nat. Commun. 10, 893 (2019)). Confirmation of binding specificity of cloned human monoclonal antibodies was confirmed by ELISA.
[0268] Protein Expression and Purification. The soluble extracellular fragment of human ACE2 (residues 19-615; GenBank: AB046569.1) was cloned into a modified pHLSec (Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D Biol. Crystallogr. 62, 1243-50 (2006)) mammalian expression vector following an N-terminal monoFc, hexahistidine tag and Tobacco Etch Virus (TEV) protease cleavages site. SARS-CoV2-Spike RBD (Dalvie, N. C. et al. Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters. bioRxiv 2021.03.03.433558 (2021)) fused to CBD (residues 276-434 of Hungateiclostridium thermocellum CipA) was cloned into the pHLmMBP-10 vector (Bokhove, M. et al. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins. J. Struct. Biol. 194, 1-7 (2016)) (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 (Meulen, J. et al. Human monoclonal antibody combination against SARS coronavirus: Synergy and coverage of escape mutants. PLoS Med. 3, e237 (2006)), 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; 104 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.
[0269] 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 (1.sup.st Base, #BIO-4000-1 kg) supplemented kanamycin (GOLDBIO, #K-120-25) was allowed to grow till OD.sub.600 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.
[0270] 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 hrs. The level of biotinylation was measured by HABA assay provided from the kit.
[0271] 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. Plate was washed three times in PBS and blocked for 2 hours 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 hour incubation. 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 hour 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, reaction was stopped with 100 μL of 1 M H.sub.2SO.sub.4 and OD.sub.450 was measured using microplate reader (Tecan Sunrise). OD.sub.450 reported was calculated by subtracting the background signal from plasma binding to the blocking buffer.
[0272] Bio-layer Interferometry (BLI). The N-terminally biotinylated monoFc-ACE2 interaction with RBD-CBD was measured on an 8-channel Octet RED96e system (Forte 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 600s of association phase. The tips were transfer back into assay buffer during 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 highest concentration of RBD-CBD. The data was processed by Octet Data Analysis software then transferred into GraphPad Prism 9 for association-dissociation non-linear regression model curve-fitting.
[0273] SARS-CoV2 pseudotyped lentivirus production. A third-generation lentivirus system, was used to produce pseduotyped viral particles expressing SARS-CoV2 S proteins via reverse transfection. 36×10.sup.6 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% CO.sub.2 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 1600 g for 60 minutes at 4° C. Lenti-X p24 rapid titer kit (Takara Bio, #632200) was used to quantify the viral titres, as per manufacturer's protocol.
[0274] Pseudovirus neutralization assay (pVNT). The ACE2 stably expressed CHO cells were seeded at a density of 5×10.sup.4 cells in 1004 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 in 37° C. with humidified atmosphere at 5% CO.sub.2 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 2×10.sup.6 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 a further incubation of 48 h. The cells were washed twice with sterile PBS. 100 μL of ONE-Glo™ EX luciferase assay reagent (Promega, #E8130) was added to each well and the luminescence values were read on the Tecan Spark 100M. The percentage neutralization was calculated as follows:
[0275] Modified ELISA-based sVNT. ACE2-Fc was conjugated to peroxidase using Peroxidase Labeling Kit—NH.sub.2 (Abnova, #KA0014) according to 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. Plate was washed and blocked as described above. 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, plate was washed three times and incubated for 1 hour 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
[0276] 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 3 layers with the 5 mm strips on the topmost layers and 6 mm strips on the second and third layers. The three layered of wax printed paper allows consistent flow of liquid at ˜10 sec 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.
[0277] 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 minutes 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/H.sub.2O.sub.2 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:
[0278] Pearson's correlation Pearson's correlation coefficiency was calculated using Microsoft Excel function PEARSON.
[0279] 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:
Example 4—an Antibody-Free Cellulose-Based Vertical Flow Assay for COVID-19 Surveillance
[0280] At the end of 2019, emergence of an unusual pneumonia in Wuhan, Hubei province, China was reported. It was later discovered to be caused by infection with the virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2), which led to the COVID-19 pandemic. COVID-19 is the largest pandemic in the modern era. It has affected more than 200 million people and claimed more than 4 million lives worldwide. Though the fatality rate of SARS-CoV-2 infection is lower than its relatives, SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus), it has high infectivity and transmissibility. Furthermore, emerging of new viral variants have imposed higher risk of infection due to the increase viral infectivity and replication rates. Vaccination programs against SARS-CoV-2 have been initiated world-wide in the late 2020. Although the vaccines provide potential promise to help limit the viral infection and spreading, recent reports revealed that infection can still occur post vaccination. Therefore, to effectively control the pandemic without closing all social activities, frequent surveillance of SARS-CoV-2 infection remains a valuable mean to timely isolate infected individuals. As such, early diagnostic and surveillance of SARS-CoV-2 infection is essential and continues to be urged.
