Enzygnost hbsag 6

The antigen-binding to BSMNPs coated with antibodies was evaluated as follows. The solution containing HBsAg was added to 100 μL of BSMNPs coated with antibodies and without antibodies separately. After 30 minutes stirring, an external magnet was applied to collect the NPs. BSMNPs were repeatedly washed with PBS buffer. Then, 0.5 mL of glycine solution (0.1 M, pH 2.8) was added to isolate the antigen from the antibodies on the BSMNPs. After five minutes, a magnet was used to collect the NPs, and the supernatant was removed. Tris buffer (1 M, pH 8) was added to the supernatant to neutralize the pH. The antigen-binding to BSMNPs coated with antibodies was evaluated using an ELISA kit (Enzygnost HBsAg 6.0, Siemens, Germany). For this step, 100 μL separated supernatant (dilution of 1/1000), as well as the standards were introduced into the wells. Then, 25 μL of the biotin-conjugated anti-HBsAg was added and incubated for 1 hour at 37°C. After washing four times, 100 μL of the peroxidase-streptavidin solution was added to each well and incubated for 30 minutes at 37°C. The plate was washed five times and then 100 μL of TMB solution was added to the wells. After 15 minutes, the reaction was stopped by adding 100 μL of 1 M sulfuric acid. Finally, the absorbance of the wells was measured at 450 nm.