key: cord-0869655-l0slp1wa authors: Rusanen, J.; Kareinen, L.; Levanov, L.; Mero, S.; Pakkanen, S. H.; Kantele, A.; Amanat, F.; Krammer, F.; Hedman, K.; Vapalahti, O.; Hepojoki, J. title: Rapid homogeneous assay for detecting antibodies against SARS-CoV-2 date: 2020-11-04 journal: nan DOI: 10.1101/2020.11.01.20224113 sha: 79c8d227558d753c62fc6e792ed414b6cf2e552a doc_id: 869655 cord_uid: l0slp1wa Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and will help to examine possible vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (S) and nucleocapsid protein (N). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Forster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal generation. We set up LFRET assays for antibodies against S and N and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described S construct and a novel N construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 S and N. We then compared the LFRET assays with these enzyme immunoassays and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90-95% vs. 90-100%) and specificity (100% vs. 94-100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91-96% vs. 82-87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-minute 'mix and read' assay, to detection of SARS-CoV-2 antibodies. labor, and deliver the results at best within hours, whereas LFAs are simple and rapid but 72 may be of substandard diagnostic performance. 73 We have previously set up rapid homogeneous (wash-free) immunoassays utilizing time- (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint 4 binding to the antibody of interest of its donor-labeled antigen and of an acceptor-labeled 87 protein L. If the patient's serum contains antibodies against the antigen, they bring the two 88 fluorophores to close proximity, generating a TR-FRET signal that indicates the presence 89 of the specific antibodies. Interestingly, a recent report describes a TR-FRET based 1-hour 90 assay for separate detection of anti-SARS-CoV-2 antibodies of diffent immunoglobulin 91 isotypes 9 . 92 SARS-CoV-2 is an enveloped (+)ssRNA virus with a non-segmented 30 kb genome and 93 four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). Protruding from the viral surface are transmembrane homotrimers of S, essential for host 95 cell entry. The glycoprotein S is proteolytically cleaved into subunits S1 and S2, of which 96 S1 contains the host cell receptor-binding domain (RBD), while S2 mediates fusion with 97 the host cell membranes 10 for SARS-CoV-2, the persistence of antibodies and the extent to which these provide 112 protective immunity remains as of yet uncertain. In this study we introduce rapid wash-free LFRET assays for detection of antibodies 114 against SARS-CoV-2 N and S antigens and compare them with ELISAs and 115 microneutralization. 116 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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint 119 LFRET assays for SARS-CoV-2 S and N were set up using Eu-labeled in-house antigens 120 and AF-labeled protein L. First, the assay conditions were optimized separately for S and Table 1 ). The development of LFRET signals over time 138 among patients with follow-up samples available is shown in Figure S1 . 140 In order to compare the performance of LFRET with classical serology, we tested the set (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 4, 2020. was 88-89%, and that between anti-S ELISA and LFRET 96-98% (Table S1 ). The samples 165 representing discordance between PCR, LFRET and/or ELISA are detailed in Table S2 . 166 The LFRET and ELISA results are compared with microneutralization titers in Figure 3 . (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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint 7 ELISA in correctly identifying microneutralization-positive and -negative samples are 173 shown in Table S3 . Receiver operating characteristic analysis 175 We also rated the performances of both LFRET assays using receiver operating 176 characteristic (ROC) curves ( Figure S4 ). The curves were plotted in RStudio (version (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 4, 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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint to 100% by two weeks from symptom onset (Table 1) , importantly for clinical use. In 212 follow-up, the LFRET signals first showed a rapid rise within three weeks from onset and 213 thereafter plateaued or slowly declined ( Figure S1 ). The agreement between ELISA and LFRET was high: ~90% for anti-N -antibodies and 215 >95% for anti-S -antibodies (Table S1 ). With the anti-N and anti-S LFRET vs. IgG ELISA 216 results combined, the overall agreement between the methods in the entire study material 217 was 94% (124/129 samples). A