key: cord-0734594-9mkew3sl authors: Lake, D. F.; Roeder, A. J.; Kaleta, E.; Jasbi, P.; Periasamy, S.; Kuzmina, N.; Bukreyev, A.; Grys, T. E.; Wu, L.; Mills, J. R.; McAulay, K.; Seit-Nebi, A.; Svarovsky, S. title: Development of a Rapid Point-Of-Care Test that Measures Neutralizing Antibodies to SARS-CoV-2 date: 2020-12-16 journal: nan DOI: 10.1101/2020.12.15.20248264 sha: 1f5d911e1dbbf8e1f837b8ec51df00b70d2bbbca doc_id: 734594 cord_uid: 9mkew3sl As increasing numbers of people recover from and are vaccinated against COVID-19, tests are needed to measure levels of protective, neutralizing antibodies longitudinally to help determine duration of immunity. We developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies in plasma, serum or whole blood. The LFA is based on the principle that neutralizing antibodies inhibit binding of the spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). The test classifies high levels of neutralizing antibodies in sera that were titered using authentic SARS-CoV-2 and pseudotype neutralization assays with an accuracy of 98%. Sera obtained from patients with seasonal coronavirus did not prevent RBD from binding to ACE2. As a demonstration for convalescent plasma therapy, we measured conversion of non-immune plasma into strongly neutralizing plasma. This is the first report of a neutralizing antibody test that is rapid, highly portable and relatively inexpensive that might be useful in assessing COVID-19 vaccine immunity. The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) continues to be transmitted by person to person spread from its origin in Wuhan, China in December 2019 [1] [2] [3] . Vaccine trials are ongoing, and preliminary results from at least 2 vaccines appear to elicit protective immunity, but durability of vaccine responses is not known 4 Almost everyone infected with SARS-CoV-2 generates antibodies against the virus. However, since not all antibodies are created equal, it is essential to know which individuals generate high levels of neutralizing antibodies (NAbs) so that they can resume normal activities without fear of being re-infected and spreading the virus to others [8] [9] [10] . The goal of COVID-19 vaccines is to induce NAbs that prevent infection/re-infection. Additionally, development of NAbs indicates which individuals might be optimal donors for convalescent plasma protocols. Although NAbs are important for elimination of the virus and protection from subsequent infection, it has been reported by several groups that up to one-third of convalescent plasma from individuals who have recovered from COVID-19 do not neutralize SARS-CoV-2 or spike pseudotype virus infection [11] [12] [13] . Viral neutralization assays measure levels of antibodies that block infection of host cells. Two main types of viral neutralization assays are utilized for SARS-CoV-2. Authentic neutralization assays measure reduction of viral plaques or infectious foci in microneutralization assays in susceptible host cells using SARS-CoV-2 under BSL3 conditions. These assays are slow, laborious, require highly trained personnel and require a BSL3 facility. Pseudotype virus neutralization assays have been developed in which SARS-CoV-2 spike protein is expressed in a virus such as vesicular stomatitis virus or lentivirus 14, 15 . These assays are faster and less dangerous than authentic neutralization assays, but still require BSL2 conditions and 24-48 hours for results. Another challenge is that both authentic and pseudovirus virus assays depend on host cells for infection which adds variability to the assay. It is known that SARS-CoV-2 uses RBD on spike protein to bind ACE2 on host cells and appears to be the principal neutralizing domain of SARS-CoV-2 16, 17 . Using this knowledge, we developed an LFA that measures levels of (neutralizing) antibodies which block RBD from binding to ACE2. Other groups have developed RBD-ACE2-based competition ELISAs 18,19 but none have developed a rapid, highly portable semi-quantitative point-of-care (POC) test. Peripheral blood, serum and plasma were collected for this study under an Arizona State University IRB approved protocol #0601000548 and Mayo Clinic IRB protocol #20-004544. Plasma was obtained by ficoll gradient separation of peripheral blood and serum was obtained by centrifugation 30 minutes after drawing blood. Plasma