[0281] Currently, there are two types of diagnostic tests available for detecting active SARS-CoV-2 infection: 1) molecular-based tests for detecting viral genomic material and 2) antigen rapid tests (ARTs) for detecting viral proteins predominantly spike protein (S protein) and nucleocapsid protein (N protein). Though molecular-based tests are known for their high accuracy and sensitivity, they are not suitable for frequent surveillance applications as lab settings and equipment are needed. Additionally, almost all molecular-based tests are approved for nasopharyngeal swab samples collected by trained personnel, a semi-invasive method that frequently leads to unpleasant experience.
[0282] Antigen rapid tests (ARTs) can be deployed as Point-of-Care (PoC) tests and can be used for frequent surveillance. Although ARTs may not have the same high level of sensitivity as the molecular-based tests, the ease of ART workflow allows for high frequency testing. It is suggested that test frequency and rapid turn-around time are more critical than the test sensitivity for COVID-19 surveillance. The larger number of individuals being screened, the more infected yet infectious patients can be identified. Positive results from ARTs suggest relatively high viral loads, hence highly transmissible virus. Therefore, the rapid identification of infected patients via ARTs provides a critical and rapid means to effectively and timely isolate individuals with high transmission potency. For this reason, high attention is drawn to ARTs for surveillance of SARS-CoV-2 infection. Many of the approved ARTs are constrained to nasopharyngeal or nasal swab specimen as the sample matrix. Despite the easy deployment and simple operating procedure, the unpleasant experience of nasopharyngeal and nasal swabs is not appealing to individuals and hence, there may be less motivation to adopt ARTs for frequent testing. In addition, most of the commercial ARTs available use the lateral flow assay (LFA) platform and hence the raw materials required for making of LFAs are in high demand globally. The high demand for LFA production leads to global supply shortage on one of the key LFA components, nitrocellulose paper. Therefore, an alternative system to traditional LFAs for antigen detection would be a great complementary test for surveillance of active SARS-CoV-2 infections.
[0283] A new method/system for detecting SARS-CoV-2 antigen to detect COVID-19 is described here. The example describes the detection of SARS-CoV-2 N (Nucleocapsid) protein using saliva as a testing matrix. Engineered scaffold binder proteins rcSso7d are used as capture and reporter reagents to detect SARS-CoV-2 nucleocapsid (N) protein. The binders are engineered using the RAPIDS process. A complementary pair of rcSso7d binders are engineered to bind to the N protein at two different epitopes. One of the binders is fused to CBD (Sso.E2-CBD) and used as a capture reagent. The counterpart binder is tagged to a spacer protein maltose binding protein (MBP) and BA (BA-MBP-Sso.E1) to allow the protein to interact with various types of reporting molecule via the BA-SA interaction. Similar to Example 3, SA-HRP is used as a reporting molecule. A complex of BA-MBP.Sso.E1/SA-HRP is used as the reporter reagent.
[0284] Saliva is used as a sample matrix due to its ease of collection. Usage of saliva offers a less invasive sample collection approach as compared to nasal and nasopharyngeal swab. To reduce viscosity of the sample, saliva may be filtered through a 5 μm filter unit prior to running the assay. In addition, because the target protein (N protein) is enclosed inside the virus, the sample is treated with detergent (1% triton X-100) to break the viral membrane and release the N protein. This filtered and detergent-treated saliva matrix is used for the rapid SARS-CoV-2 antigen assay development.
[0285] The test is performed by incubating the prepared saliva samples with capture (Sso.E2-CBD) and reporter (BA-MBP-Sso.E1/SA-HPR) reagents for 1 minute. The reaction is then applied onto the cellulose test strip. A washing step is introduced to minimize non-specific binding on the cellulose surface. Finally, TMB substrate is applied to generate colorimetric signal. The colorimetric reaction is left to develop for 3 minutes before the signal is measured. In this example, cyan intensity is used to measure the blue colorimetric signal from HRP/TMP reaction on the cellulose surface. Results showed that when there was an increase in recombinant N protein or SARS-CoV-2 concentration, the cyan intensities increased proportionally, indicating that the test successfully detected the SARS-CoV-2 biomarker and can be used to diagnose COVID-19. Cross-reactivity study showed that the test produced no cross-reactivity to flu-causing pathogens spiked in saliva. These results suggest that the test is highly specific to SARS-CoV-2 (see Results section below for more details).
