key: cord-0713847-cedoi5jr
authors: Bray, R. A.; Lee, J.-H.; Brescia, P.; Kumar, D.; Nong, T.; Shih, R.; Woodle, E. S.; Maltzman, J. S.; Gebel, H. M.
title: Development and validation of a multiplex bead based assay for the detection of antibodies directed against SARS-CoV-2 proteins
date: 2020-09-03
journal: nan
DOI: 10.1101/2020.09.02.20185199
sha: 13123b9991644b12ef6099e856c1d6595d20963e
doc_id: 713847
cord_uid: cedoi5jr

Transplant recipients who develop COVID-19 may be at increased risk for morbidity and mortality. Determining antibody status against SARS-CoV-2 in candidates and recipients will be important to understand the epidemiology and clinical course of COVID-19 infection in this population. There are multiple antibody tests to detect antibodies to SARS-CoV-2, but their performance varies according to their platforms and the antigenic targets, making interpretation of the results challenging. Additionally, currently available serological tests do not exclude the possibility that positive responses are due to cross reactive antibodies to community coronaviruses. This study describes the development and validation of a high throughput multiplex bead based antibody detection assay with the capacity to identify, simultaneously, patient responses to five distinct SARS-CoV-2 proteins. The antibody response to these proteins are SARS-CoV-2 specific as antibodies against four community coronaviruses do not cross-react. Assay configuration is essentially identical to the single antigen bead assays used in the majority of histocompatibility laboratories around the world and could easily be implemented into routine screening of transplant candidates and recipients. This new assay provides a novel tool to interrogate the spectrum of immune responses to SAR-CoV-2 and is uniquely suitable for use in the transplant setting.

Transplant recipients who develop COVID-19 may be at increased risk for morbidity and mortality. Determining antibody status against SARS-CoV-2 in candidates and recipients will be important to understand the epidemiology and clinical course of COVID-19 infection in this population. There are multiple antibody tests to detect antibodies to SARS-CoV-2, but their performance varies according to their platforms and the antigenic targets, making interpretation of the results challenging. Additionally, currently available serological tests do not exclude the possibility that positive responses are due to cross reactive antibodies to community coronaviruses. This study describes the development and validation of a high throughput multiplex bead based antibody detection assay with the capacity to identify, simultaneously, patient responses to five distinct SARS-CoV-2 proteins. The antibody response to these proteins are SARS-CoV-2 specific as antibodies against four community coronaviruses do not crossreact. Assay configuration is essentially identical to the single antigen bead assays used in the majority of histocompatibility laboratories around the world and could easily be implemented into routine screening of transplant candidates and recipients. This new assay provides a novel tool to interrogate the spectrum of immune responses to SAR-CoV-2 and is uniquely suitable for use in the transplant setting.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint

Following the outbreak of the COVID-19 pandemic, assays using lateral flow, ELISA, chemiluminescence and fluorescence technologies were developed to detect antibodies to SARS-CoV-2 antigens (1) (2) (3) (4) . Target proteins include the SARS-CoV-2 nucleocapsid (NC) phosphoprotein (5) , the full-length spike glycoprotein (6) , or the receptor-binding domain (RBD) of the spike protein (7) . The underlying premise is that such antibody assays can be used to evaluate the epidemiology of COVID-19 and, potentially, identify individuals and communities who would be protected from reinfection and have higher rates of herd immunity, respectively. However, it remains unclear which antibodies are actually protective or how long antibody levels are sustained (8) . Notably, the majority of tests currently in use have reported sensitivities and specificities ranging from 96%-98%, which, while acceptable, are especially concerning when evaluating individuals from regions with <5% prevalence of disease (9, 10) . Under those circumstances, a positive result has a high probability to be falsely positive, especially in a situation of low population prevalence. Since the majority of FDA approved serological assays only detect one viral target per test, even a true positive result has limited meaning, as only certain antibodies to SARS-CoV-2 appear to have viral neutralizing activity (11). Thus, simply considering a patient as SARS-CoV-2 antibody positive provides, at best, incomplete and at worst, misleading information regarding the clinical implications of those antibodies.

The limitations of the current assays to detect antibodies to SARS-CoV-2 become even more apparent when considering the spectrum of clinical responses to COVID-19. Clearly, certain patient characteristics are associated with an increased risk . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint of morbidity and mortality (12) . For example, recipients of solid organ allografts who become infected with SARS-CoV-2 are at especially high risk for adverse consequences compared to matched controls, presumably a consequence of being immunosuppressed and other underlying health issues (13) . Knowing their SARS-CoV-2 antibody status before and after transplant could affect patient care.

