Rapid SARS-CoV-2 Antigen Detection Assay

Purified anti-spike protein monoclonal antibodies (SpMA-01 and SpMA-02) were diluted in 50 mM PBS buffer (pH 7.4), 2 µg of capture mAb (SpMA-02) and 1 µg of goat anti-mouse antibody (Bethyl Lab, USA) and dropped onto a nitrocellulose membrane at the reading window to give the T and C, respectively. To 10 µL of colloidal gold conjugated anti-spike mAb (SpMA-01), an equal volume of 10% alkali-treated casein was mixed and placed onto a conjugate pad. The membranes were then dried at room temperature to immobilize antibodies. Samples of SARS-CoV-2 and its variants, or viruses and bacteria from the FDA pathogen panel were either diluted in PBS or treated with 100 mM TERGITOL-NP (prepared by mixing 334μL 100 mM Tergitol NP-9 with 666μL 100 mM Tergitol NP-10) followed by dilution with 150 µL of PBS. The samples were then placed onto sample application wells. Driven by capillary forces, the immunocomplex migrated up the membrane into the absorbent pad and after 10 to 15 minutes, the test results were evaluated visually. The selection of the optimal concentrations of the spike protein or pathogen antigens were visually inspected for Test (T-) and Control (C-) results. To determine the analytical sensitivity of spike protein in saliva or nasal samples, 200 ng of recombinant (S) was spiked into swabs and a 150μL extract was tested in the assay.