[0286] Advantageously, embodiments of the test can be easily implemented on children and adults for ‘painless’ sample collection, and is thus suitable for frequent testing of SARS-CoV-2 infection for all age ranges. Embodiments of the test provides a short turn-around-time of 5-10 min, up to 4 times shorter than the currently available ARTs which require up to 15-20 min. Using the unique protein engineering technology, the test is designed to be compatible with cellulose paper, an alternative, cost-effective and readily available material. Usage of cellulose paper avoids the risk of nitrocellulose supply chain shortage, an issue that currently hampers the production of many LFA tests. In addition, thermostable rcSso7d proteins are used instead of antibodies for capture of SARS-CoV-2 N protein. Therefore, the reagents may be produced using bacterial system, which is a more cost-effective method as compared to the mammalian cell line system required for antibody production. In addition, a unique vertical flow assay (VFA) with short flow path is developed as an alternative approach to LFA.
Results
[0287] Generation of Affinity Paired rcSso7d Binders Targeting SARS-CoV-2 N Protein
[0288] To develop the rapid vertical flow test for detecting SARS-CoV-2 antigen from saliva, a pair of rcSso7d binders binding to different epitopes of N protein were engineered. The RAPIDS screening process (
[0289] Through the RAPID screening process, rcSso7d binder pair which bind complementarily to different epitopes of SARS-CoV-2 N protein was successfully engineered. These binders are (i) BA-MBP-Sso.E1 and (ii) Sso.E2-CBD whereby the former will be used as the reporting binder and the latter will be used as the capture binder. The proteins can be easily scaled-up and purified to homogeneity as shown in SDS-PAGE analysis (
[0290] Both Sso.E1 (SEQ ID NO: 4: MATVKFTYQGEEKQVDISKIKIVRRGGQWISFWYDEGGGAYGAGYVSEKDAP KELLQMLEKQ) and Sso.E2 (SEQ ID NO: 5: MATVKFTYQGEEKQVDISKIKNVGRWGQIIDFDYDEGGGAIGIGAVSEKDAPK ELLQMLEKQ) interact with SARS-CoV-2 N protein with excellent binding affinities at the dissociation constants (K.sub.D) of 3.2 nM and 4.2 nM, respectively. To determine the binding epitopes of the binder pair, Bio-layer Interferometry (BLI) was performed using truncated N-terminal domain (NTD) and C-terminal domain (CTD) of N protein as ligands. For facile and orientation-specific loading of the Sso binders onto the BLI streptavidin (SA) probes, BA-MBP tag was fused to Sso.E1 and Sso.E2 resulting in BA-MBP-Sso.E1 and BA-MBP-Sso.E2, respectively. BLI results showed that Sso.E1 bound to CTD and Sso.E2 bound to NTD of the N protein, respectively and solely (
Integration of a Vertical Flow Assay (VFA) on Cellulose Platform
[0291] Cellulose paper was selected as the sensor material due to its advantages in price, availability, and high affinity toward CBD. Cellulose is more suitable for the vertical flow assay (VFA) format than the lateral flow assay (LFA) format due to the rather large pore size, for example, as compared to nitrocellulose which is a common material used for LFA. Therefore, a vertical flow assay (VFA) was adopted for development of the rapid SARS-CoV-2 test. To optimize for the VFA, four different assay approaches were tested to determine the workflow that give the best assay performance (
[0292] For a diagnostic test, a control reaction is required to ensure the active reagent activity and reliable test results, especially when a negative test result (no signal) is observed. A few criteria were considered for constructing of the control reaction including 1) assay reaction shall be able to apply to both test and control reactions to avoid extra steps of sample preparation and 2) control reaction needs to report ‘active’ reagent signal independent of the presence of absence of analyte/antigen in the test sample.
