Fig. 4

Properties of the IgG fraction purified from de serum of vaccinated patients (IgG comp). a Binding of IgG to VEGF-coated wells. Test samples were added to VEGF-coated wells and IgG bound to wells was detected with HRP-conjugated goat anti-human IgG antibody. This assay used as positive controls a human serum positive for VEGF-specific IgG antibodies (PCS) and bevacizumab (bev). Assay negative controls were a human serum negative for VEGF-specific IgG antibodies (NCS) and a purified human IgG isolated from pooled normal human serum (IgG neg) (b) Inhibition of the VEGF/VEGFR2 interaction. Test samples and VEGFR2-Fcγ were added to VEGF-coated wells, and VEGF/VEGFR2-Fcγ binding was determined with biotin-conjugated anti-VEGFR2 antibody followed by streptavidin-peroxidase conjugate (c) Inhibition of the VEGF/VEGFR1 interaction. Test samples and VEGFR1-Fcγ were added to VEGF-coated wells, and VEGF/VEGFR1-Fcγ binding was determined with biotin-conjugated anti-VEGFR1 antibody followed by streptavidin-peroxidase conjugate (d) Inhibition of the VEGF/bevacizumab interaction. Test samples and biotinylated bevacizumab (bev _biot) were added to VEGF-coated wells, and VEGF/Bev _biot binding was determined with streptavidin-peroxidase conjugate. Discontinued lines represent cut-off values that define the positivity for VEGF/VEGFR2, VEGF/VEGFR1 or VEGF/Bev blockade. The ability of test samples to block these three interactions was expressed as percentage of reduction of the maximum binding. Bev was used as inhibition positive control. IgG comp and IgG neg were evaluated in all ELISAs at the same total IgG concentration. All figures show the response obtained for IgG comp that exhibited the best ratio IgG comp/IgG neg. Column bars represent the means of three replicates and error bars represent standard deviations. p-values were calculated according to unpaired t-test. Statistical significance was considered as p < 0.05