key: cord-0910654-j3pr23xt authors: Kubo, S.; Ohtake, N.; Miyakawa, K.; Jeremiah, S. S.; Yamaoka, Y.; Murohashi, K.; Hagiwara, E.; Mihara, T.; Goto, A.; Yamazaki, E.; Ogura, T.; Kaneko, T.; Yamanaka, T.; Ryo, A. title: Development of an automated chemiluminescence assay system for quantitative measurement of multiple anti-SARS-CoV-2 antibodies date: 2020-11-06 journal: nan DOI: 10.1101/2020.11.04.20225805 sha: 0d57e8d7de2cd85c6fb0104af5a83cfcf3dd6aee doc_id: 910654 cord_uid: j3pr23xt Objective: Serological tests for COVID-19 have been instrumental in studying the epidemiology of the disease. However, the performance of the currently available tests is plagued by the problem of variability. We have developed a high-throughput serological test capable of simultaneously detecting total immunoglobulins (Ig) and immunoglobulin G (IgG) against two of the most immunologically relevant SARS-CoV-2 antigens, nucleocapsid protein (NP) and spike protein (SP) and report its performance in detecting COVID-19 in clinical samples. Methods: We designed and prepared reagents for measuring NP-IgG, NP-Total Ig, SP-IgG, and SP-Total Ig (using N-terminally truncated NP ({Delta}N-NP) or receptor-binding domain (RBD) antigen) on the advanced chemiluminescence enzyme immunoassay system TOSOH AIA-CL. After determining the basal thresholds based on 17 sera obtained from confirmed COVID-19 patients and 600 negative sera. Subsequently, the clinical validity of the assay was evaluated using independent 202 positive samples and 1,000 negative samples from healthy donors. Results: All of the four test parameters showed 100% specificity individually (1,000/1,000; 95%CI, 99.63-100). The sensitivity of the assay increased proportionally to the elapsed time from symptoms onset, and all the tests achieved 100% sensitivity (153/153; 95%CI, 97.63-100) after 13 days from symptoms onset. NP-Total Ig was the earliest to attain maximal sensitivity among the other antibodies tested. Conclusion: Our newly developed serological testing exhibited 100% sensitivity and specificity after 13 days from symptoms onset. Hence, it could be used as a reliable method for accurate detection of COVID-19 patients and to evaluate seroprevalence and possibly for surrogate assessment of herd immunity. Moreover, serological assays enable to estimate the prevalence of existing immunity to SARS-CoV-2 in a population (2) . In general, antibody testing not only helps to indirectly diagnose an infection but also reflects the individual immune response (3) . This is fundamentally different from reverse transcription polymerase chain reaction (RT-PCR) or antigen detection tests, which measure viral nucleic acids and antigens, respectively. Unlike other infections, COVID-19 exhibits concurrent elevation of immunoglobulin (Ig) G and IgM in the second week after disease onset, with IgM reaching peak titers in the second week and IgG in the third week of the disease (4, 5) . Since the transition of antibody titers over time 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint is different in different types of immunoglobulin, it is recognized that the measurement of multiple antibody titers can holistically assess the immune status in infected individuals. Various antigens of SARS-CoV-2 can be utilized by serodiagnostic kits for the detection of virus-specific antibodies. These include viral surface antigens such as membrane proteins, envelope proteins, and spike protein (SP) and non-surface antigens such as the nucleocapsid protein (NP) (6, 7). Of these, the SP and the NP are the most widely employed for serological diagnosis (8) . The SP mediates viral entry into host cells through the receptor-binding domain (RBD) and antibodies against RBD are predicted to represent the protective immunity to SARS-CoV-2 infection (9, 10). The NP not only participates in ribonucleic acid (RNA) packaging and virus particle release but also plays a principal role in orchestrating various intracellular events required for viral replication after entry (11) . The NP shares antigenic similarity with other human coronaviruses and may exhibit cross-reactivity when used in serological tests as the full-length protein, but is highly specific for SARS-CoV-2 in the absence of its N-terminal amino acid residues (12) . High-throughput COVID-19 serology testing is required for epidemiological 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint surveillance. The most common method for measuring antibody titers is enzyme-linked immunosorbent assay (ELISA). Although ELISA has many advantages, it is a time-consuming and labor-intensive procedure with a higher likelihood of technical errors than other automated platforms (13) . To overcome these problems, we used the automated chemiluminescent enzyme immunoassay system AIA-CL (TOSOH, Tokyo, Japan), to develop a serodiagnostic test that quantitatively detects SARS-CoV-2 antibody titers with a short turn-around-time of 10 minutes. Apart from lower manual errors, this platform