[0293] With these considerations, the control reaction was constructed by pre-immobilizing the cellulose control spot with SARS-CoV-2 N protein. To immobilize the N protein on the cellulose surface, the protein was fused to CBD at its C terminus (NP-CBD). The NP-CBD on the cellulose control spot is designed to capture the reporting molecule (BA-MBP-Sso.E1) presented in the sample mixture, followed the CP-F test format (
[0294] The control spot performance was tested with pooled healthy control saliva samples spiked with either N protein or PBS. Results showed that the control spots produced cyan intensity signals higher than 0.2 for both testing conditions (
Assay Construction and Optimization
[0295] A test strip contains a ‘test’ and a ‘control’ spot (
[0296] Saliva is the desired testing matrix for this assay development. Therefore, pooled saliva from healthy control subjects was used as a testing matrix for the assay optimization. The saliva was filtered through a 5 μm filter to reduce viscosity. N protein is enclosed inside the virus. Therefore, the sample is treated with a detergent to break the viral membrane to allow the release of the N protein. To mimic the final format of the testing sample matrix, the filtered saliva sample used for optimization was treated with 1% v/v triton X-100.
[0297] VFA workflow follows the CP-F assay format (
[0298] To achieve an even higher signal-to-noise ratio, enzymatic reaction time allowing for optimal colorimetric signal development was evaluated. Using the CP-F workflow with one washing step, the colorimetric reactions were left to develop for different durations following the TMB substrate application. Images of the cellulose test and control spots were taken at different development times and cyan intensities were analyzed. The highest signal-to-noise ratio was achieved with 3 minutes of the color development reaction (
[0299] To further simplify the assay workflow, testing reagent was prepared as a single tube reaction by mixing capture molecule (Sso.E2-CBD) and reporting molecules (BA-MBP-Sso.E1/SA-HRP) together into a single tube. Based on the assay optimized procedures, the assay workflow can be describe as shown in
Assay Performance
[0300] With the fully assembled VFA, performance was first tested with different concentrations of SARS-CoV-2 N protein spiked in filtered saliva containing 1% v/v triton X-100 to determine the test limit of detection (LoD). LoD was determined as the lowest concentration that gave signals higher than three times standard deviation values of the background (no antigen) signal (negative+3σ). Results showed that the VFA achieved a LoD at 2.5 nM (
[0301] To further assess the assay performance, the specificity of the rapid SARS-CoV-2 VFA to 2 pneumoniae bacterial strains and 16 viral strains (Table 6) that were spiked into filtered saliva containing 1% triton X-100 was investigated. These pathogens were chosen due to their flu-like symptoms upon infection, including fever and sore throat, symptoms that are also observed in SARS-CoV-2 infection. As shown in
TABLE-US-00006 TABLE 6 List of non-specific pathogens used for the cross-reactivity study Test Remarks/expire Number Strain Source Lot # Concentration date 1 Human ATCC VR-3 70033218 4 × 10.sup.6 adenovirus 3 TCID.sub.50/mL 2 Human ATCC VR-5 70024114 4 × 10.sup.7 adenovirus 5 TCID.sub.50/mL 3 Coronavirus- ATCC VR-740 70035459 4 × 10.sup.4 229E TCID.sub.50/mL 4 Coronavirus- ATCC VR-1558 70036255 4 × 10.sup.4 OC43 TCID.sub.50/mL 5 Human ATCC VR-1572 58527797 1.25 × 10.sup.5.5 adenovirus 4 TCID.sub.50/mL 6 Coronavirus ZEC. 325222 1.17 × 10.sup.3 HI (15 Oct. 2023) NL63 0810228CFHI TCID.sub.50/mL 7 RSV Type A ZEC. 324924 1.25 × 10.sup.4 HI (25 Aug. 2023 0810040ACFHI TCID.sub.50/mL 8 RSV Type B ZEC. 323000 1.25 × 10.sup.4 HI (4 Sep. 2022 0810040CFHI TCID.sub.50/mL 9 HMPV3 Type ZEC. 325204 0.97 × 10.sup.3 HI (7 Oct. 2023) B1 0810156CFHI TCID.sub.50/mL 10 Rhinovirus ZEC. 316699 2.5 × 10.sup.4.1 HI (N/A) A16 0810285CFHI TCID.sub.50/mL 11 Influenza A AMR in house 3 × 10.sup.7 culture CFU/mL 12 Influenza B AMR in house 3 × 10.sup.7 culture CFU/mL 13 Dengue 1 AMR in house NA Doing plaque culture assay 14 Dengue 2 AMR in house 2.5 × 10.sup.3 culture PFU/mL 15 Dengue 3 AMR in house NA Doing plaque culture assay 16 Dengue 4 AMR in house NA Doing plaque culture assay 17 Klebsiella AMR in house 4 × 10.sup.6 pneumoniae culture CFU/mL 18 Streptococcus AMR in house 3 × 10.sup.7 pneumoniae culture CFU/mL
Translation of the Rapid SARS-CoV-2 VFA to PoC Application
[0302] Following the assay optimization and verification, the next step is to translate the rapid SARS-CoV-2 VFA into PoC applications. To do so, there has to be a portable device which could measure cyan intensity signals. Two approaches to translate the VFA to PoC test were tested: 1) adopting of mobile phone camera to capture and interpret the test signals and 2) developing of a portable reader for analysis of the colorimetric signals.
