key: cord-0900306-qbf3wg7m
authors: Oshiro, Satoshi; Tabe, Yoko; Funatogawa, Keiji; Saito, Kaori; Tada, Tatsuya; Hishinuma, Tomomi; Mizutani, Naeko; Akiwa, Makoto; Sekiguchi, Jun-ichiro; Miida, Takashi; Kirikae, Teruo
title: Development of an immunochromatographic kit to detect severe acute respiratory syndrome coronavirus 2
date: 2021-05-11
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114183
sha: 512a66564f2b71e1122a5ac2779252b3177573aa
doc_id: 900306
cord_uid: qbf3wg7m

BACKGROUND: The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the worldwide coronavirus disease-19 (COVID-19) pandemic, starting in late 2019. The standard diagnostic methods to detect SARS-CoV-2 are PCR-based genetic assays. Antigen-antibody-based immunochromatographic assays are alternative methods of detecting this virus. Rapid diagnosis kits to detect SARS-CoV-2 are urgently needed. STUDY DESIGN: Three monoclonal antibodies against SARS-CoV-2 nucleocapsid (N) protein were used to develop an antigen-antibody-based immunochromatographic kit to detect SARS-CoV-2. These assays were evaluated using nasopharyngeal swab specimens collected from patients suspected of having COVID-19. RESULTS: These assays detected recombinant SARS-CoV-2 N protein at concentrations >0.2 ng/ml within 10 minutes after protein loading, but did not detect the N proteins of Middle East respiratory syndrome coronavirus (MERS-CoV), human coronaviruses OC43 (HCoV-OC43) and 299E (HCoV-229E) and other pathogens causing respiratory infections. Nasopharyngeal swab specimens obtained 1～3, 4～9, and ≥ 10 days after symptom onset from COVID-19 patients diagnosed by RT-PCR showed positivity rates of 100%, >80%, and <30%, respectively. CONCLUSIONS: Kits using this immunochromatographic assay may be a rapid and useful tool for point-of-care diagnosis of COVID-19 when samples are obtained from patients 1～9 days after symptom onset.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the worldwide coronavirus disease suspected human cases). Other genetic methods have also been described, including nested RT-PCR (Shirato et al., 2020) , single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) (Baek et al., 2020) and modified rRT-PCR methods (Loeffelholz et al., 2020) Antigen-antibody-based immunochromatographic assays are alternative methods to detect SARS-CoV-2. Immunochromatographic assays have become point-of-care diagnostic methods to detect seasonal influenza (Sturenburg et al., 2009) , as well as outbreaks of emerging infectious diseases, including severe acute respiratory syndrome (Kogaki et al., 2005) , Middle East respiratory syndrome (MERS; Song et al., 2015) and pandemic H1N1 (2009) influenza virus (Miyoshi-Akiyama et al., 2009; Kawachi et al., 2009) . Rapid diagnosis kits to detect SARS-CoV-2, perhaps using immunochromatographic assays, are urgently needed.

This study describes the development of a novel immunochromatographic assay using three monoclonal antibodies against recombinant SARS-CoV-2 nucleocapsid (N) protein to detect SARS-CoV-2.

Nasopharyngeal swab samples were collected from patients suspected of having COVID-19 who had symptoms such as fever, dry cough, fatigue, loss of taste/smell, nasal congestion, conjunctivitis, sore throat, headache, muscle/joint pain, skin rash, nausea/vomiting, diarrhea and/or chills/dizziness. Nasopharyngeal swab samples were collected during 18 March 2021 to 20 August 2021 at a university hospital in Tokyo. A total number of samples were 98. All samples were subjected to RT-PCR for the detection of SARS-CoV-2 according to a protocol published by the National Institute of Infectious Disease, Japan (Manual for the detection of pathogen 2019-nCoV Ver2.6). Of them, 45 were RT-PCR positive. Of the 45 samples, 11 were collected 1-3 days after symptom onset, 16 were 4-6 days after symptom onset, 8 were 7-9 days after symptom onset and 7 were 10 days after symptom onset, respectively. Regarding the remaining 3 samples, the collection days after symptom onset were not available. In addition, nasopharyngeal swab samples were collected from 21 healthy medical staffs without the above symptoms during 28-30 January 2021 at a hospital in Iwate prefecture, Japan. HCoV-229E (accession no. NC_002645), respectively. The genes encoding the recombinant N proteins of these coronaviruses were cloned into an expression vector using the primers listed in Table S1 ; following their expression in E. coli, the proteins were purified as described above. The gene encoding the recombinant N protein derived from the SARS-CoV-2 501Y variant 2 (also called B.1.1.7, 20B/501Y.V1 and VOC-202012/01) was amplified using a pair of primers listed in Table S1 ; relative to the original N protein, the recombinant N protein had two amino acid substitutions, D3L and S235F (Leung et al., 2021) .

Wistar rats were immunized with full-length recombinant SARS-CoV-2 N protein.

