Ab7153

Streptavidin Coated pre-blocked plates (Thermofisher, 15124) were washed 3 times with 200 μL of phosphate buffered saline with 0.1% tween (PBS-T) prior to coating with 100 μL of biotinylated anti-ApoA-I antibody (abcam, ab27630) diluted 1:1400 in PBS-T. Plates were incubated at 37 °C for 2 hours and subsequently washed 6 times with PBS-T. Plasma samples diluted 1:200 in PBS with 0.05% casein (PBS-C) was added to the wells and incubated at 37 °C for 30 min. Following 6 washes with PBS-T, 100 μL of horseradish peroxidase (HRP)-conjugated anti-Human IgG (abcam, ab7153) was added at a dilution of 1:4000 in PBS-C and incubated for 30 min at 37 °C. After a final 6 washes with PBS-T, 100 μL of room temperature tetramethylbenzidine (TMB) (Rockland Immunochemical) was added and allowed to react for 30 min at room temperature in the dark. The reaction was stopped using 100 μL of 0.5 M H₂SO₄ and absorbance was measured at 450 nm (Biotek, Synergy Hybrid Reader). All samples were analyzed in duplicate.

High binding plates were washed with Na₂CO₃ buffer 3 times and coated with 50 μL of anti-IgG (abcam, ab102421) diluted 1:500 in Na₂CO₃ buffer. The plates were incubated for 1 hour at 37 °C, then washed 6 times with PBS-T before blocking for 1 hour at 37 °C with PBS-C. After blocking, the plates were washed 2 times with PBS-T and 100 μL of plasma diluted 1:1,000,000 in PBS-C was added and incubated for 30 min at 37 °C. After 6 washes with PBS-T, 100 μL of a 1:4000 dilution of anti-human IgG HRP (abcam, ab7153) in PBS-C was added to each well and incubated for 30 min at 37°C. The plates were then washed, developed with TMB, quenched with H₂SO₄ and the absorbance measured at 450 nm. Samples were analyzed in duplicate with a standard curve on each plate containing human IgG (Gene Script, A01006).