also has the inherent advantage of being more precise and sensitive than ELISA (14) . We evaluated the performance of the AIA-CL, and analyzed chronological change of antibody titers in patients. Previously, we showed that the cross-reactivity of NP is due to conserved residues in its N-terminus; accordingly, truncated N-terminal NP (ΔN-NP) is highly specific for detection of SARS-CoV-2. Δ N-NP (121-419 aa) was synthesized using the wheat germ 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint cell-free protein synthesis system and purified using Ni-Sepharose beads (15) . The complementary deoxyribonucleic acid (cDNA) sequence corresponding to the RBD (residues 319-541) of SARS-CoV-2 spike protein was chemically synthesized and inserted into in-house Strep-tagged mammalian expression vector. Strep-tagged RBD protein was transient expressed in a mammalian cell (Expi293, Thermo Fisher Scientific), purified by Strep-tag purification system (IBA) (16) . We developed four types of serological test reagents (TOSOH NP-IgG, NP-Total Ig, SP-IgG, SP-Total Ig test) to detect anti-SARS-CoV-2 antibodies on the AIA-CL. The serological test comprises of an automated immunoassay for the quantitative determination of either serum IgG or Total Ig (IgG, IgM, IgA, IgE, etc.) levels against the Δ N-NP or RBD antigen. Micro-magnetic beads coated with Δ N-NP or RBD antigen were added to the first reaction layer, and alkaline phosphatase-conjugated anti-human IgG or Δ N-NP antigen or RBD antigen was added to the second reaction layer and prepared by an immediate lyophilization in a test cup. The AIA-CL system performs an automated 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint process that includes specimen dispensing, incubation of the reaction cup, bound/free washing procedure, dispensing of in-house chemiluminescent substrates (DIFURAT), chemiluminescence detection, and reporting of results (up to 240 tests/hour/device). (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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint in Yokohama City University Hospital (YCUH) and Kanagawa Cardiovascular and Respiratory Center (KCRC) were used. The severity of symptoms on admission was determined according to National Institutes of Health (NIH) classification (17) . If the patient was in the recovery phase (more than 30 days after symptoms onset and the symptoms were improving on admission), categorized as "others". To ensure the quality of the clinical specimens, one patient with a history of anticancer therapy was excluded. Mann-Whitney Rank Sum test was performed to identify the correlations of health donors and COVID-19 patients. In the initial evaluation, we set the cutoff values of NP-IgG, NP-Total Ig, SP-IgG and SP-Total Ig titers by maximizing the overall prediction performance with the Youden's J index (J = sensitivity + specificity − 1) based on the receiver operating characteristic (ROC) analysis, and defined them as 1.0 index. Next, the sensitivity, specificity and 95% confidence interval (95% CI) of the clinical samples was calculated using ROC curve. A p-value of <0.05 was considered to be indicative of 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint statistical significance. All statistical analyses were performed by using GraphPad Prism 6 software. All healthy donors provided written informed consent for use of their clinical and biological data for the purpose of the scientific research. All patients were recruited using the opt-out approaches. The study was performed according to protocols approved by our institutional review board. Reagents for the NP-IgG, NP-Total Ig, SP-IgG and SP-Total Ig tests were prepared using ∆ N-NP and RBD antigen respectively. Schematic illustration of the assay principles used in the TOSOH AIA-CL system is depicted in Figure 1 . After confirming within-run reproducibility, we measured the intensity of the chemiluminescent signals of 600 healthy donor samples and 17 COVID-19 antibody-positive samples ( Figure S1 A-D) . Using the ROC curve, we determined the cutoff values at 3,889 counts of intensity for NP-IgG, 1,705 counts for NP-Total Ig, 6,700 counts for SP-IgG, and 1,000 counts for SP-Total Ig, 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint and defined them as 1.0 index ( Figure S1 E-H) . Next, we evaluated a total of 202 samples collected after 7 days from symptoms onset from 43 symptomatic patients with COVID-19 diagnosed by RT-PCR, using the NP-IgG, NP-Total Ig, SP-IgG, SP-Total Ig reagents. The median age of COVID-19 patients was 61.3 years in YCUH and 64.5 years in KCRC. 2 patients were mild cases, 17 were moderate cases, 16 were severe cases, 7 were critical cases, and 2 were categorized as "others" on admission (Table S1) . We compared antibody titers in the following cohorts: 7-9 days, 10-12 days, 13-20 days, 21-30 days, and >31 days (up to 96 days) after onset of the disease. Clinical sensitivity of NP-IgG was 59.3% at 7-9 days, that of NP-Total Ig was 74.5%, that