[0303] For the first approach, a complementary ‘light box’ was created to ensure the consistency of lighting condition and the phone camera distance (
[0304] For the second approach, a spectrophotometer was designed to measure the red absorbance at 650 nm. The device equipped with a complementary software for signal analysis was outsourced to Attonics Systems Pte. Ltd., Singapore (
Discussion
[0305] Amidst the available options of SARS-CoV-2 vaccines, the COVID-19 pandemic continues. The importance of early detection and surveillance of SARS-CoV-2 infection has been emphasized and highlighted. Rapid, cheap, and easy-to-use diagnostic tests are still urged and employed as critical tools to help controlling the spreading of the virus.
[0306] The inventors have successfully developed a rapid SARS-CoV-2 VFA, on a cellulose platform using unique rcSso7d binder pair, that could detect SARS-CoV-2 N protein from saliva samples. During the lab-based verification, the system achieved LoD of 2.5 nM (
[0307] The group have developed, in parallel, two types of rapid SARS-CoV-2 VFA, one being the ‘bottom readout’ test and the second one being the test described in this example. Building upon the first developed ‘bottom readout’ test where the cellulose test strips required the ‘flipping’ action to apply TMB and for detection the colorimetric signals, the current test simplified the workflow by omitting the ‘flipping’ step. This workflow provides simpler operating steps at PoC settings as well as paves the way for ease of high throughput applications, where the cellulose test strips can be easily treated by different solutions via a liquid handling system.
[0308] Saliva has gained much interest as the alternative sample matrix for diagnosis and health monitoring as it provides easy mean of sampling and is non-invasive. Usage of saliva for diagnosing of SARS-CoV-2 infection has been proven feasible by many studies, in which the amount of viral load in saliva was shown to be in a comparable range to the viral load in naso- or oropharyngeal swabs. With an intention to improve the usability of ARTs for SARS-CoV-2 which currently employed nasal or nasopharyngeal swabs as a sample matrix, saliva was selected as a sample matrix due to its ‘painless’ and non-invasive sample collection method. Though the sampling of saliva is easy and straightforward, the sample processing was rather challenging. As the high viscosity of saliva caused by mucous content was problematic for controlling of fluid flow rate. This issue was resolved by filtering of saliva prior to performing the assay. Filtration of saliva may seem trivial during the test development phase, however to implement the test for PoC applications, an easy-to-operate device for saliva filtration at PoC settings has to be considered and sorted.
[0309] In conclusion, the inventors have successfully developed a rapid SARS-CoV-2 VFA that detects clinically relevant concentrations of viral N protein present in saliva. Thus far, this test offers the shortest turn-around-time of 5-10 minute from sample to results. Results obtained from this study indicated that the test has a great potential to be practically translated into a PoC diagnostic test for rapid detection of COVID-19. With more frequent testing, the control and isolation of COVID-19 infected individuals could be done more effectively, facilitating safe social interactions within the new community norms.
Materials and Methods
Materials
[0310] All chemicals were of analytical grades. Sources of standard chemicals that were not listed in the method sections were purchased from Merck/Sigma-Aldrich, Singapore.