Hybridomas were screened with an enzyme-linked immunosorbent assay (ELISA) using full-length recombinant SARS-CoV-2 N protein. Briefly, 100 µl aliquots of recombinant SARS-CoV-2 N protein in 0.05M carbonate buffer (pH 9.0) were placed in the wells of 96-well plates. The plates were incubated for 1 hour at room temperature, washed three times with phosphate-buffered saline (PBS)-0.1% Tween 20, and incubated with SuperBlock TM Blocking Buffer in PBS (Thermo Scientific., Swedesboro USA) (150 µl/well) for 30 min at room temperature. To each well was added 100 µl of the culture supernatant of a hybridoma, followed by incubation for 1 hour at room temperature. A 100 µl aliquot of peroxidase-conjugated anti-rat IgG (1:10,000 dilution) (SouthernBiotech; Birmingham, AL, USA) was added to each well, and the plates were incubated for 30 min at room temperature. A 50 µl aliquot of TMB Peroxidase ELA Substrate Kit (Bio-Rad, Hercules, CA, USA) was added to each well, followed by incubation at room temperature for 10 min and the addition of 50 µl 1 M sulfuric acid to stop the peroxidase reaction. The absorbance of each well at 450 nm, and at 620 nm as reference, was measured using an Infinite F50 microplate reader (Tecan, Mannedorf, Switzerland).

Monoclonal antibodies (mAbs) were purified using Spin Column Based Antibody Purification kits (Cosmo Bio USA; Carlsbad, CA, USA). To determine the binding regions of the mAbs, direct ELISA was performed using full-length (aa 1-419) and three partial fragments (aa 1-120, 111-220 and 210-419) of SARS-CoV-2 N protein.

Sandwich ELISAs were performed using these mAbs as first or capture antibodies and horseradish peroxidase-conjugated mAbs as second or detection antibodies. The mAbs had been labeled with peroxidase using Ab-10 Rapid Peroxidase Labeling Kit (Cosmo Immunochromatographic assays (ICAs) were developed by coating nitrocellulose membranes with capture antibodies (N1J7 and N1K1) and control goat anti-rodent IgG antibody. Detection antibody (N1K2)-conjugated colloidal golds were immersed into glass fiber. Sample pad, absorbent pad, nitrocellulose membrane with the capture antibodies, and the grass fiber containing N1K2-conjugated colloidal gold were laminated and pasted onto the polystyrene self-adhesive baseplate. The assembled on the baseplate was divided into strips. Nasopharyngeal swab specimens, recombinant proteins, and various pathogens were applied to assess the strip in the ICA. The strip was vertically immersed into sample in test tube, ensuring the sample pad of strip was fully reacted with the sample. Line formation on the strip was assessed within 10 minutes after immersing it. Table S2 , and ≥10 days, respectively after symptom onset; of these, 11 (100%), 13 (81.3%), seven (87.5%), and two (28.6%) samples, respectively, were ICA-positive (Table 1) .

The threshold cycle (Ct) values of RT-PCR correlated inversely with ICA-positive rates, with 100% (3/3), 96.4% (29/30), 88.5% (23/26), and 60.0% (18/30) samples with Ct ≤15, 15<Ct≤20, 20<Ct≤25, and Ct>25, respectively, being ICA-positive (Table 2) .

Twenty-one nasopharyngeal swab samples from 21 healthy medical staffs without any symptoms were tested for the ICA. All the samples were negative for both RT-PCR and ICA (data not shown). Table 3 (Table S2 ). All pathogens listed in Table S2 causing respiratory infections were ICA negative.

The present study showed that the developed immunochromatographic kit, KBM Linecheck nCoV, will likely be a powerful tool for COVID-19 diagnosis during the early phase of the disease. All 11 samples obtained within 3 days after symptom onset were positive on these assays, as were 31 (88.6%) of 35 samples obtained within 9 days after symptom onset. However, only 60% of the SARS-CoV-2 positive samples with Ct>25 were positive values. Although RT-PCR methods have been reported able to detect 25 copies of SARS-CoV-2 RNA (Shirato et al., 2020) , RT-PCR targeting RNA J o u r n a l P r e -p r o o f and ICA targeting N protein cannot be directly compared, with the sensitivity of the ICA kit being lower than that of RT-PCR. These findings suggest that these kits, when combined with RT-PCR methods, will be effective in diagnosing COVID-19.

The amino acid sequences of N proteins of SARS-CoV-1 and SARS-CoV-2 are well conserved (Kang et al., 2020) . Nevertheless, SARS-CoV-2 variants have emerged and are spreading worldwide (Leung et al., 2021) , with numerous mutations resulting in amino acid substitutions in spike proteins (Guruprasad 2020) . In contrast, few mutations have been observed in SARS-CoV-2 N proteins, with only two mutations leading to amino acid substitutions (D3L and S235F) reported to date in SARS-CoV-2 N501Y variant 2 (also called B.1.1.7, 20B/501Y.V1 and VOC-202012/01), which was first identified in the United Kingdom in December 2020 (Leung et al., 2021) . The KBM Linecheck nCoV kit was able to detect the recombinant N protein of this variant.

In conclusion, these findings indicate that the KBM Linecheck nCoV ICA kit will be useful for detection of SARS-CoV-2 in point-of-care diagnosis of COVID-19 with high specificity. The kit can rapidly detect SARS-CoV-2 within 10 minutes and is easily handled without any special equipment.

This study was supported by a grant from the Japan Agency for Medical Research and Development (grant number 20he0622015h0001) and a joint research fund from Kohjin Bio Co., Ltd. 

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* Ct values: threshold cycle values generated by SARS-CoV-2 RT-PCR

** To assess samples with various Ct values generated by of SARS-CoV-2 RT-PCR

RT-PCR positive samples and 44 swab samples to which SARS-CoV-2 culture supernatants were added at various concentrations. The 44 swab samples were prepared by adding various doses of SARS-CoV-2 2019-nCoV/JPN/TY/WK-521 culture to RT-PCR-negative swab samples