Lateral flow assay

As previously described by Chan et al., the Easy Check COVID-19 IgM/IgG™ test is an immunochromatographic assay for the detection of SARS-CoV-2 IgM and/or IgG antibodies in human plasma specimens [26]. The test uses the spike RBD and the nucleocapsid protein as antigens. Control antibody, anti-human IgG and streptavidin (test line for IgM) are immobilized onto a nitrocellulose membrane to form three distinct lines: the control line, IgG test line and IgM test line (Fig 1). The nitrocellulose membrane is attached onto a plastic backing card and combined with other reagents and pads to construct a test strip. The test strip is encased inside a plastic device (Fig 2).

Sample is loaded into sample well and wicks into test strip. Lines can be visualized in the result window. A band will appear at C (control line) if the test is valid. A line will appear at the SARS-CoV-2 IgG (G) test line if the sample is positive for anti-SARS-CoV-2 IgG antibodies. A line will appear at the SARS-CoV-2 IgM (M) test line if the sample is positive for anti-SARS-CoV-2 IgM antibodies.

Sample is loaded into the sample port and wicks into the test strip via capillary flow. The sample specimens migrate sequentially through filter pad, conjugate pad, nitrocellulose membrane, and absorbent pad. Present on the conjugation pad are SARS-CoV-2 antigens conjugated to gold nanoparticles, gold conjugated chicken IgY antibody, and biotin conjugated anti-human IgM antibodies. Anti-SARS-CoV-2 IgM antibodies present in the sample will bind to the biotin conjugated anti-human IgM antibodies and gold conjugated antigens at the conjugate pad before binding to the streptavidin present at the “M” test line on the nitrocellulose membrane. Anti-SARS-CoV-2 IgG antibodies present in the sample will bind to the gold conjugated antigens present at the conjugate pad before migrating further through the strip and binding to the anti-human IgG antibody at the “G” test line on the nitrocellulose membrane. Sample flowing through the test strip will carry gold conjugated chicken IgY antibodies present at the conjugate pad through the test strip where they will bind to the anti-chicken IgY antibody located at the “C” line on the nitrocellulose membrane.

The test results were visually interpreted 10 minutes after starting the test. Any colored lines in the test region were considered as positive regardless of line intensity. The presence of two lines marked by “C” and “G” indicates SARS-CoV-2 IgG positivity. The presence of two lines marked by “C” and “M” indicates SARS-CoV-2 IgM positivity. The presence of three lines “C”, “G” and “M” indicates positivity for both SARS-CoV-2 IgG and IgM. The appearance of only the control line “C” indicates a correctly performed test, but also that the sample is negative for SARS-CoV-2 IgM and IgG.