ELISA for evaluation of the NE-A1AT complexes in the plasma samples:
Standard NE-A1AT complexes were prepared by combining 2.5 μM NE with 10 μM A1AT in 100ul of PBS, and incubated at 370C for 30 and 60 min. After the incubation, aliquots were snap frozen and kept at -800C until further use. For ELISA, 96-well plates were coated with the anti-NE antibody at 1 μg/ml and incubated overnight at 40C. On the following day, the plates were washed once with PBS Tween-20, and blocked with 1 % BSA in PBS for 1 hour at RT. The plates were then incubated with plasma samples collected from non-COVID-19 or COVID-19 patients for 2 hours at RT, followed by 3 washes, and further incubation with rabbit polyclonal anti-A1AT antibody at 1:10000 dilutions for 1 hour. Next, the plates were washed 3 times, and incubated with anti-rabbit-IgG-AP for 1 hour at 1:2000, followed by 3 washes, and further incubation with para-Nitrophenylphosphate substrate for 30min at RT in the dark. The color developed was read at 405 nm.44,45 The NE-A1AT known standard complexes were used to quantitatecomplexes present in the COVID-19 and non-COVID-19 plasma samples.