Detection of SARS-CoV-2 omicron variants by immunochromatographic kit

An immunochromatographic kit using antibodies against recombinant N protein of an omicron B.1.1.529 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed to detect SARS-CoV-2 omicron variants. The kit detected omicron variants (BA.1.18, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.4.1, BA.4.6, BE.1, BA.5.2.1, XE, BF.7, BF.7.4.1, XBB.1, XBB.1.5 and BQ.1.1) as well as Wuhan strain and a delta variant.

To prepare monoclonal antibodies, rats were immunized with recombinant SARS-CoV-2 N protein from omicron B.1.1.529variant (hCoV-19/Japan/TY38-873/2021).The monoclonal antibodies were purified using Spin Column Based Antibody Purification kits (Cosmo Bio USA, Carlsbad, CA, USA) from the culture supernatants of antibody-producing hybridomas.Antibody N3J13 was found to be the most effective capture antibody and N3J15 the most effective detection antibody in sandwich ELISA tests against the recombinant N protein of SARS-CoV-2 omicron B.1.1.529,and recognized aa 1-220 and 210-419 regions, respectively (data not shown).The ICA kit [KBM Linecheck nCoV (device-type)] was developed by coating nitrocellulose membranes with a capture monoclonal antibody against SARS-CoV-2 (N3J13) and with a goat anti-rat IgG antibody as described [4].A detection monoclonal antibody against SARS-CoV-2 (N3J15) was conjugated with colloidal gold nanoparticles and immersed into glass fiber.A nasopharyngeal swab sample (50 μl) obtained from a COVID-19 patient was added to 200 μl of Tris-based buffer (pH 7.6), of which 90 μl (three droplets) was added to a sample well of the ICA (Fig. 1 A and B).Line formation on the strip was assessed within 15 min after immersing it.
A total of 69 nasopharyngeal swab samples were collected from 44 COVID-19 patients and 25 volunteers during March 2021 to July 2022 in Tokyo.All 44 samples from COVID-19 patients were from Juntendo University Hospital and not arbitrarily chosen, while all 25 samples from volunteers were from Nansho Hospital, Iwate, Japan.Positive diagnosis of the 44 patients was confirmed by RT-qPCR using Ampdirect 2019-nCoV detection kit according to manufacturer's protocol (SHIMADZU Corporation, Kyoto, Japan).The Ct values of the 44 samples ranged from 12.0 to 19.9 (mean 17.0 ± 2.1, median 17.3).No RT-qPCR-positive sample with Ct > 20 was obtained.SARS-CoV-2 omicron and delta variants were identified by RT-qPCR using VirSNiP SARS-CoV-2 spike T478K and VirSNiP SARS-CoV-2 spike S371L S373P according to manufacturer's protocols (TIB MolBio, Berlin, Germany).
In this study, the ICA kit effectively detected several omicron variants of SARS-CoV-2 demonstrating it is useful to diagnose COVID-19 patients infected with current prevalent strains, albeit with less sensitivity than that of RT-qPCR (Table 1).We previously developed an ICA kit to detect SARS-CoV-2 which recognized regions of N protein in Wuhan strain (aa 1-120 and aa 111-220) [4], whereas, the present kit recognized different regions of N protein in an omicron variant (aa 1-220 and aa 210-419).Both ICA kits can detect SARS-CoV-2 Wuhan strain and omicron variants (data not shown).This ICA kit has the potential to detect variants of SARS-CoV-2 in future waves of the pandemic, because the amino acid sequence of N protein is relatively conserved compared to that of the spike (S)  protein; i.e. compared to the original Wuhan strain, the current most prevalent omicron variant BA.5 has eight amino acid substitutions in the N protein (Table 3) but 34 in the S (https://gisaid.org/GISAID).Assessment and improvement of the ICA kit will be continued.
The ICA kit seems to detect SARS-CoV-2 omicron variants as well as Pango Lineage A and delta B.1.617.2 at the same sensitivity.Preliminary analysis was unable to determine the epitopes of these monoclonal antibodies using synthesized overlapped peptides with 20 amino acids of N protein (date not shown).Further detailed epitope analysis is necessary in further studies to identify suitable and stable epitopes of N protein of SARS-CoV-2 to improve ICA kits.

Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MA and JS are employees of Kohjin Bio Co., Ltd.

Fig. 1 .
Fig. 1.Details of the immunochromatographic assay kit (KBM LineCheck nCoV (device-type).Samples showing two lines, one each at the control and test position for SARS-CoV-2 were positive for SARS-CoV-2 (A).Samples showing a single line at the control position were negative for SARS-CoV-2 (B).

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