Protein Expression and Purification
[0311] All cloning and protein expression work were done in the Escherichia coli DH5α and BL21 (DE3) strains, respectively. N protein NTD and CTD domain boundaries were scouted with the help of NTU Protein Production Platform. Coding sequences were cloned into the pET28b plasmid backbone. For protein expression, relevant plasmids were transformed into E. coli BL21 (DE3). Single colonies were obtained on LB agar plates supplemented with 50 μg/mL kanamycin (Sigma-Aldrich, Singapore) and inoculated in LB media supplemented with 50 μg/mL kanamycin. Each starter culture was grown overnight with shaking at 37° C. and added to 1 L of LB media for protein expression. Each expression culture was allowed to grow at 37° C. with shaking until the OD.sub.600 reached 0.6-0.9. The culture was then supplemented with 0.5 mM IPTG (Sigma-Aldrich, Singapore) to induce protein production and was grown overnight (16-20 hours) with shaking at 16° C. When expressing the reporting binder BA-MBP-Sso.E1, 0.2 mM of D-biotin (Sigma-Aldrich, Singapore) was also added to the culture prior to IPTG supplementation.
[0312] Cell pellet was harvested by centrifugation and subjected to lysis by sonication in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM Imidazole, pH 7.6) supplemented with protease inhibitor cocktail (Nacalai Tesque Inc., USA). The lysate was then clarified by high-speed centrifugation at 25,000 g. The soluble fraction (supernatant) was collected and incubated with HisPur Ni-NTA resin (Thermo Fisher Scientific, USA) for metal-affinity purification. Protein-bound resin was washed with buffers containing increasing imidazole concentrations before the desired protein was eluted by using elution buffer containing 300 mM imidazole. All constructs except SARS-CoV-2 N protein were collected at this step. Proteins collected from each fraction were ran on SurePAGE™ precast gels (GenScript, Hongkong, China), and stained using Brilliant Blue R Staining Solution (Sigma-Aldrich, Singapore) for visualization. Fractions containing pure protein of interest were concentrated and buffer-exchanged to PBS with Vivaspin centrifugal concentrators (Sigma-Aldrich, Singapore) and stored at −80° C. SARS-CoV-2 NP was further subjected to size exclusion chromatography with HiLoad 16/60 Superdex 75 (Sigma-Aldrich, Singapore) and eluted with gel filtration buffer. Pure SARS-CoV-2 N protein fractions were pooled and concentrated with Vivaspin centrifugal concentrators and stored at −80° C.
Processing of Saliva Samples
[0313] Pooled saliva was collected from healthy donors by passive drool saliva into collection tubes. To reduce the sample viscosity, collected saliva was filtered with 5 μm syringe filter (Sartorius, USA) and treated with 1 v/v % triton X-100 (Sigma-Aldrich, Singapore). This saliva sample matrix was used for all experiments, otherwise stated.
Preparation of SARS-CoV-2 Virus Sample
[0314] African green monkey kidney cells Vero E6 (CRL-1586; American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 8% fetal bovine serum (FBS) and penicillin-streptomycin at 37° C. in 5% CO.sub.2. SARS-CoV-2 (hCoV-19/Singapore/2/2020) virus was previously isolated (Anderson et al., 2020). and the genome sequence is deposited in GISASID (Accession ID EPI_ISL_407987). The virus stock was propagated in Vero E6 cells using DMEM supplemented with 2% FBS. The virus was titrated in ten-fold serial dilutions on 96-well plates of Vero E6 cells to obtain a 50% Tissue Culture Infectious Dose (TCID.sub.50). After 4 days incubation, the TCID.sub.50 of stock virus was calculated based on eight replicates by the Reed and Muench method (Reed & Muench). The viral stock was pre-diluted in FBS-free DMEM culture media to 107 TCID.sub.50/mL. Then, five-fold (2×10.sup.5, 4×10.sup.4, 8×10.sup.3, 1.6×10.sup.3 TCID.sub.50/mL) and two-fold dilutions (3.2×10.sup.2, 1.6×10.sup.2, 0.8×10.sup.2, 0.4×10.sup.2 TCID.sub.50/mL) were prepared using DMEM media. Three replicates of SARS-CoV-2 virus samples were prepared at each dilution for the VFA assay.
Cellulose Test Strips Preparation
[0315] Whatman Grade 1 Chr filter paper (GE Healthcare, USA) was used for fabrication of the test strip. Each test strip contained 1 test spot and 1 control spot, each with a reactive circular testing area of 4 mm diameter. To define the reactive testing area that allows liquid to pass through, wax ink was printed (ColorQube 8570, Xerox, USA) on to the cellulose paper to create hydrophobic boundary around the testing areas. The printed paper was baked for 1 minute at 150° C. to allow the wax ink to diffuse throughout the paper thickness, forming continuous hydrophobic boundary throughout the paper layer. The test strip was folded in a zig-zag motion to create a 3 layered test strip, generating the final strip ‘length×width’ dimension of 30×15 mm.sup.2. The top layer of the wax ink printed test strip was treated with 10 μL of 5% BSA dissolved in PBS. The treated papers were air dried and stored at 4° C. until used.