closer look at the discordance (Table S2) as negatives (91% for S and 96% for N) were higher than those of IgG ELISAs (87% for 237 S and 82% for N) (Table S3 ). This may be due to lower analytical sensitivity of LFRET, (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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint identification of samples containing neutralizing antibodies, with a sensitivity of 98% and 242 a specificity of 91%. (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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint ampicillin and 50 µg/ml Zeocin (ThermoFisher Scientific). After overnight incubation at Protein expression and purification 299 We initially attempted producing SARS-CoV-2 S protein in Expi293F cells utilizing the (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 4, 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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint We labeled the SARS-CoV-2 S and N proteins with the donor fluorophore europium (Eu) 347 using QuickAllAssay Eu-chelated protein labeling kit (BN Products and Services Oy) 348 according to the manufacturer's instructions to generate Eu-labeled S (Eu-S) and N (Eu-349 N). We also labeled recombinant protein L (Thermo Scientific) with the acceptor 350 fluorophore Alexa Fluor 647 to generate AF647-labeled protein L (AF-L), as reported 5 . IgG-free bovine serum albumin (BSA) was from Jackson ImmunoResearch Inc. 353 The LFRET assay was done as described 5 and as briefly illustrated by the flowchart in Enzyme linked immunosorbent assays (ELISAs) 365 We set up the SARS-CoV-2 S protein ELISA as described 25 with the following 366 amendments. We coated the plates (ThermoScientific NUNC-immuno 446442 polysorp 367 lockwell C8) with 50 ul/well of antigens diluted 1 µg/ml into 50 mM carbonate-bicarbonate 368 buffer pH 9.6 (Medicago AB) and used 1-Step Ultra TMB-ELISA Substrate Solution 369 (ThermoFisher Scientific). As secondary antibodies we made use of polyclonal rabbit anti- (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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint The authors wish to acknowledge Drs. Tomas Strandin and Eliisa Kekäläinen for helping (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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint Coronavirus disease (COVID-19) Weekly Epidemiological Update Testing for SARS-CoV-2 antibodies Time-resolved FRET -based approach for antibody detection -408 a new serodiagnostic concept A protein L-based immunodiagnostic approach utilizing time-410 resolved Förster resonance energy transfer Rapid 412 homogeneous immunoassay based on time-resolved Förster resonance energy 413 transfer for serodiagnosis of acute hantavirus infection Competitive Homogeneous Immunoassay for Rapid 416 Serodiagnosis of Hantavirus Disease Immunoassay for serodiagnosis of Zika virus infection based on 418 time-resolved Förster resonance energy transfer LFRET, a novel rapid assay for anti-tissue transglutaminase 420 antibody detection Rapid 'mix and read' assay for scalable detection of SARS-CoV-2 Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Antibody responses to SARS-CoV-2 in patients of novel coronavirus 427 disease 2019 Antibody responses to SARS-CoV-2 in patients with COVID-19 Humoral 431 Immune Response to SARS-CoV-2 Evaluating the Association of Clinical Characteristics With 433 Neutralizing Antibody Levels in Patients Who Have Recovered From Mild 434 COVID-19 in Severe Acute Respiratory Syndrome Coronavirus 2-Specific 436 Antibody Responses in Coronavirus Disease Patients Safety and immunogenicity of the ChAdOx1 nCoV-19 439 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, 440 randomised controlled trial Potent neutralizing antibodies from COVID-19 patients 442 define multiple targets of vulnerability Middle East Respiratory Syndrome Coronavirus Infection 444 Dynamics and Antibody Responses among Clinically Diverse Patients 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 IgG and IgA ELISAs using coronavirus disease (COVID-19) patient samples CoV-2 infections Detection of 2019 novel coronavirus (2019-nCoV) by real-457 time RT-PCR A serological assay to detect SARS-CoV-2 seroconversion in 459 humans SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol 461 for a Serological Assay, Antigen Production, and Test Setup Snake Deltavirus Utilizes Envelope Proteins of Different 464 Viruses To Generate Infectious Particles. mBio 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 4, 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 4, 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 4, 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 4, 2020. ; https://doi.org/10.1101/2020.11.01.20224113 doi: medRxiv preprint