Tmb substrate solution

Three mL of plasma was isolated by centrifuging the tubes at 250 g for 10 minutes. The wells of a 96-well microplate were coated with capture antibody prolactin (PRL) (R&D, America), placenta growth factor (PIGF) (R&D, America) or nerve growth factor receptor (NGFR) (R&D, America) with 30 µL of coating buffer (0.1 M sodium carbonate-sodium bicarbonate, PH 9.5; 1.59 g Na₂CO₃ and 7.13 g NaHCO₃ in 1 L dH₂O) overnight at 4 °C, and were removed the coating buffer for adding 50 µL blocking buffer (1 mL PBS) at RT BSA%. Protein samples (30 µL) were pre-mixed with dilution buffer (PBST, 30 µL PBS, PH 7.4, containing 0.05% Tween 20, 1% BSA and 5 µg protein) for 1 h. The mixture was loaded and incubated for 1 h at RT, and the plate was washed three times with PBST. Next, 30 µL of the detection antibody (R&D, America) in the dilution buffer was separately added. The plates were incubated for 1 h at RT and washed three times with PBST. The reaction was observed by adding 30 µL/well of TMB substrate solution (R&D, America) at RT for 30 minutes. The reaction was stopped with 30 µL/well of stop solution (NaH₂SO₄). ELISA plates were detected at 450 nm using an Epoch microplate spectrophotometer (Bio-Tek).