Transplant candidates and recipients are routinely monitored to evaluate their HLA antibody status pre-and post-transplantation. The majority of histocompatibility laboratories around the world use a Luminex platform to detect and identify the HLA antibodies these patients possess (14) . The HLA Luminex-based test is a multiplex, solid-phase antibody detection assay where up to 100 different microparticles coated with individual class I or class II HLA alleles, respectively, are simultaneously tested with patient sera. Due to its sensitivity, specificity and high throughput, this multiplex bead assay revolutionized the field of organ transplantation (15) . In a similar manner, a Luminex-based multiplex test designed to identify antibody responses to unique SARS-CoV-2 targets could dramatically enhance our understanding of the immune response to COVID-19. In this study, we describe the development and validation of a multiplex high throughput solid phase assay that simultaneously determines the presence/absence of antibodies to five distinct SARS-CoV-2 viral proteins.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted September 3, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted September 3, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted September 3, 2020. 

Validation of SARS-CoV2 coated microparticles: Microparticles coated with the SARS-CoV-2 proteins, Full Spike, S1, S2, RBD and NC as well as non-SARS-CoV-2 proteins from community coronaviruses (CoV-229E-S1, HCoV-HKU1-S1, HCoV-NL63-S1, HCoV-OC43-S1), and novel coronaviruses (MERS-S1, and SARS-S1), were evaluated with commercial polyclonal antibodies in a matrix fashion as shown in Figures   1A and 1B. For the SARS-CoV-2 proteins ( Figure 1A ), antisera specific for the NC showed positive reactivity with the NC-coated bead while failing to react with the other 4 SARS-CoV-2 proteins. The antisera directed against the Full Spike protein reacted with the Full Spike, S1 and RBD coated beads and was non-reactive with the S2 and NC.

This reactivity pattern was in agreement with the manufactures published specifications for this antibody. The antisera directed against the SARS-CoV-2 S2 protein showed positive reactivity against both the Full Spike and S2 proteins as anticipated based on the manufacturers published specifications for this antibody. The antisera specific for . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint the SARS-CoV-2 Spike protein showed cross-reactivity against SARS-CoV-2 Full Spike, S1 and RBD. The antisera specific for SARS NC protein also showed cross-reactivity with the SARS-CoV-2 NC protein consistent with the published specifications for this antibody. Figure 2A shows data from a representative experiment wherein serum samples, collected from 96 randomly selected and pre-COVID-19 (October, 2018) kidney transplant candidates were tested with the COVID Plus assay. As the data illustrate, with the exception of the S1 spike protein, all samples had MFI levels <5000. For the Full Spike protein, the average value was 675 + 816; range 44 -4540 MFI. For the spike S2 protein, the average value was 138 + 97; range 42 -908 MFI. For the spike RBD protein, the average value was 224 + 363; range 23 -3000 MFI. For the NC protein, the average value was 388 + 441; range 89 -2593 MFI. For the S1 protein, background levels were higher but all samples showed MFI values <7,500. For the S1 spike, the average background value was 1226 + 1411; range 133 to 7439 MFI. Based on these data, responses <5000 MFI for the Full Spike, S2, RBD and NC coated beads were considered negative, while values <7500 MFI were considered negative for S1. Figure 2B displays the responses for the 96 pre-COVID-19 samples against a panel of other coronavirus targets including SARS-CoV and MERS. The data demonstrate that while most individuals have significant IgG responses to community coronaviruses, all pre-COVID-19 samples tested negative against the SARS-CoV-2 targets as well as to SARS and MERS S1 proteins.

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The copyright holder for this preprint this version posted September 3, 2020. . Specifically, MFI values ranged from 10,000 -60,000 MFI for the Full Spike protein, 5,000 -50,000 MFI for S1, 2,000 -8,000 for S2, 2,000 -30,000 for RBD and 3,000 -15,000 for the NC protein. All 42 patients had a >5,000 MFI response to the Full Spike, while 33/42, 38/42, and 39/42 patients had >5,000 MFI response to S2, RBD and NC, respectively. For S1, 38/42 samples showed a response >7,500 MFI.

The spectrum of response to SARS-CoV-2 proteins is illustrated in Figure 4 . In figure 4A , the semi-quantitative MFI differences between and within individuals is plotted. Figure 4B illustrates the proportional difference among the COVID-19 positive patients. The data show that, on average, the Full Spike protein constituted the majority of the response in any given individual. However, the proportional differences between the three components of the Full Spike S1, S2 and RBD varied greatly between individuals. Of note, the proportional response to the RBD protein varied from 2% to 26% of the total MFI response. For cumulative MFI values <40,000, the RBD response ranged between 2 and 10% with a . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 1 1 mean response proportion of 5.5%. However, for patients with a cumulative MFI value >40,000, the RBD response ranged between 12-26% with an average of 20%. 

