Table 2 List of the 15 tumor-associated antigens used in the study. ELISA methodology ELISA was used to measure the humoral immune response in the serum or plasma of participating women to the various peptides or whole proteins antigens (Table 2). At each location, a specific standardized read FAQ ELISA protocol was followed (described below) on local samples to ensure assay consistency across sites. Each sample was given a barcode identifier in the laboratory to ensure a blinded analysis. White ��Maxiorp�� 96 wells plates (Nunc, Roskilde, Denmark) were coated with commercial antigens at concentrations ranging 2�C6 ��g/mL for proteins, and 0.25�C1 mg/mL for peptides in phosphate-buffered saline (PBS) and blocked with Well Champion reagent (Kem-En-Tec, Taastrup, Denmark) according to the manufacturer��s instructions.
Serum or plasma samples (100 ��L) were loaded in 6 serial dilutions starting at 1:40�C1:320 in 1% skim milk in PBS (Fluka, St. Louis, MO, USA) for each of the coated antigens in the plates and incubated at 37oC for 1.5 h with gentle agitation. The plates were washed 8 times with 300 ��L of Dulbecco��s PBS, 0.05% Tween 20 (PBST), and 1:10,000 horseradish peroxidase conjugated goat anti-human IgG (Chemicon, Temecula, CA, USA) was added for 1 h at 37oC, followed by 4 washes with 0.025% PBST. EZ-ECL (Biological Industries, Beit-Haemek, Israel) was used for luminescent development according to the manufacturer��s instructions. Luminescence was measured with Luminoscan Ascent (Thermo Scientific, Waltham, MA, USA) using Ascent software (Thermo Scientific).
Results were loaded into an internet database in a secure server according to the barcodes. Statistical methods All statistical analyses were performed using STATA 12 SE (StataCorp, College Station, TX, USA). All P-values were two-sided. P-values below 0.05 were considered significant. No corrections for multiple comparisons were performed. The initial data for each sample consisted of 6 measurements of AAb relative luminescence units (RLU) for each antigen in 6 consecutive dilutions. In the first step, we fitted the log10[RLU] as a linear function of the log10[dilution]. If the goodness of fit was not satisfactory, we excluded one outlier and refit the data by linear regression with the remaining 5 points. Reference values of the dilution were fixed for each AAb.
If the Dacomitinib goodness of fit was high, the fitted value at the reference point was calculated for each AAb. Otherwise the value was classified as ��missing�� for this antigen only, meaning that the data did not pass the quality control. A missing value was only given to a specific antigen and not to a sample. Thus, each sample was left with a set of maximum 15 values of AAb log10[RLU] for all antigens, each at the reference dilution point chosen for the antigens.