of SP-IgG was 40.7% and that of SP-Total Ig was 37.0%, which indicated that NP-Total Ig was higher sensitivity than the other antibodies in early phase of COVID-19. (Figure 2A ). All the four antibody titers increased around 13 days after symptoms onset respectively ( Figure 2B-E) . Furthermore, chronological observation of antibody titers of serial samples 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint collected from nine representative patients are shown in Figure 3 . NP-IgG titer was slightly decreased at more than 30 days after symptoms onset ( Figure 3A ), but NP-Total Ig, SP-IgG and SP-Total Ig titers were increased continuously for up to 21-30 days after symptoms onset and tended to be sustained ( Figure 3B-D) . There was no obvious difference of the transition of each antibody titer among moderate, severe and critical cases ( Figure 3A-D) . We compared antibody titers of samples from 1,000 healthy donors and 153 samples from COVID-19 patients collected 13 days after symptoms onset and analyzed using the NP-IgG, NP-Total Ig, SP-IgG and SP-Total Ig reagents ( Figure 4A-D) . Subsequently, we determined the sensitivity and specificity of our antibody assays by ROC analysis. Each of the four antibodies precisely identified all the true negatives and true positives thereby achieving 100% sensitivity and specificity ( Figure 4E-H) . Given its excellent performance, our newly developed assay may be suitable for large-scale antibody detection. 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint We next wanted to compare the performance our test against the commercially successful chemiluminescent Immunoassay platforms Roche-Total Ig and Abbott IgG. As these platforms utilize only the NP, we examined the correlations between the data acquired using the NP-IgG and NP-Total Ig tests from this study and the data acquired using the above mentioned commercial kits. For this purpose, we used 44 COVID-19 antibody-positive samples. Results from NP-IgG were strongly correlated with the results from Roche-Total Ig (Pearson's correlation coefficient, r=0.97) and Abbott IgG (r=0.962), as were the results from NP-Total Ig (r=0.989 and 0.875, respectively) (Table) . Because our antibody tests can measure both NP-IgG and NP-Total Ig with high reliability and additionally SP-IgG and SP-Total Ig, they have a practical scope for COVID-19 serological analysis. COVID-19 being a global public health problem, surveillance to estimate infection rates 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint based on percentage of antibody positivity and chronological analysis of antibody titers are actively being conducted (18). This is done either as a qualitative assessment using a rapid diagnostic kit or as a quantitative evaluation by antibody titer measurement using ELISA. In particular, the large-scale evaluation of seroprevalence and herd immunity against SARS-CoV-2 requires a high-throughput and quantitative screening as it is more rapid and accurate measurement of viral antibody levels. In this study, we established a combination of NP-IgG, NP-Total Ig, SP-IgG and SP-Total Ig tests for use with the TOSOH AIA-CL system. The sensitivity and specificity of each of the four antibody assay individually was 100% in COVID-19 patient samples collected 13 days after symptoms onset. Moreover, the NP-IgG and NP-Total Ig assays correlated strongly with Abbott and Roche assays for COVID-19 serodiagnosis (19, 20, 21) . Considering its excellent performance, this assay could play important roles in generating authentic data during public surveillance of COVID-19. All the four types of antibody titers began to rise 7-9 days after symptoms onset and were positive in 100% of patients after 13 days, which is consistent with previous reports (4, 22, 23) . In addition, antibodies against NP had higher sensitivity than 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint antibodies against SP in the early phase of COVID-19 infection and NP-Total Ig showed the highest sensitivity among the four antibodies. It has been reported that the positivity of NP antibodies is higher than SP antibodies in acute phase of SARS-CoV-2 infection (24, 25). As in common viral infections, COVID-19 infection also has polyclonal elevation of Immunoglobulin such as IgM and IgA, not only IgG. Although many antibody kits can measure only IgG or/and IgM, it has been reported that measuring IgA in addition to IgG or IgM increases the sensitivity of the assay (8) . Since our assay measures total-Ig in addition to IgG, it can possibly be more precise than assays that only detect a particular Ig type. Antibodies against spike antigen, especially RBD, have the ability to prevent the entry of the virus into the host cells (26, 27). According to previous reports, while NP-IgG declined slowly over the course of approximately half a year and turned negative in some cases, SP-IgG declined slowly but sustained its antibody titer and rarely turned negative (28). In this study, all