Herein, we describe the development of a novel multiplexed Luminex based immunoassay that simultaneously and semi-quantitatively assesses patient sera for antibodies to multiple SARS-CoV-2 proteins. This assay builds on >25 years of experience with the Luminex platform to detect and identify antibodies to HLA antigens present in the serum of transplant candidates and recipients. The strengths of this . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint multiplexed, solid phase assay compared to other platforms (e.g., ELISA) include the ability to assay multiple viral targets simultaneously (and thereby provide internal assay controls for coronavirus specificity), the capacity to incorporate additional SARS-CoV-2 proteins in the future, a relatively short assay time, high throughput and semiquantitative assessment of the antibody response. Furthermore, these data demonstrate that the antibody responses to common community coronaviruses do not cross react with the SARS-CoV-2 proteins in the COVID Plus assay (Figure 2 ). An additional benefit is that evaluation and monitoring of samples from transplant candidates and recipients for antibodies to SARS-CoV-2 is readily adaptable into the routine clinical practice of histocompatibility laboratories that support solid organ and/or stem cell transplant programs.

In the United States, the need for robust, accurate and reproducible serological tests to identify individuals who developed antibodies to SARS-CoV-2 was recognized by mid-March 2020. In response to demand, the FDA authorized emergency use authorization of a multitude of such tests to be developed, implemented and used throughout the US. It was anticipated that the tests would identify individuals 1) who had developed humoral immunity in response to exposure; 2) would be resistant to reinfection with SARS-CoV-2 and 3) from whom convalescent plasma could be collected and used as a therapeutic or prophylactic for patients diagnosed with COVID-19 (19) . However, recent studies reveal that a positive antibody test is not necessarily adequate to determine current or future immunity to SARS CoV-2. Indeed, recent data by Sutar et al (18) documented the importance of appropriate timing for serological testing relative to PCR testing and/or symptom onset after infection. Additional studies . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint by Chi et al revealed that although antibodies to the RBD of SARS CoV-2 are the antibodies most likely to be neutralizing, so, too, could antibodies to other viral targets (20) . Thus, understanding which antibodies are protective and being able to detect them are both essential components of serological testing.

The genome of SARS-CoV-2, a single stranded, enveloped RNA coronavirus, encodes four major structural proteins, namely, spike, envelope, membrane and nucleocapsid as well as more than a dozen non-structural proteins (21) . Based on their immunogenicity and predicted neutralizing potential, either the NC, spike (full length, S1 or S2) or the RBD are the individual designated targets for the vast majority of antibody assays in use. Sensitivity and specificity for such assays typically ranges from 96-98%, which, while reasonable, can unfortunately lead to false positive and false negative results (9,10). An additional important limitation of such single target assays is that they fail to assess the breadth of a patient's response. In fact, recent studies reveal that patient antibody responses to SARS CoV-2 are not uniform. For example, while convalescent plasma donors and patients who have recovered from SARS-CoV-2 infection have detectable RBD antibodies that are neutralizing, RBD antibodies detected in pediatric COVID-19 patients who developed multisystem inflammatory syndrome are not neutralizing (11).

The collective published data illustrate the gap in our understanding the complexities of the humoral immune response to COVID-19. Assays that interrogate a response to a single viral antigen limit our ability to understand fully the immune responsiveness to SARS-CoV-2. This is evidenced by the response of subject #8 who showed strong reactivity against the Full Spike protein but was negative for S1, S2 and . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint RBD. The low level of NC protein reactivity also did not exceed the 3 SD cutoff to consider the reaction as positive. Since S1, S2 and RBD proteins are elements of the Full Spike protein; this reactivity could reflect responsiveness to a unique cross-reactive epitope not related to SARS-CoV-2 infection. This reactivity pattern was not observed in any other SARS-CoV-2 negative sample either pre-or post-COVID-19. Furthermore, the reactivity pattern of subject #8 extended well back into the global pre-COVID-19 era.

The exact nature of this reactivity is under further investigation. When the results of all confirmed SARS-CoV-2 positive samples and negative samples obtained from samples prior to the COVID-19 pandemic are considered, the specificity and sensitivity of the assay is 98.6% and 100%, respectively. Excluding the data from the one subject positive only with the Full Spike protein, assay specificity and sensitivity are both 100%.

One recently described assay simultaneously evaluated the response to both nucleocapsid and spike proteins with a reported sensitivity of 100% and 99.9% sensitivity (22) , However, that assay required patient samples to be diluted 1/200 before testing, which suggests that the signal to noise ratio of undiluted samples was less than optimal. Furthermore, the assay time was close to 20 hrs. In the multiplex assay described here, the responses to five different viral targets were simultaneously tested and the total assay time was <4 hours. Furthermore, 96 samples can be assessed at the same time in a single tray.