and serum samples obtained from excess clinical samples at Mayo Clinic were pre-existing, de-identified and leftover from normal workflow. COVID-19 samples ranged from 3 to 84 days post PCR diagnosis. Titers were obtained from 60 COVID-19 patient sera using a VSV spike protein pseudotype assay as previously reported 14 . Inhibitory concentrations for which 50% of virus is neutralized by serum antibodies (IC50 values) were obtained using a different set of 38 COVID-19 sera in an authentic SARS-CoV-2 neutralization assay using a recombinant SARS-CoV-2 that expresses mNeonGreen (SARS-CoV-2ng) during replication in permissive cells 20 . Sixty µl aliquots of SARS-CoV-2ng were pre-incubated for 1 h in 5% CO2 at 37ºC with 60 µl serial 2-fold serum dilutions, and 100 µl were inoculated into Vero-E6 monolayers in black polystyrene 96well plates with clear bottoms (Corning). Each serum was tested in duplicates. The final amount of the virus was 200 PFU/well, and the starting serum dilution was 1:20. Cells were maintained in Minimal Essential Medium (ThermoFisher Scientific) supplemented by 2% FBS (HyClone) and 0.1% gentamycin in 5% CO2 at 37ºC. After 2 days of incubation, fluorescence intensity of infected cells was measured at a 488 nm wavelength using a Synergy 2 Cell Imaging Reader (Biotek). The signal readout was normalized to virus control aliquots with no serum added and was presented as the percentage of neutralization. IC50 was calculated with GraphPadPrism 6.0 software. Work with infectious SARS-CoV-2ng was performed in a BSL-3 biocontainment laboratory of the Galveston National Laboratory. The LFA contains a test strip composed of a sample pad and blood filter, conjugate pad, nitrocellulose membrane striped with test and control lines, and an absorbent pad to wick excess moisture (Axim Biotechnologies Inc, San Diego, CA). Test strips are secured in a cassette that contains a single sample port (Empowered Diagnostics, Pompano Beach, FL). For procedural control purposes, the LFA also contains a control mouse antibody conjugated to red gold nanospheres and corresponding anti-mouse control line. Thus, a control line should be visible in every assay. In the absence of a developed control line, results are not valid, and the test should be repeated. The test leverages the interaction between RBD-conjugated green gold nanoshells (Nanocomposix, San Diego, CA) and ACE2, to detect RBD-NAbs in COVID-19 convalescent plasma (CCP). As indicated by the competitive nature of this assay, test line density is inversely proportional to RBD-NAbs present within the sample. Thus, an absent or faint test line indicates high levels of RBD-NAbs, whereas a dark or strong test line suggests lack of RBD-NAbs within a given plasma sample. To demonstrate the ability of the test to measure NAbs in whole blood, 10µg of a neutralizing monoclonal antibody (mAb) based on the sequence of B-38 21 (Axim Biotechnologies, Inc, San Diego, CA) was mixed with 10µl of normal donor whole blood collected in a heparinized blood collection tube. Two-fold dilutions were made in whole blood to a final concentration of 0.625µg neutralizing mAb. Then, 10µl of each dilution were transferred to LFA cassettes, chased with 50µl running buffer, and read with an LFA reader after 10 minutes. LFAs were performed at ambient temperature and humidity on a dry, flat surface and left to run undisturbed for 10 minutes prior to reading results. First, 10µL of plasma or whole blood were transferred to the cassette sample port and immediately followed by two drops (~50µL) of chase buffer. After 10 minutes, each test was placed in a Detekt RDS-2500 LFA reader (Austin, TX, USA) and the densities of both test and control lines were recorded electronically. To convert NIHP into strongly neutralizing plasma (SNP), plasma from a convalescent donor who demonstrated the ability to block RBD from binding to ACE2 (M21) was mixed with NHP collected prior to December 2019. For example, for a 1% mixture, 1ul of SNP was mixed with 99ul NIHP; for a 5% mixture 5ul of SNP was admixed with 95 ul NIHP, etc. We performed the test with 10ul of 1%, 5%, 10%, and 20% SNP admixed into NIHP. To establish and compare the validity of the LFA and the VITROS® Anti-SARS-COV-2 IgG assay (Ortho Clinical Diagnostics), regression analysis using IC50 values was performed to evaluate consistency 18 