the four antibodies were positive in all cases after 13 days from symptoms onset, and NP-Total Ig, SP-IgG, and SP-Total Ig have been sustained during the period of observation. However, NP-IgG titers tended to decrease after 30 days 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 November 6, 2020. It should be noted that there were some limitations of this study. First, most cases in this study were of high severity. It is known that COVID-19 antibody titers tend to be lower in less severe cases (29, 30). Although there was no obvious difference in the antibody titers between moderate, severe and critical cases in this study, it was insufficient to evaluate the different in antibody titers due to the severity of COVID-19 because the number of samples in moderate cases were smaller than that of severe or critical cases. Therefore, there is the possibility that the cutoff value set in this assay may not be appropriate for detecting the antibody titers in lower severity cases, such as mild or asymptomatic patients. Second, the newly developed reagents can only measure IgG and total Ig, but not other immunoglobulin levels, such as IgM, IgA. Also, this study showed that NP-Total Ig was sustained but NP-IgG decreased after 30 days since symptoms onset. The reason for this reduction in IgG titers is unclear and the other immunoglobulin which 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint elevated to sustain the total Ig levels also remains to be identified with further investigations. In conclusion, the automated TOSOH AIA-CL assay that can simultaneously quantify anti-SARS-CoV-2 NP-IgG, NP-Total Ig, SP-IgG and SP-Total Ig which was developed in this study, performed to be a highly accurate and specific serodiagnostic test This work was in part supported by Rapid Research and Development Projects 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 November 6, 2020. 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint A Review of Coronavirus Disease-2019 (COVID-19) COVID-19 herd immunity: where are we? Protective Adaptive Immunity Against Severe Acute Respiratory Syndrome Coronaviruses 2 (SARS-CoV-2) and Implications for Vaccines Antibody responses to SARS-CoV-2 in patients with COVID-19 specific IgM and IgG responses in COVID-19 patients. Emerg Microbes Infect 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 The Molecular Biology of Coronaviruses The global spread of 2019-nCoV: a molecular evolutionary analysis Serum IgA, IgM, and IgG responses in COVID-19 Convergent antibody responses to SARS-CoV-2 in convalescent individuals Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients The coronavirus nucleocapsid is a multifunctional protein Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) Development of Fully Automated Low-Cost Immunoassay System for Research Applications Chemiluminescent immunoassay technology: what does it change in autoantibody detection? Whole nucleocapsid protein of SARS-CoV-2 may cause false positive results in serological assays The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins 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 Humoral Immune Response to SARS-CoV-2 in Iceland Evaluation of Roche Elecsys Anti-SARS-CoV-2 serology assay for the detection of anti-SARS-CoV-2 antibodies. Public Health England Performance Characteristics of the Abbott Architect SARS-CoV-2 IgG Assay and Seroprevalence in Detection of IgM and IgG antibodies in patients with coronavirus disease 2019 Sensitivity in Detection of Antibodies to Nucleocapsid and Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Coronavirus Disease 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint 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 November 6, 2020. ; https://doi.org/10.1101/2020. 11 Communities and Reveal Durable Humoral Immunity. Immunity. 2020.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 November 6, 2020. (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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint antigen (right panel). Alkaline phosphatase, used as the labeling enzyme, was detected using in-house chemiluminescent substrates (DIFURAT).NP: nucleocapsid protein, SP: spike protein, AIA-CL: automated chemiluminescent enzyme immunoassay system, RBD: receptor-binding domain. (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 November 6, 2020. Center.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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv 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 preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv 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 preprint this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint 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 November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint 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 pr this version posted November 6, 2020. ; https://doi.org/10.1101/2020.11.04.20225805 doi: medRxiv preprint