Assessment of an immunochromatographic kit for detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses.

An immunochromatographic kit was developed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses (A and B) on two detection positions of a single strip. The sensitivity and specificity for SARS-CoV-2 were 97.4 % and 100 %, respectively, and those for influenza viruses were 100 %, respectively.

WHO (2022a) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started in December 2019 and continues at the time of writing in 2021 (https://www.who.int/emergencies/diseases/novel-coronavirus-2019) with no predictable endpoint (Telenti et al., 2021). A worrisome problem is that an influenza epidemic or pandemic may occur during the CODIV-19 pandemic because both viruses cause respiratory tract infections and are transmitted by respiratory droplets and/or aerosols (Telenti et al., 2021). We describe here production of an immunochromatographic assay (ICA) to detect both SARS-CoV-2 and influenza A and B (A/B) viruses.

Monoclonal antibodies were prepared by immunization of rats with recombinant SARS-CoV-2 nucleocapsid (N) protein (Oshiro et al., 2021) or N proteins of influenza A/B viruses (Flarebio Biotech, LLC, MD, USA and Sino Biological Inc, Beijing, China, respectively). The ICA was developed by coating nitrocellulose membranes with capture antibodies for SARS-CoV-2 (N1J7−1), and influenza A/B viruses (K2A4 and K3B1, respectively) and control goat anti-rodent IgG antibody. Detection antibody-conjugated colloidal gold nanoparticles for SARS-CoV-2 (N3J3−1), and influenza A/B virus (K2A7 and K3B2, respectively) were immersed into glass fiber. The ICA kit, KMB LineCheck nCoV/Flu, was a single-strip, lateral-flow device comprising three detection positions on the strip, including a test position for SARS-CoV-2, a test position for influenza A/B viruses, and a control position (Fig. 1 ). To examine the stability of the ICA kit, the sensitivity and specificity of the kits were regularly determined using SARS-CoV-2 and influenza A/B virus antigens after stored at room temperature (5−30 °C). The sensitivity and specificity did not changed after 6 months storage. Further examination is now in progress. A nasopharyngeal swab sample (50 μL) obtained from a patient suspected COVID-19 or seasonal influenza was added in elution buffer (200 μL Tris-based buffer, pH 7.6). Three droplets of the solution were added into the sample well. To determine the detection limit of the ICA, culture supernatants of SARS-CoV-2 [2019-nCoV/JPN/TY/WK-521, 4.2 × 105 tissue culture infectious dose 50 (TCID50)/mL], influenza A virus [A/New Jersey/8/76(H1N1), 1.24 × 104 plaque forming unit (PFU)/mL] or influenza B virus (B/Taiwan/2/62, 1.72 × 104 PFU/mL) were diluted in PBS. An aliquot of diluted supernatant (10 μL) was further diluted in 240 μL of Tris-based buffer (pH 7.6), and 90 μL of the product was used to test the ICA.

Fig. 1

Details of the immunochromatographic assay kit (KBM LineCheck nCoV/Flu). Samples showing a single line at the control position were negative for SARS-CoV-2 or influenza A and B viruses (a), whereas samples showing two lines, one each at the control and test position for SARS-CoV-2 or influenza A and B viruses, were positive for SARS-CoV-2 (b) or influenza A and B viruses (c).

A total of 100 nasopharyngeal swab samples were collected from patients suspected of COVID-19 with 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; during September 2020 to February 2021 at a university hospital in Tokyo. All samples were subjected to RT-PCR for the detection of SARS-CoV-2 according to protocol (Shirato et al., 2020). Of the 100 samples, 39 were RT-PCR-positive for SARS-CoV-2 (Table 1 ) among which 38 were ICA-positive for SARS-CoV-2. All 61 RT-PCR-negative samples were ICA-negative for SARS-CoV-2 (Table 1). All 100 samples were ICA-negative for influenza (Table 1). The sensitivity and specificity of ICA for SARS-CoV-2 detection were 97.4 % and 100 %, respectively. The lower detection limit of ICA for SARS-CoV-2 was 7.81 × 102 TCID50/mL. The ICA for influenza viruses was negative even at the highest concentration of 2.5 × 104 TCID50/mL of SARS-CoV-2 (data not shown). The samples in elution buffer containing SARS-CoV-2 at less than the lowest detection limit of ICA (4.88 × 102 TCID50/mL) were RT-PCR-positive for SARS-CoV-2.