while Bland-Altman plots were constructed to assess agreement and bias 22, 23 . Regression analysis was performed using Microsoft Excel (Redmond, WA); Bland-Altman plots were visualized using IBM SPSS (Armonk, NY). For two-group analysis, IC50 values corresponding to >240 were categorized as titer of ≥1:320 (neutralizing), whereas IC50 values ≤240 were categorized as ≤1:160 (non-neutralizing). Receiver operating characteristic (ROC) analysis was performed to assess classification accuracy, sensitivity, and specificity of the LFA and Ortho methods in assessing neutralizing capacity; optimal cutoffs for each method were established to maximize area under curve (AUC) 24, 25 . ROC analysis was conducted using R (version 3.6.2). The lateral flow assay reported here is a rapid 10-minute POC test. As shown in the schematic in Figure 1 , this test utilizes serum, plasma or whole blood to semi-quantitatively measure levels of NAbs. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; Figure 1 . Schematic of Neutralization LFA. Below each graphic is a representative image of a lateral flow strip demonstrating actual line density. Addition of non-COVID19-immune serum or plasma (top) does not block binding of RBD-beads to ACE2 resulting in the RBD-bead-ACE2 complex creating a visible line. Addition of moderate titer NAbs to the sample pad creates a weak line (middle). Addition of high titer NAbs (> 1:640) blocks binding of RBD-beads to ACE2 such that no line is observed at the test location on the strip (bottom). Red control line represents capture of gold nanospheres coupled to a mouse monoclonal antibody. Neutralizing Ab Nanoshells coupled to RBD All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20248264 doi: medRxiv preprint An example of strong, moderate and non-neutralizing sera is shown in Figure 2 . As diagrammed in Figure 1 , levels of NAbs in serum or plasma are reflected by the intensity of the test line which can be read on a hand-held densitometer or compared visually to a scorecard (Supplemental Figure 1) . Figure 2A is representative of highly neutralizing sera, while Figure 2B and 2C represent moderately neutralizing sera. Figure 2D represents sera that do not contain NAbs or have undetectable levels of Nabs. We tested serum samples with known neutralization titers from a VSV-based spike pseudotype assay in our LFA. De-identified serum samples were sent from Mayo Clinic Rochester to our laboratories. Personnel running the LFA were blinded to the titers of each sample. After de-coding, the LFA tracked with serum titers, especially at higher titers and correctly distinguished all non-neutralizing serum ( Figure 3A) . To further support the application of our lateral flow test to measure levels of antibodies that neutralize SARS-CoV-2, we tested a different set of 38 serum samples that were assigned preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20248264 doi: medRxiv preprint IC50 values in an authentic SARS-CoV-2 neutralization assay 20 . Again, the experiment was performed in a blinded manner such that personnel running either the LFA or the microneutralization assay did not know the results of the comparator test. When line densities from the LFA are plotted against IC50 values determined in the SARS-CoV-2 microneutralization assay, serum samples with strong neutralization activity also demonstrate low line densities which indicates inhibition of RBD from binding to ACE2 (Figure 3B) . preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; We evaluated how the Ortho assay and our LFA compared to IC50 values determined in an authentic SARS-CoV-2 neutralization assay using 38 COVID-19 sera (different from the 60 sera in Figure 3A with known titers). FDA guidance indicates that an Ortho VITROS SARS-CoV-2 IgG assay of ≥12 meets a threshold for convalescent plasma use in patients with COVID-19. To determine the agreement between our lateral flow assay and the Ortho VITROS SARS-CoV- All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20248264 doi: medRxiv preprint significant relationship with each other (r = -0.572, p < 0.001). To evaluate bias between the assays, mean differences and 95% confidence intervals (CIs) were calculated and plotted alongside limits of agreement (Figure 6 ). Both LFA and Ortho values showed high agreement with titer, although Ortho showed a tendency