key: cord-1042592-444ve60t authors: Fong, C. H. Y.; Dissanayake, T. K.; Chen, L.-L.; Choi, C. Y.-K.; Wong, L.-H.; Ng, A. C.-K.; Pang, P. K. P.; Ho, D. T.-Y.; Poon, R. W.-S.; Chung, T. W.-H.; Sridhar, S.; Chan, K.-H.; Chan, J. F.-W.; Hung, I. F.-N.; Yuen, K.-Y.; To, K. K.-W. title: Improved detection of antibody against SARS-CoV-2 by microsphere-based antibody assay date: 2020-05-26 journal: nan DOI: 10.1101/2020.05.26.20113191 sha: 89f0c21e25e6afb734d0486c86329ec6e1e408b9 doc_id: 1042592 cord_uid: 444ve60t Objective: Currently available COVID-19 antibody tests using enzyme immunoassay (EIA) or immunochromatographic assay have variable sensitivity and specificity. Here, we developed and evaluated a novel microsphere-based antibody assay (MBA) for the detection of immunoglobulin G (IgG) against SARS-CoV-2 nucleoprotein (NP) and spike protein receptor binding domain (RBD). Method: We developed a microsphere-based assay (MBA) to determine the levels of IgG against SARS-CoV-2 NP and spike RBD. The seropositive cut-off mean fluorescent intensity (MFI) was set using a cohort of 294 anonymous serum specimens collected in 2018. The specificity was assessed using serum specimens collected from organ donors or influenza patients before 2020. Seropositive rate was determined among patients with COVID-19. Time-to-seropositivity and signal-to-cutoff (S/CO) ratio were compared between MBA and EIA. Results: MBA had a specificity of 100% (93/93; 95% confidence interval [CI], 96-100%) for anti-NP IgG and 98.9% (92/93; 95% CI 94.2-100%) for anti-RBD IgG. The MBA seropositive rate for convalescent serum specimens of COVID-19 patients were 89.8% (35/39) for anti-NP IgG and 79.5% (31/39) for anti-RBD IgG. The time-to-seropositivity was shorter with MBA than that of EIA. When compared with EIA, MBA could better differentiate between COVID-19 patients and negative controls with significantly higher S/CO ratio for COVID-19 patients and lower S/CO ratio with negative controls. MBA also had fewer specimens in the equivocal range (S/CO 0.9-1.1) than EIA. Conclusion: MBA is robust and simple, and is suitable for clinical microbiology laboratory for the accurate determination of anti-SARS-CoV-2 antibody for retrospective diagnosis, serosurveillance, and vaccine trials. for convalescent serum specimens of COVID-19 patients were 89.8% (35/39) for anti-NP IgG 48 and 79.5% (31/39) for anti-RBD IgG. The time-to-seropositivity was shorter with MBA than that 49 of EIA. When compared with EIA, MBA could better differentiate between COVID-19 patients 50 and negative controls with significantly higher S/CO ratio for COVID-19 patients and lower 51 S/CO ratio with negative controls. MBA also had fewer specimens in the equivocal range (S/CO 52 0.9-1.1) than EIA. 53 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 4 Conclusion: 54 MBA is robust and simple, and is suitable for clinical microbiology laboratory for the 55 accurate determination of anti-SARS-CoV-2 antibody for retrospective diagnosis, 56 serosurveillance, and vaccine trials. 57 58 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) has caused the first 60 severe coronavirus epidemic, leading to more than 8000 cases mainly in Asia 1,2 . In 2019, a 61 novel coronavirus, now known as the severe acute respiratory syndrome coronavirus 2 (SARS-62 CoV-2) has become the first coronavirus to cause a global pandemic 3 . Unlike the 2003 SARS-63 CoV, the novel SARS-CoV-2 transmits efficiently among humans, possibly due to high viral 64 load at presentation 4 and efficient binding to the human receptor angiotensin receptor 2. 65 Antibody assays play a major role in clinical management, contact tracing, vaccine 66 studies and the understanding of the epidemiology and pathogenesis of COVID-19 5,6 . Antibody 67 testing allows the retrospective diagnosis of an infection by comparing the antibody titer at the 68 acute and at the convalescent phase of the illness. This is especially important for patients whose 69 viral load is too low to be detected by virus detection assays. Furthermore, antibody testing is the 70 preferred method for identifying subclinical infections. 71 Several assays have been developed to detect antibody against SARS-CoV-2. Enzyme 72 immunoassay is a commonly used antibody assay for the detection of SARS-CoV-2 7-9 . We have 73 previously used enzyme immunoassay to determine the serial antibody profile of COVID-19 74 patients 7 and to determine the seroprevalence of SARS-CoV-2 in Hong Kong and in Hubei 75 province 10 . Lateral flow immunochromatographic assay allows rapid detection, but 76 Currently available antibody testing assays for SARS-CoV-2 mainly relies on enzyme 77 immunoassay or lateral flow immunochromatographic assaysm but the sensitivities of these 78 assays are relatively low 11 . 79 With the advance in technology, microsphere-based antibody assay (MBA) using flow 80 cytometers have been developed for different clinical applications. The use of multiplex 81 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 6 microsphere-based assays have been reported for respiratory viruses 12,13 , viruses that cause 82 childhood exanthems 14 , and arthropod-borne viruses 15, 16 . There are several advantages with 83 MBA. First, since a large number of microspheres can be coated in a single reaction, the coating 84 would be expected to be more uniformed than those of EIA, in which each well are coated 85 separately. Second, the signal from MBA is detected inside a flow cytometer, which avoids 86 potential external sources that may affect the measurement of the signal. For example, scratches 87 on microtiter plates can affect the value for EIA. Third, MBA can be easily modified into a 88 multiplex and high-throughput platform for simultaenous detection of different antigens in 89 multiple specimens 13, 17 . Finally, there are fewer steps and reagents involved for MBA than EIA 90 (Supplementary Figure S1 ). In this study, we developed and evaluated an in-house MBA for the 91 detection of IgG against SARS-CoV-2 nucleoprotein (NP) and spike protein receptor binding 92 domain (RBD). 