Table 1

Detection of SARS-CoV-2 by KBM LineCheck nCoV/Flu in 100 nasopharyngeal swab samples from COVID-19 suspected patients.

			RT-PCR
			SARS-CoV-2 (influenza virus)a
			Positive	Negative	Total
ICA	ICA for SARS-CoV-2 (influenza virus)b	Positive	38 (0)	0 (0)	38 (0)
		Negative	1 (0)	61 (100)	62 (100)
		Total	39 (0)	61 (100)	100 (100)

Numbers of RT-PCR samples for SARS-CoV-2 (samples of RT-PCR-positive or negative for influenza virus).

Numbers of samples ICA-positive or negative for SARS-CoV-2 (samples ICA-positive or negative for influenza virus).

A total of 152 nasopharyngeal swab samples were collected from patients suspected of seasonal influenza with symptoms such as fever, cough, headache, muscle and joint pain, severe malaise, sore throat and a runny nose (WHO (2022b); during January to April 2016 in seven clinics in Fukuoka, Japan. All samples were subjected to RT-PCR for the detection of influenza A/B viruses. Of them, 42 were RT-PCR-positive for influenza A and 50 were positive for influenza B (Table 2 ) of which all were ICA-positive for influenza viruses. All RT-PCR-negative samples were ICA-negative for influenza viruses (Table 2). All the 152 samples were ICA-negative for SARS-CoV-2 (Table 2). The sensitivity and specificity of KBM LineCheck nCoV/Flu for influenza viruses were 100 %, respectively. The lower detection limits of ICA for influenza A/B viruses were 1.21 × 101 PFU/mL and 1.68 × 101 PFU/mL, respectively. The ICA for SARS-CoV-2 was negative even at the highest concentrations of 1.55 × 103 PFU for influenza A and 2.15 × 103 PFU for influenza B viruses, respectively (data not shown). The samples in elution buffer containing influenza viruses at less than the lowest detection limit of ICA (6.05 PFU/mL and 8.4 PFU/mL, respectively) were RT-PCR-positive for influenza viruses.

Table 2

Detection of influenza A and B viruses by KBM LineCheck nCoV/Flu in 153 nasopharyngeal swab samples from influenza suspected patients.

			RT-PCR
			Influenza viruses (SARS-CoV-2)a
			Positive	Negative	Total
ICA	Influenza viruses (SARS-CoV-2)b	Positive	92 (0)	0 (0)	92 (0)
		Negative	0 (0)	61 (153)	61 (153)
		Total	92 (0)	61 (153)	153 (153)

Numbers of samples RT-PCR-positive or negative for influenza viruses (those of sampels RT-PCR-positive or negative forSARS-CoV-2).

Numbers of samples ICA-positive or negative for influenza viruses (those of sampels ICA-positive or negative for SARS-CoV-2).

As shown in Table S1, recombinant N proteins of SARS-CoV-1 and SARS-CoV-2 were ICA-positive for SARS-CoV-2 and ICA-negative for influenza virus. Influenza A virus (H1N1) [A/Virginia/ATCC1/2009, A/Swine/1976/31, A/Swine/Iowa/15/30], influenza A virus (H3N2) A/HongKong/8/68(TC-adapted) and influenza B virus, B/Lee/20 were ICA-positive for influenza virus and ICA-negative for SARS-CoV-2 (Table S1). Three recombinant N proteins of MERS-coronavirus, human coronavirus OC43 and human coronavirus 299E, and 59 pathogens causing respiratory infections were ICA-negative for both SARS-CoV-2 and influenza viruses (Table S1).

KMB LineCheck nCoV/Flu is a useful kit to test for COVID-19 and influenza simultaneously, especially during an influenza season amid the COVID-19 pandemic.

Authors’ contributions

MA, JS and SO developed the kit. JS and YT acquired clinical samples. SO, NM and KS assessed the kit and analyzed the data. KF, MA and JS conducted RT-PCR. YT, TM, TT and TK supervised the study. All authors approved this final version manuscript.

Funding

This study was supported by a grant from the (grant number20he0622015h0001).

Declaration of Competing Interest

MA and JS are employees of Kohjin Bio Co., Ltd.