Supplementary data: A novel click chemistry based peptide ELISA protocol: development and technical evaluation

Supplementary Figure 1: estimated mass (kDa) of the BSA-peptide conjugate on the y-axis plotted against the number of coupled spacer+linker+peptide group on the x-axis. Blue line shows the estimated masses for coupling of azido-PEG4 and spacer-peptide-propargylglycine to 1 to 146 available -NH₂ groups based on the published sequence of BSA (lysine, arginine, glutamine and asparagine were considered as primary -NH₂ group providers). Orange line shows the estimated masses of coupled azido-PEG4 coupled to BSA.

Supplementary Figure 2: 10 peptide BSA conjugates were selected for Western Blot investigation of coupling confirmation. (A) shows the total protein staining of the membrane after transfer, (B) the anti-FLAG detection using anti-FLAG HRP antibody.

Supplementary Figure 3: (a) dilution series of peptides plotted for blank-uncorrected absorption values at 450nm. Two peptides (EBV1, EBV2) on the three evaluated surfaces are shown. (b) absorbance values for blocked (1% BSA in PBST) and unblocked wells obtained for dilution series of unmodified, azide modified carrier molecule (BSA, azido BSA) and IgG incubated with 0.15 mg/mL IgG. Uncoated wells are shown as “buffer”, “blank” wells represent BSA (unmodified) coated wells incubated with 0.15 mg/mL IgG.