93 To set the cut-off for the EIA and MBA, we retrieved 294 archived anonymous serum 97 specimens from the clinical biochemistry laboratory collected between April and June 2018, 98 which were used in our previous study 18 . For assessment of specificity, we retrieved 93 sera 99 collected before 2020, including 53 sera collected from potential organ donors between 2016 and 100 2018 in a study on hepatitis E in HKSAR, and from 40 influenza patients between January and 101 September 2019. 102 For COVID-19 patients, serum specimens were collected 39 recovered COVID-19 103 patients during the convalescent phase at the infectious disease out-patient follow-up clinic at 104 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 Queen Mary Hospital. To assess the time-to-seropositivity, we retrieved 161 serial serum 105 specimens obtained from 33 of these 39 patients during hospitalization. This study has been 106 approved by the HKU/HA HKW Institutional Review Board . 107 Written informed consent was obtained from all COVID-19 patients. 108 109 Cloning, purification, and biotinylation of recombinant NP and spike protein RBD of 110 Cloning and purification of SARS-CoV-2 NP and spike RBD were performed as we 112 described previously 7 . The purified NP and spike protein RBD were biotinylated with EZ-link TM 113 with 500 μ l of phosphate buffered saline (PBS) and 1% bovine serum albumin (BSA), and were 123 centrifuged at 14,000 rpm at 4 o C 15 minutes. Then, the microspheres were sonicated using 124 sonicator for 30 seconds. The microspheres were coated with biotinylated NP or spike RBD, and 125 were incubated overnight at 4 o C with shaking. The microspheres were distributed onto V-bottom 126 96 well plates and centrifugated at 1,500 rpm and 4 o C for 5 mins to remove the uncoated 127 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020. Statistical analysis was performed using PRISM 6.0. We compared categorical variables 140 using Fisher's exact test and continuous variables using Mann-Whitney U test. The S/CO ratio 141 was compared between MBA and EIA by Wilcoxon matched-pairs signed rank test. A P value of 142 less than 0.05 was judged statistically significant. 143 Establishing the MBA 146 First we detemined the optimal microsphere-protein ratio for MBA. For NP, increasing 147 the microsphere-protein ratio from 1:1 to 1:4 resulted in higher MFI values ( Figure 1A ). 148 However, since a microsphere-protein ratio of 1:2 could already result in a high MFI value, this 149 ratio was selected ( Figure 1B ). For RBD, there was no significant difference when increasing the 150 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.26.20113191 doi: medRxiv preprint 9 microsphere-protein ratio from 1:1 to 1:4 ( Figure 1C ). Therefore, we have selected a 151 microsphere-protein ratio of 1:1 for RBD ( Figure 1D) . 152 Next, we tested MBA at a ratio of 1:2 for NP and 1:1 for RBD with different negative 153 controls and a positive control (Supplementary Figure S3) . The negative controls include 154 microspheres without biotinylated NP or RBD (green), diluent only control (orange), and a 155 serum specimen collected in 2018 (blue). The positive control was a serum specimen from a 156 COVID-19 patient (red). All 3 negative controls had an MFI of <35, while the serum specimen 157 from the COVID-19 patient had an MFI of 1695 for anti-SARS-CoV-2 RBD IgG and 843 for 158 anti-SARS-CoV-2 NP IgG. 159 Next, we determined the optimal serum dilution for MBA and EIA using serially-diluted 160 serum specimens from a COVID-19 patient. With both NP and RBD, the MFI plateaued at 1:400 161 for MBA and 1:100 provided the highest OD values with EIA ( Figure 2 ) . Hence, we have used 162 1:400 dilution for MBA and 1:100 for EIA for subsequent evaluation. 163 To determine the seropositive cutoff value for MBA, microsphere-based NP and RBD 164 IgG assay was performed on 294 anonymous archived serum specimens collected in 2018. These 165 anonymous serum specimens encompass all age groups from the pediatric population to those 166 aged over 80 years (Supplementary Table S1 ). We first excluded outliers with >3SDs above the 167 mean of the 294 archived anonymous serum specimens described previously 16 (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 preprint this version posted May 26, 2020. . https://doi.org/10. 