Discovery of anti-idiotypic antibody against 14c10 hG1.

(A) Schematics of phage library panning which consists of four main steps; the binding of Fab expressing phage to the coated target antigen (14c10 Fab), the washing away of the unbound phage, followed by the elution and amplification of the bound phage. The cycle then repeats for the enrichment of the specific phage for the target antigen. (B) Polyclonal ELISA showing the enrichment of the successive rounds of phage panning against 14c10 Fab. The wells were coated with 14c10 Fab, other irrelevant Fabs (3H5 and D29) and blocking buffer (skim milk). Negative control was the addition of skim milk instead of the polyclonal phage to the coated antigens. Results represent one independent experiment. (C) Monoclonal ELISA showing the specificity of the monoclonal phage from pan three. Controls were included to ensure that the antigens were properly coated; negative control and positive control were the addition of skim milk instead of the monoclonal phage clones and detected with αM13- HRP (negative) and α-histag-HRP (positive) respectively. Results represent one independent experiment. (D) ELISA to test the specificity of phage clone E1 for 14c10 hG1 in serum. The antigens (14c10 hG1, 3H5 hG1 and D29 hG1) were coated. PEG precipitated phage clone E1 was used at 1 in 10 dilution either in human serum (diluted 1 in 4 with skim milk) or skim milk. Results represent one independent experiment.