Optimization of Different VFA Test Formats
[0316] For the test conditions that required immobilization of Sso.E2-CBD on the test strip, the top layered of the test strip was treated with 10 μL of 2 μM Sso.E2-CBD diluted in PBS. To ensure that the proteins were immobilized on the cellulose surface, the cellulose test strip was suspended in the air, allowing the protein to remain on the test strip for ˜2 min, subsequently excess liquid was absorbed away using multi-purpose towel paper. The test strip was blocked with 10 μL 5% BSA in PBS, air dried and stored in 4° C. until used. For the condition which did not require pre-immobilization of Sso.E2-CBD, the cellulose test strip was treated with only 5% BSA. The test strips were constructed following the protocol described in the ‘VFA Test Assembly’.
[0317] PBS was used as the ‘sample matrix’ base for this experiment. The sample matrix was prepared by spiking PBS or 5 nM N protein into the PBS mixture reaction. Reporting molecule was prepared by mixing 500 nM of BA-MBP-Sso.E1 with 62.5 pM of streptavidin (SA) horse radish peroxidase (SA-HRP), (Biolegend, USA) in PBS. The reaction was kept incubated for at least 15 min on ice to ensure effective complex formation of BA-MBP-Sso.E1/SA-HRP prior to use. For the condition which did not require Sso.E1-CBD to be pre-immobilized onto the cellulose surface, 500 nM of Sso.E1-CBD was used for the mixture reaction. All mixture reaction volumes were prepared at 40 μL per reaction. This reaction volume maintained the hypothetical equimolar concentration of Sso.E1 in the solution and on the pre-immobilized cellulose surface, which was equivalent to 2×10.sup.−18 moles. 40 μL of 3,3′,5,5″-tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Singapore) was applied to generate colorimetric signal. After 3 minutes of the color development Image was taken using Epson Perfection V750 Pro Scanner (Epson, Singapore). Images were stored as .jpg files for subsequent colorimetric signal analysis.
VFA Test Assembly
[0318] The 3 layered wax ink printed cellulose test paper was prepared according to the protocol described in ‘Cellulose Test Strip Preparation’. 1.5 mm-thick cellulose filter paper (Whatman gel blotting papers grade GB005, Sigma-Aldrich, Singapore) was cut to 30×15 mm.sup.2 and was used as a material for absorbent pads. Two pieces of the absorbent material were placed underneath the 3 layered wax ink printed paper. To hold all the cellulose test strip components together, plastic manifolds were created using 2 mm thickness acrylic sheet. The acrylic sheet was cut into two pieces using a laser cutter (Epilog Fusion Edge Laser System, USA), each piece with a dimension of 30×50×2 mm.sup.3. An opening dimension 20×10 mm.sup.2 was cut through on one of the acrylic pieces, providing access to the test strips (
[0319] The test strip used for the custom-made spectrophotometer (Attonics Systems, Singapore) was assembled using re-usable cassettes made of aluminum, dimensioned 30×50×3.2 mm.sup.3. The cassette contained two circular openings, each with 3.8 mm diameter, for accessing of test and control spots. Following the principle set by the acrylic manifolds, the height in the inner chamber of the cassette was maintained at 3 mm for a consistent pressure applied to the test strip components.
Image Analysis
[0320] Images of all test strips were taken using Epson Perfection V750 Pro Scanner (Epson, Singapore), otherwise stated. All images were saved in .jpg format. Cyan intensity was analyzed from each image using an open-source ImageJ software from The US National Institute of Health (NIH). Image was separated into single channel of CYMK color space. Cyan intensity was calculated based on a mean value of the defined area in an image.
[0321] For assessment of live virus performed on the VFA, test result images were taken using an iPhone 5S instead of the scanner. The phone was used instead of the scanner due to restrictions of the BSL3 facility employed to perform the test. Images from the phone were transferred to a PC and analyzed using ImageJ software. The analysis was performed using a similar process applied to images acquired from the scanner. Despite the different image acquisition devices, cyan intensities were found to be within a comparable range.
