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

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posted on 02.03.2022, 13:56 by Lisa Milchram, Regina Soldo, Valerie Regele, Silvia Schönthaler, Markus Degeorgi, Sophie-Kathrin Baumgartner, Elena Kopp, Andreas Weinhäusel

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.

Funding

This work received funding from FFG Research Studios Austria (5th call, grant agreement no. 859182, project PepPipe) and H2020 Research and Innovation (grant agreement no. 633937, project ULTRAPLACAD). The funding organization was not involved in the design of the experiments. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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