key: cord-0826915-d0hu9neb
authors: Conzentino, M. d. S.; Forchhammer, K.; Souza, E. M.; Pedrosa, F. d. O.; Nogueira, M. B.; Raboni, S. M.; Rego, F. G. M.; Zanette, D. L.; Aoki, M. N.; Nardin, J. M.; Fornazari, B.; Morales, H. M. P.; Celedon, P. A. F.; Lima, C. V. P.; Mattar, S. B.; Lin, V. H.; Morello, L. G.; Marchini, F. K.; Bueno, L. B.; Reis, R. A.; Huergo, L.
title: Antigen production and development of an indirect ELISA based on the nucleocapsid protein to detect human SARS-CoV-2 seroconversion
date: 2021-02-23
journal: nan
DOI: 10.1101/2021.02.19.21252100
sha: 8cfe8ffe6d9ad49c7939b52fb1f9b93f8ea07433
doc_id: 826915
cord_uid: d0hu9neb

Serological assays are important tools to identify previous exposure to SARS-CoV-2, helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 infections and/or immunization to future vaccines. Here the SARS-CoV-2 nucleocapsid protein was expressed in Escherichia coli and purified to homogeneity and high yield using a single chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 PCR-confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with a commercial ELISA kit. Antigen coated plates were stable for up to 3 months at 4oC). The ELISA method described is ready to mass production and will be an additional toll to track COVID-19 cases.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.19.21252100 doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

Abstract 27 Serological assays are important tools to identify previous exposure to SARS-CoV-2, 28 helping to track COVID-19 cases and determine the level of humoral response to SARS-CoV-2 29 infections and/or immunization to future vaccines. Here the SARS-CoV-2 nucleocapsid protein was 30 expressed in Escherichia coli and purified to homogeneity and high yield using a single 31 chromatography step. The purified SARS-CoV-2 nucleocapsid protein was used to develop an 32 indirect enzyme-linked immunosorbent assay for the identification of human SARS-CoV-2 33 seroconverts. The assay sensitivity and specificity were determined analyzing sera from 140 PCR-34 confirmed COVID-19 cases and 210 pre-pandemic controls. The assay operated with 90% 35 sensitivity and 98% specificity; identical accuracies were obtained in head-to-head comparison with 36 a commercial ELISA kit. Antigen coated plates were stable for up to 3 months at 4 o C). The ELISA 37 method described is ready to mass production and will be an additional toll to track COVID-19 38 cases. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint COVID-19 cases (Petherick, 2020). The structural Nucleocapsid (N) protein is known to be a major 59 antigen of coronavirus producing a strong humoral response in humans (Cheng et al., 2007) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The purified N protein was diluted in buffer and used to coat 96 well polystyrene plates 75 overnight at 4 0 C. Plates were blocked with skimmed milk and serum from a COVID-19 positive 76 case and a negative control serum were serial diluted and placed into the wells of pre-coated plates.

The presence of IgG reacting with the SARS-CoV-2 N protein was revealed by incubating with 78 secondary anti-human IgG -HPR followed by chromogenic substrate TMB. The COVID-19 79 positive serum showed strong reaction with the N protein even at high dilutions (Fig. 1B) . On the 80 other hand, the negative control serum showed only minor background cross reaction in low 81 dilutions ( Fig. 2A) . The OD450nm response vs serum dilution of the positive serum was linear 82 between the 1,600 to 25,600 dilutions with R 2 = 0.995 (Fig. 1C ). This positive serum was used as a 83 positive control throughout this study, the inter assay CV% was 7.2% (same sample run in 12 84 different plates in duplicate). The mean intra assay CV% was 7.6% (mean CV% was obtained from 85 duplicate 96 samples x 12 plates). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Trap column (Cytiva), which had been previously equilibrated with buffer 1. The column was is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 23, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 23, 2021. To validate our ELISA assay to detect SARS-CoV-2 seroconversion in human a cohort 157 consisting of 140 PCR-confirmed COVID-19 cases and 210 pre-pandemic negative controls were 158 used. The pre-pandemic control sera produced only background signal which were clearly 159 distinguished from the PCR-confirmed COVID-19 cases (Fig. 2A) . The age of the negative cohort is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.19.21252100 doi: medRxiv preprint 8 ranged from 21 to 86 years of age (mean = 62). It is likely that most of these subjects had 161 experienced prior infections with common coronavirus (229E, NL63, OC43, and HKU which 162 usually cause mild upper-respiratory tract disease, like the common cold). These data suggest that 163 our ELISA assay shows negligible cross reactivity for IgG raised against common coronavirus.

However, it is worth mentioning that we detected 4 reactive pre-pandemic samples whose signals 165 were above the established cutoff thereby being classified as false positives ( Fig. 2A) . Such Indeed, our analysis confirmed that the IgG titer was significantly lower in mild versus hospitalized 178 COVID-19 patients (Fig. 2C) . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 23, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.19.21252100 doi: medRxiv preprint

A Peptide-Based Magnetic 218

Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Coronavirus Disease

Severe acute respiratory 221 syndrome coronavirus as an agent of emerging and reemerging infection

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative 224 Detection of Human SARS-CoV-2 Antibodies

SARS-CoV-2-Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach

Accounting for Accurate Seroprevalence. J Infect Dis . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted February 23, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted February 23, 2021. ; https://doi.org/10.1101/2021.02.19.21252100 doi: medRxiv preprint