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.), 10µl/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 incubated 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 in duplicate and incubated 4 hours at room te mperature. The plate is washed and incubated with 200 μl/well 8M Urea for 3 minutes. The plate is washed and 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% BS A and incubated for 45 minutes at room temperature (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. Avidity Index is expressed as a percentage, where absorbance reading after urea wash is divided with absorbance reading without urea wash multiplied by 100. 3. Quantitative ELISA for the detection of tetanus toxoid antibodies and antibody avidity: Patients have been immunized with a DT at enrollment. 96 well Immulon 4 plates (Dynatech Laboratories) are incubated with tt protein (Lederle Labs) at a concentration of 1:1000 diluted in carbonate buffer alternating with rows of PBS/1% BSA. W ells set up as a standard curve are incubated with purified human IgG (Sigma BioSciences, St. Louis, MO) in serial dilutions ranging from 0.0217 μg/ml to 2.78 μg/ml overnight at 4°C. After incubation, all wells were blocked with PBS and 1% bovine serum al bumin (BSA) (Sigma Chemical Co.), 100 µl/well for 1 hour at room temperature. The protein -coated plate is incubated with experimental sera diluted 1:400, 1:800, 1:1600 and 1:3200 in duplicate for 2 hours at room temperature. One set of duplicate is treat ed with 8M Urea for 5 minutes. Goat anti -human IgG (gamma) horseradish peroxidase (HRP) (Zymed, So. San Francisco, CA) are added to the wells at a 1:5,000 dilution in PBS/1% BSA and incubated for 45 minutes at room temperature (Amersham Co., Arlington Heig hts, 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 650nm until the standard curve well containing 0.174 µg/ml reaches 0.3 OD. More di lute sera will be used if values are at the top or off of the standard curve. The reaction is stopped with 1N HCl and the optical density read at 450nm. The OD of each serum dilution is calculated as the OD of the protein coated wells minus the OD of the PBS/1% BSA coated wells. Values are calculated in µg/ml based on the standard curve. To calculate the % avidity index, the absorbance reading after urea wash is divided with absorbance reading without urea wash and multiplied by 100. 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 deviations of the experimental runs. Validation runs will be tabulated and discussed by the IMC and a quality assurance report filed with the data manager.