to underestimate neutralizing capacity while the LFA method showed no bias. As can be seen in Figure 7B and 7D, the LFA misclassified one non-neutralizing sample (Neg-1:160) as neutralizing (≥1:320). In contrast, Ortho misclassified that same non-neutralizing sample as neutralizing, in addition to incorrectly classifying five additional neutralizing samples as non- All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20248264 doi: medRxiv preprint neutralizing. Importantly, our LFA method exhibited a greater dynamic range of differential values as compared to Ortho (Figure 6) , translating to superior classification accuracy of the LFA method in detecting neutralizing samples (titer ≥ 1:320) compared to Ortho (Figure 7A and 7C) . Univariate ROC analysis was performed to assess the performance of the newly developed LFA and Ortho assay to classify non-neutralizing (Neg-1:160), and neutralizing groups (≥1:320) (Figure 7) . Our LFA showed high accuracy for classification of neutralizing A B All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. The principle of infusing convalescent plasma from recovered individuals into patients fighting COVID19 is to transfer NAbs from the donor to the recipient as has been done for several other diseases [26] [27] [28] . The rapid test described here, could quickly and efficiently classify COVID19 convalescent plasma (CCP) units so that the sickest patients might receive the most potently neutralizing plasma. Although levels of neutralizing antibody to achieve in patients receiving CCP remains undefined, our point-of-care test might be useful at the bedside to monitor a patient receiving highly neutralizing CCP as he/she begins to demonstrate NAbs in circulation, as shown in Figure 8A . It could help in deciding whether to administer another unit of highly neutralizing CCP to a patient fighting COVID19. The same scenario might also be applied to hyperimmune gammaglobulin or neutralizing monoclonal antibody infusion. To demonstrate the utility of our LFA to measure NAbs in whole blood, we used a lateral flow strip with a blood filter and performed an experiment in which a neutralizing monoclonal antibody based on the B-38 21 sequence was titrated into whole blood as shown in Figure 8B . Density units were not obtained in these experiments but both Figures 8A and 8B demonstrate All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20248264 doi: medRxiv preprint the semi-quantitative nature of this test to visually distinguish different levels of NAbs in plasma and whole blood. For both types of cassettes, plasma or blood sample was immediately chased with 50µl sample buffer. Densities were read and cassettes were imaged after 10 minutes development. Serologic tests that detect responses to infection are an important population surveillance tool during pandemics because they provide data on pathogen exposure, especially Whole Blood + 5.0 ug B38 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020.12.15.20248264 doi: medRxiv preprint when a subset of the population is asymptomatic and would not have been diagnosed by molecular methods 29, 30 . While over 90% of individuals diagnosed with COVID19 generate antibody responses 29 we are only beginning to learn about the prevalence and duration of NAbs induced by a natural infection. This lack of knowledge is mainly due to the fact that virus-based neutralization assays require i) BSL2 or BSL3 facilities, ii) highly skilled personnel, iii) permissive cells and quantified virus, and iv) take longer than 24 hours to obtain results. Since every viral vaccine, including COVID19 vaccines, administered to humans is designed to elicit antibodies that neutralize virus by blocking host cell infection, development of NAbs is a hallmark of protection from disease. Therefore, there is a need for rapid, accurate tests that measure levels of NAbs. We developed a rapid, 10-minute lateral flow test that measures levels of NAbs in serum and plasma. As shown in Figure 3 , the lateral flow test correlates well with serologic titers determined using a VSV-based pseudotype assay, and IC50 values in an authentic SARS-CoV-2 microneutralization assay, especially when serum sample titers are ≥640 and IC50 values are >250. Samples with less potent NAbs in both viral assays correlated with decreased ability to block RBD from binding to