Direct ELISA analysis of Alpha2-Macroglobulin was performed by coating wells of a 96-well plate with 100ul per well of Alpha2- Macroglobulin recombinant protein diluted in carbonate/bicarbonate buffer, at a concentration of 1 µg/mL overnight at 4C. Wells of the plate were washed, blocked with StartingBlock blocking buffer, and incubated with 100ul per well of a mouse Alpha2-Macroglobulin monoclonal antibody starting at a concentration of 2 µg/mL and serially diluting it to a concentration of 0.03125 µg/mL for 2 hours at room temperature. The plate was washed and incubated with 100ul per well of an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:10,000 for one hour at room temperature. Detection was performed using an Ultra TMB Substrate for 30 minutes at room temperature in the dark. The reaction was stopped with 0.16M sulfuric acid and absorbances were read on a spectrophotometer at 450-550 nm.