Example 5—Rapid Detection of SARS-CoV-2 Neutralizing Antibodies from Non-Processed Whole Blood Samples and its Application on Monitoring of Immunity Status Against SARS-CoV-2 Variants
[0322] Stemming from the application in Example 3, the inventors further developed a test better suited to Point-of-Care (PoC) applications by using non-processed whole blood samples (fingertip or venous) instead of plasma or serum samples which require sample processing steps in a laboratory. Similar to the application in Example 3, this test utilizes the interaction between RBD/ACE2 to determine the level of NAb. However, HRP cannot be used as a reporting molecule in whole blood sample due to the presence of peroxidase in red blood cells that interferes with the colorimetric signal generation. Instead of using HRP, the ACE2 protein was conjugated to fluorescent molecules (ACE2-FI) i.e., AF594 and used as a reporting reagent (
[0323] A modified workflow was applied to this assay application to improve the test sensitivity. Whole blood sample was first incubated with RBD-CBD for 3 minutes, subsequently ACE2-FI was added to the reaction and incubated for another 5 minutes (
[0324] In a similar fashion to Example 3, absence or low concentrations of NAb led to high fluorescent intensities (
[0325] Because the positive NAb signals are derived from the absence of fluorescent intensity, a control spot is critical to ensure that the loss of signals are due to presence of NAb and not the malfunction of reagents. A control spot was designed to produce high fluorescent signal regardless of presence or absence of NAb. To do so, high concentration of RBD-CBD was immobilized on the cellulose surface at the control spot to capture ACE2-FI from the sample mixture (
[0326] Using this assay workflow, the inventors demonstrated that the test can detect different levels of NAb from samples that present different stages of COVID-19 vaccination (
[0327] In addition, the inventors have also demonstrated that the rapid cellulose-based SARS-CoV-2 NAb test can be used to monitor immunity status against the SARS-CoV-2 variants. Recombinant RBD proteins from each of the SARS-CoV-2 variant were fused to CBD (RBDv-CBD) and used as capture reagents. Using non-infected and non-vaccinated blood as sample matrix, the results showed that most of the RBD from the variants produced higher fluorescent intensities as compared to the wild-type (WT) SARS-CoV-2 (
Applications
[0328] Embodiments of the method and system disclose herein relates to a cellulose pull down (CP) technology that can capture target analytes more effectively using (i) pre-mixing solution and (ii) cellulose binding domain fused to an analyte binder. Cellulose binding domain (CBD) has a very high affinity toward cellulose substrate. It can be captured on to the cellulose substrate within less than a second. By fusing the analyte binder to CBD, embodiments of the method and system take advantage of the CBD to pull the assay complex down to a cellulose substrate.
[0329] In embodiments of the method and system, the sample, the reporter agents and optionally the capture agents are pre-incubated in a solution off site of a cellulose substrate. The pre-incubation step allows excess time for the assay complex (e.g. reporter agent-analyte complex or reporter agent-analyte-capture complex) to form (typically 1-3 min is sufficient). Once the complex is formed, it is applied to the cellulose substrate. The whole assay complex can be instantaneously pulled down to the cellulose paper via high affinity CBD-cellulose interaction, resulting in high sensitivity of signal production. In a scenario where a full assay complex incubation is not favorable, half complex pre-incubation (analytes with reporter reagents) can be applied. The capture agents comprising CBD can be pre-immobilized on to the cellulose substrate prior to performing the assay. The half complex formation during the pre-incubation allows most of the target analytes captured to generate signals, therefore promoting high sensitivity of signal production.
[0330] Rapid diagnostic tests often compromise sensitivity for rapid detection time. Embodiments of the method and system disclosed herein generally improve the performances of RDTs to the next level of sensitivity while maintaining the rapid detection time. Embodiments of the cellulose pull down (CP) technology allows sufficient time for full sandwich complex formation and effective capture of the full sandwich complex on to cellulose substrate. As a result, embodiments of the method allow high sensitivity signal detection in a short period of time. In addition, the use of low capture agent concentration could save up to 8 times on raw materials which could further lower down the production cost significantly.
[0331] In addition to RTD, embodiments of the method and system can be applied to wide array of biomedical and life science applications including protein separation. Altogether, embodiments of the method have great potential to be expanded to many applications. Examples of such applications include, but are not limited to, the rapid detection of SARS-CoV-2 neutralizing antibodies from human plasma/serum samples, the rapid detection of SAR-CoV-2 antigen from saliva samples and the rapid detection of SARS-CoV-2 neutralizing antibodies from non-processed whole blood samples and its application on monitoring of immunity status against SARS-CoV-2 variants.
[0332] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.