key: cord-289247-qc3to2xj
authors: Yamaoka, Yutaro; Jeremiah, Sundararaj S; Miyakawa, Kei; Saji, Ryo; Nishii, Mototsugu; Takeuchi, Ichiro; Ryo, Akihide
title: Whole nucleocapsid protein of SARS-CoV-2 may cause false positive results in serological assays
date: 2020-05-23
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa637
sha: 
doc_id: 289247
cord_uid: qc3to2xj

nan

M a n u s c r i p t 2 TO THE EDITOR -

The nucleocapsid protein (NP) of SARS-CoV-2 is one of the widely used antigens in serodiagnostics of the novel Coronavirus disease . We appreciate Guo and colleagues for shedding new light on the humoral response profile of COVID-19 [1] . They have generated recombinant whole nucleocapsid protein (rNP) of SARS-CoV-2 using Escherichia coli expression system and used it to develop an enzyme linked immunosorbent assay (ELISA) to detect anti-SARS-CoV-2 antibodies in plasma. The rNP ELISA was negative in the plasma of all the 285 non COVID-19 individuals tested.

The authors have also shown that rNP does not have cross reactivity with antibodies of other common Coronaviruses; NL63, 229E, OC43, and HKU1. However, we have observed controversial findings and suggest the advantage of N-terminally truncated nucleocapsid protein (ΔN-NP).

Although the amino acid sequences of the entire nucleocapsid proteins (NP) of SARS-CoV and other common Coronaviruses are dissimilar to that of SARS-CoV-2 [1] , the conserved residues at the Nterminal domain of NP show a high degree of similarity (Figure-1A) . This was pointed out by Yu et al in SARS-CoV, who observed cross reactivity of whole NP with other Coronaviruses and encountered high rates of false positivity while testing healthy donor sera [2] . They had overcome this problem by using ΔN-NP which was devoid of the homogenous conserved residues at the N-terminal region [2, 3] .

Since SARS-CoV-2 NP has over 90% homology to SARS-CoV NP [1] and their conserved residues are almost identical (Figure-1A) , we expected the possibility of cross reactions with whole/full length NP (FL-NP). We expressed both FL-NP and ΔN-NP of SARS-CoV-2 in wheat germ cell free protein production system [4] and designed an IgG detection ELISA to compare their performance. Upon after the third week [5] . In the time-course analysis, the ΔN-NP ELISA detected the lower level of antibodies that began to increase from second week onwards while FL-NP ELISA at the same cut-off would have falsely reported these as negative (Figure-1C) .

Detection of anti-viral antibodies can be used both for diagnosis and population surveillance, ideally with the former possessing high sensitivity and the latter high specificity. We aimed to enhance the sensitivity to detect the low level of IgG in patients during relatively earlier stages of the disease.

With higher sensitivity, FL-NP based ELISA gives a higher background but this occurs to a much lesser extent with ΔN-NP. We presume this is probably due to the cross reactivity of FL-NP with antibodies to other human coronaviruses and this should be studied further. We conclude that ΔN-NP is better suited than FL-NP to develop high sensitivity diagnostic assays for COVID-19. A c c e p t e d M a n u s c r i p t

Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)

Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay

Recombinant Truncated Nucleocapsid Protein as Antigen in a

Novel Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Severe Acute Respiratory Syndrome Coronavirus Infection

Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen

Antibody Detection and Dynamic Characteristics in Patients with COVID-19

We thank Mayuko Nishi and Chizu Suzuki for their technical assistance. This study was approved by the Institutional Review Board of Yokohama City University (IRB No. B200200048, B160800009), and the protocols used in the study were approved by the ethics committee. Written informed consent was obtained from the patient (or family/guardian)

Research and Development (AMED) to AR (19fk0108110h0401).

The authors have no conflicts of interest directly relevant to the content of this article. Y.Y. is a current employee of Kanto Chemical Co., Inc.

A c c e p t e d M a n u s c r i p t