Antigen-binding activity of Nluc-ch2C5 in a double-antibody sandwich ELISA

Next, a double-antibody sandwich ELISA was performed to determine the sensitivity of the Nluc-ch2C5 antibody for SARS-CoV-2 or S-RBD. The capture antibody MW06 (2 μg/ml) was coated overnight at 4 °C onto a white 96-well plate. After blocking with PBST/3% BSA, inactivated SARS-CoV-2 (10⁵pfu/ml–39pfu/ml, serially diluted five-fold) or S-RBD (1 μg/ml–0.06 ng/ml, serially diluted four-fold) was added to the wells for 1.5 h at 37 °C. Inactivated H7N9 and 3% BSA were used as negative controls, and 0.1% BAS-PBST was used as a blank control. After washing, Nluc-ch2C5 (10 ng/ml) was added to the wells at 37 °C for 1.5 h. After washing five times with PBST, 100 μl of Nano luciferase substrate was added. The average value of the negative control group plus three standard deviations was set as the cutoff value; samples showing values higher than the cutoff value were deemed positive. The ratio of the average luminescence intensity in each test sample to the cutoff value (S/C) was calculated. An S/C value > 1 was taken as a positive threshold for results analysis.