Ring) at a concentration of 1µg antibody per ml . Wells set up as a standard curve are incubated with purified human IgG (Sigma BioSciences, St. Louis, MO) in serial dilutions ranging from 0.03µg/well to 0.0002µg/well. After incubation, all wells are blocked with PBS and 1% bovine serum albumin (BSA) (Sigma Chemical Co.), 100µl/well for 4 hours at room temperature. The plate is washed with PBS/0.1% Tween and lysates of SKBR3, a human breast cancer cell line which overexpresses the HER2 protein, are added in alternating rows with PBS/1% BSA and incubat ed overnight at 4°C. After washing, the plate is incubated with human sera at the following dilutions: 1:25, 1:50, 1:100, 1:200. The sera is diluted in PBS/1% BSA/1% FBS/25 µg/ml mouse IgG/0.01% NaN 3 and then serially into PBS/1% BSA. 5µl of diluted sera is added per well and incubated 1 hour at room temperature. Goat anti -human IgG (gamma) horseradish peroxidase (HRP) (Zymed, So. San Francisco, CA) is added to the wells at a 1:50,000 dilution in PBS/1% BSA and incubated for 45 minutes at room temperatu re (Amersham Co., Arlington Heights, IL). Following the final wash, TMB (Kirkegaard and Perry Laboratories, Gaithersburg, MD) developing reagent is added. Color reaction is read at an optical density of 650 nm until the standard curve well containing 0.04 µg reaches 0.3 OD. The reaction is stopped with 1N HCl and the optical density read at 450nm. The OD of each serum dilution was calculated as the OD of the HER2 coated wells minus the OD of the PBS/1% BSA coated wells. A normal human serum is run on e ach plate as a negative control. Values are calculated in µg/ml based on the standard 6304p Protocol 118 04/06/2016 curve. The final value expressed is the mean and standard deviation of quadruplicate dilutions. All positive responses will be evaluated by Western Blot. A population of 150 volunteer blood donors is run to establish the baseline of the assay. Random normal blood donors (n=10) are run every 100 plates, or quarterly, whichever comes first, and the standard curve and antibody values compared to mean and standard deviati ons of the experimental runs. Validation runs will be tabulated and discussed by the IMC and a quality assurance report filed with the data manager.