Sandwich ELISA of Transferrin was performed by coating wells of a 96-well plate with 100ul of an transferrin chicken polyclonal antibody diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer overnight at 4C. Wells were blocked with 100ul of StartingBlock T20 (TBS) Blocking Buffer for 2 hours, and 100ul of recombinant transferrin protein ranging from 1.6-1000ng/ml were incubated for 1 hour at room temperature. The plate was washed with 1X TBST, and 100ul per well of an Transferrin mouse monoclonal antibody diluted to a concentration of 1 µg/mL was added to each well for 1 hour at room temperature. The plate was washed, and 100ul per well of a Streptavidin-HRP was incubated for 30 minutes. Detection was performed using 1-Step Ultra TMB Substrate, followed by Stop Solution. Absorbances were read on a spectrophotometer at 450-550nm.