Autoantibodies were detected by ELISA according to previously described methods [34] (link). The BirA control was included to allow subtraction of any assay signal due to nonspecific binding.
In brief 41 TAAs/antigenic fragments (see supporting information), and 2 assay controls (BirA and buffer only) were adsorbed to ELISA plates in duplicate and at 2 concentrations of antigen (100 nM and 50 nM). These proteins were then incubated with serum samples from cancer, high-risk and healthy control cohorts (Table 1 ) diluted 1 in 110 in blocking buffer. The presence of an IgG response to the antigens was detected with horseradish peroxidase-conjugated rabbit anti-human IgG (Dako) and 3,3′,5,5′-tetramethylbenzidine, as previously described [29] . All assays were conducted on a semi-automated robotic system and cancer, high-risk and healthy control samples were interspersed. Incubations with anti-His monoclonal antibody (AbCam) and, where available antigen-specific monoclonal antibodies (Sigma, AbCam, Santa-Cruz), were carried out to validate antigen plate coating. SDS-PAGE analysis of TAA plate-coating solutions was also carried out to verify plate layouts and protein dilutions.
In brief 41 TAAs/antigenic fragments (see supporting information), and 2 assay controls (BirA and buffer only) were adsorbed to ELISA plates in duplicate and at 2 concentrations of antigen (100 nM and 50 nM). These proteins were then incubated with serum samples from cancer, high-risk and healthy control cohorts (
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