1101 To determine the specificity of the MBA, we retrieved 93 archived serum from organ 174 donors collected between 2016 and 2018 (n=53), and from patients with influenza virus infection 175 between January and September 2019 (n=40) (Figure 3) and 9 (27.3%) patients had anti-RBD detected earlier by MBA, compared to 4 (12%) by EIA. 196 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.26.20113191 doi: medRxiv preprint The time-to-seropositivity of MBA was shorter than that of EIA for both anti-NP (median time-197 to-sepositivity, 10 vs 12 days; hazard ratio for time-to-seropositivity, 1.41; 95% confidence 198 interval, 0.89-2.47, P=0.1546) and anti-RBD (median time-to-sepositivity: 13 vs 14 days; hazard 199 ratio for time-to-seropositivity: 1.05; 95% CI, 0.62-1.79, P=0.8655), though not reaching 200 statistical significance (Figure 4) . CoV-2 NP and spike protein RBD. MBA was found to be highly specific, and had a high 217 seropositive rate for patients with COVID-19. The time of seropositivity was shorter for MBA 218 than that of EIA. Furthermore, when compared with EIA, MBA had a significantly higher S/CO 219 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020. Virus neutralization assay can detect antibodies that prevent virus from infecting cells. 241 Since neutralization assays require the use of live SARS-CoV-2 virus, they can only be 242 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 13 performed in biosafety level 3 laboratory. Hence, neutralization assays cannot be performed in 243 most clinical laboratories. In contrast, the detection of IgG with recombinant virus antigens can 244 be performed safely in biosafety level 2 clinical microbiology laboratories. Previous studies have 245 shown that IgG correlates well with neutralizing antibody titer 9,24 . 246 A recent study measures anti-SARS-CoV-2 IgG level using a magnetic bead based assay 247 20 . However, since a magnetic chemiluminescence analyzer is required, this may not be feasible 248 in most clinical laboratories. In this study, our in-house MBA only requires a simple flow 249 cytometer that is available in most clinical laboratories. Our technique can be easily applied to 250 any laboratory with a standard flow cytometer. After simple gating and optimization, no further 251 adjustments are required. Our method can be easily extended to other protein antigens. 252 253 First, we only recruited adult patients. Further evaluation should be performed in 255 pediatric patients. Second, as for all serology assays, cross-reactivity may affect the results 9 . 256 Even for the pre-pandemic serum, some samples can be seropositive for SARS-CoV-2 because 257 of cross reaction with other human coronaviruses, especially from lineage B betacoronavirus. 258 Third, samples were not tested for virus neutralization and therefore neutralizing activities of the 259 detected IgG antibodies are unknown. 260 261 In this study, we have demonstrated that our novel flow-cytometry based MBA allowed 263 earlier detection of anti-SARS-CoV-2 antibody among COVID-19 patients than EIA. MBA also 264 had fewer equivocal results than EIA. A rapid and accurate diagnosis of the SARS-CoV-2 is 265 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 crucial for clinicians to provide appropriate treatment to patients, to limit further spread of the 266 virus and ultimately to eliminate another peak of pandemic risk to the public. Furthermore, our 267 assay can be used to investigate the immune response in COVID-19 patients, establishing 268 retrospective diagnosis especially for patients with immune-mediated diseases, determining 269 seroprevalence in epidemiological studies, and assessing the efficacy of novel vaccines. All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 15 KKWT, CHYF designed the study, KKYT, CHYF and LLC acquired the data. KKWT, CHYF 288 carried out the statistical analysis. All authors interpreted the data, revised the manuscript 289 critically for important intellectual content and approved the final report. The Lancet Microbe 308 2020;Accepted and in Press 309 310 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 All rights reserved. No reuse allowed without permission. (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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101 Coronavirus as a possible cause of severe acute 293 respiratory syndrome Severe acute respiratory syndrome coronavirus 295 as an agent of emerging and reemerging infection A familial cluster of pneumonia associated with the 298 2019 novel coronavirus indicating person-to-person transmission: a study of a family 299 cluster Development and Evaluation of a Multiplexed 332 Immunoassay for Simultaneous Detection of Serum IgG Antibodies to Six Human 333 Development 335 of a bead-based multiplex immunoassay for simultaneous quantitative detection of IgG 336 serum antibodies against measles, mumps, rubella, and varicella-zoster virus Multiplex microsphere immunoassays for the 339 detection of IgM and IgG to arboviral diseases Use of Bead-Based Serologic Assay to Evaluate 341 Development and 343 validation of HIV-1 Multiplex Serology Assessment of population susceptibility to upcoming 346 seasonal influenza epidemic strain using interepidemic emerging influenza virus strains Clinical Characteristics of Coronavirus Disease Antibody responses to SARS-CoV-2 in patients with 351 COVID-19. Nature medicine All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. anti-NP anti-RBD IgG 0.54. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.(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 preprint this version posted May 26, 2020. 2018 (blue). The positive control was a serum specimen from a patient with COVID-19 (red). All rights reserved. No reuse allowed without permission.(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 preprint this version posted May 26, 2020 . . https://doi.org/10.1101