ACE2 in the LFAs. The LFA and Ortho methods showed a strong, significant correlation with each other (r = -0.572, p < 0.001), displaying an appreciable degree of linear relation 31 . Importantly, LFA accounts for 52% of observed IC50 variance (R 2 = 0.5187) while, in comparison, Ortho accounts for 27% (R 2 = 0.2725). Although absolute quantitation of a construct demands an excellent coefficient of determination (R 2 ≥ 0.99) 32 , variables with R 2 ≥ 0.5 are highly predictive in univariate regression models while measures with R 2 < 0.5 are recommended for use in multivariate models in combination with complementary measures to increase predictive accuracy 33, 34 . Additionally, Bland-Altman analysis (Figure 6 ) showed Ortho to be prone to underestimation of IC50 values. In contrast, the LFA method did not exhibit any over-or underestimation bias. Furthermore, across mean values for both methods, LFA showed discrete differential values while Ortho struggled to differentiate samples with high neutralizing capacity (IC50 values). Since the Ortho Vitros IgG assay uses S1, which includes RBD, it is likely that antibodies reactive with other parts of S1-even some that may neutralize via N-terminal domain-are responsible for increased reactivity to S1 when they are not neutralizing in our RBD-ACE2 competition assay. Advantages of our POC test is that it can be inexpensively and rapidly deployed in studies to determine levels of NAbs that protect against re-infection and limit transmission of the virus. Moreover, rapid inexpensive tests can be used longitudinally to evaluate duration of protective immunity in both naturally infected and many more vaccinated individuals than could ever be evaluated using BSL2 or BSL3-based neutralization assays. In the setting of much debated CCP, a rapid test could be used to measure levels of NAbs in a CCP product prior to infusion, or potential donors who recovered from COVID-19. Limitations of the LFA are that it uses only the RBD portion of SARS-CoV-2 spike protein. Although the vast majority of reports indicate that the principle neutralizing domain is RBD portion of spike protein, mAbs have been reported that neutralize SARS-CoV-2 by binding All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted December 16, 2020. ; https://doi.org/10.1101/2020. 12.15.20248264 doi: medRxiv preprint to the N-terminal domain of spike protein 35, 36 . Also, since the spike protein assumes several conformations during viral binding and entry 37 , neutralizing epitopes exist on the quaternary structure of spike 36 . Although RBDs on the nanoparticles may associate, it is unlikely they assume a native quaternary conformation. Other limitations are the binary nature of the data analysis (neutralizing and nonneutralizing) of a continuous assay. Clearly, line densities demonstrate moderate levels of neutralization. Since blood draws and subsequent assays are a "snapshot in time" of neutralizing antibody activity, levels were undoubtedly increasing in some patients and decreasing in others. LFAs are generally inexpensive and highly portable compared to other laboratory-based tests, so neutralizing antibody levels could be measured using the LFA to longitudinally assess protective neutralizing antibody immunity. Another limitation is that the LFA does not differentiate high affinity anti-RBD NAbs from an abundance of lower affinity anti-RBD NAbs. In related experiments, we have observed patient sera that bind strongly to RBD, but do not demonstrate neutralizing activity (data not shown). This test may prove very useful in monitoring COVID-19 vaccine recipients. Although vaccines have now been approved for distribution and administration, durability of protective immunity elicited by any COVID-19 vaccine is unknown. It is the goal of all COVID-19 vaccines to induce protective NAbs. However, since clinical trials of vaccines have enrolled 30,000 to 60,000 participants, it is not logistically possible to draw a tube of blood from each vaccine recipient longitudinally to determine duration of protective NAbs. Application of our test in vaccine recipients using a drop of blood obtained from a finger-stick as shown in Figure 8B might lead to more comprehensive longitudinal monitoring of increases and decreases in protective humoral immunity. 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