Urea dissociation test of GICA.

Sera (100 μl) were added into 1-ml sample diluents (phosphate-buffered saline [PBS], NaCl, and Tween 20) and mixed, and then 100 μl of the diluted sample was put into the sample hole of the test card. The liquid was chromatographed upward under the control of the capillary effect; when the liquid was about to reach the upper absorbent paper, 100 μl PBS solution containing 6 mol/liter urea was added into the sample hole of the test card; the results were observed after 20 to 25 min. The SARS-CoV-2 IgM in the sample bound first with the anti-human-IgM labeled by colloidal gold and then with the SARS-CoV-2 recombinant antigen at the test line (T) position to form a complex of SARS-CoV-2 antigen, SARS-CoV-2 IgM, and anti-human IgM labeled by colloidal gold. A complex of goat polyclonal IgG and anti-human IgM labeled by colloidal gold was formed at the control line (C) position. For the positive standard, colloidal gold color reactions occur at both the T-line and C-line positions; for the negative standard, the colloidal gold color reaction occurs only at the C-line position.