Interestingly, the proportional responses to the five antigens differed among the patients. For example, some individuals displayed a higher MFI to NC vs RBD (e.g., sample P18; 15,641 vs 8662, respectively), while other subjects displayed a higher response to the RBD target than to the NC (e.g., sample P36; 33,491 vs 14,703, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . 1 5 respectively). It is intriguing to speculate whether such observations would explain differences in clinical manifestations and outcomes. The differences could potentially be derived from immune deviation because of underlying disease or genetics. There may also be influences due to inter-individual variations in B cell repertoires, differences in disease progression, and the amount of time from which patients were initially diagnosed until the time they were tested for antibodies. Interrogation of antibody responses from well-characterized patients grouped according to age, severity of disease, and response to therapy will be improved by simultaneously evaluating the response to multiple SARS-CoV-2 proteins. Other important questions such as an individual's antibody response to vaccination, viral neutralization titers and whether some antibodies enhance disease progression will be better addressed with this technology.

The semi-quantitative nature of the response offered by this assay will also likely be of greater value rather than the simple "yes" or "no" result obtained from lateral flow assays. The titer of antibodies to SARS-CoV-2 correlates with their ability to limit viral infectivity. Our preliminary data suggest that due to the increased dynamic range of the Luminex FLEXMAP 3D ® instrument, multiplex testing for SARS-CoV-2 antibodies allows for a level of quantification not possible with lateral flow, ELISA or chemiluminescence assays unless serial dilutions are performed for each sample.

While this first iteration of this multiplex Luminex based assay has five target proteins, our intention is to add additional targets to the panel. Indeed, the SARS-CoV-2 virus has 29 total proteins any of which could be immunogenic and stimulate an antibody response. Antibodies to non-structural and accessory proteins of SARS-CoV-. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. .

2, namely ORF9b and NSP5, have been identified in the convalescent sera of infected patients infected with SARS-CoV-2 (23). Although viral targets not expressed on the cell surface are not typically considered to generate antibodies that are neutralizing, that is not always true. For example, for influenza, antibodies to ORF proteins confer protective immunity based on passive transfer studies and experiments with B cell knockout mice (24, 25). As the current version of COVID Plus only uses a total of thirteen individual Luminex addresses, there is ample real estate for expansion.

From a more practical standpoint, the SARS-CoV-2 multiplex assay described here is very similar to the multiplex assays used to identify HLA antibodies routinely deployed by HLA laboratories around the world. As such, incorporating the SARS-CoV-2 assay into routine testing of transplant candidates and recipients would be seamless, and provide useful information to the surgeons and clinicians caring for patients during the COVID-19 pandemic.

Limitations of this study include the source of our negative and positive control sera. Negative control samples were obtained from patients with chronic kidney disease. As such, the patients may be limited in their ability to produce antibodies.

However, we observed that all the sera from this group of subjects had antibodies to community CoV and, furthermore, sera from two patients that had tested negative prior to the COVID-19 pandemic did develop antibodies to all five SARS-CoV-2 at a time infections were surging. With regard to sera from confirmed positive cases, we were unable to collate sample date with disease status (e.g., acute vs convalescent). A final limitation to the multiplex assay (and all other antibody detection assays) is that it does . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint 1 8 for presenting CME certified lectures. All other authors have no conflicts of interest as defined by the American Journal of Transplantation.

RAB: designed the study, collected the data, analyzed the data, interpreted the data, drafted the article and revised the article critically.

J-H Lee: designed the study, collected the data, analyzed the data, interpreted the data, drafted the article and revised the article critically.

PB: collected and analyzed the data.

DK: designed the study, interpreted the data and revised the article critically TN: collected and analyzed the data.

RS: collected and analyzed the data.

ESW: designed the study, interpreted the data and revised the article critically.

JSM: designed the study, collected the data, analyzed the data, interpreted the data, drafted the article and revised the article critically.

HMG: designed the study, collected the data, analyzed the data, interpreted the data, drafted the article and revised the article critically.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. Samples were obtained from candidates on the kidney wait list during the month of May 2020. Note that patients #14 and #51 would be scored as positive for antibody. Patient #8 represents an apparent false positive with reactivity against only the Full Spike protein.

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The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.02.20185199 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted September 3, 2020. 1  3  5  7  9  11  13  15  17  19  21  23  25  27  29  31  33  35  37  39  41  43  45  47  49  51  53  55  57  59  61  63  65  67  69  71  73  75  77  79  81  83  85  87  89  91  93  95 HCoV-HKU1-S1 0   5000   10000   15000   20000   1  3  5  7  9  11  13  15  17  19  21  23  25  27  29  31  33  35  37  39  41  43  45  47  49  51  53  55  57  59  61  63  65  67  69  71  73  75  77  79  81  83  85  87  89  91  93  95 HCoV-NL63 

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