Example of a LRC-TriCEPS experiment

Case study Dualsystems Biotech AG and Biaffin GmbH & Co KG

Example of a LRC-TriCEPS™ (CaptiRec) experiment performed at Dualsystems to identify the target receptor of Anti-EGFR-Antibody on human breast cancer cells (MDA-MB-231):
Positive control ligand (included in the kit): Insulin
Ligand of interest: Anti-human EGF Receptor Antibody (mouse mAb, clone 225, SIGMA-ALDRICH #E2156)
Cells: MDA-MB-231 human breast cancer cells

epidermal growth factor receptor (EGFR)

Figure 1:

Quality control 1: Expression of human epidermal growth factor receptor (EGFR)  in human breast cancer cells (MDA-MB-231) was monitored by FACS using primary labeled Anti-EGFR antibodies (green) with the corresponding isotype control antibodies (grey).

MDA-MB-231

Figure 2:

Quality control 2: Expression of Insulin receptor (INSR)and Insulin-likegrowth factor 1 receptor (IGF-1R) in human breast cancer cells (MDA-MB-231) cells was monitored by FACS using primary labeled Anti-IGF1R antibodies (orange) and Anti-INSR antibodies (blue) with the corresponding isotype control antibodies (grey).

Streptavidin-HRP

Figure 3:

Quality control 3: Dot Blot to monitor coupling of ligands to TriCEPS. Detection by Streptavidin-HRP (horseradish peroxidase) that binds to the biotin part of TriCEPS. TriCEPS itself does not bind to the nitrocellulose (see Glycine control).

Upper lane: positive control Insulin coupled to TriCEPS
Middle lane: ligand of interest (Anti-EGFR Antibody) coupled to TriCEPS
Lower lane: negative control for the Dot Blot, Glycine coupled to TriCEPS
Dilutions as indicated in the figure.

Volcano-Plot-on-protein-level

Figure 4: 

Volcano Plot on protein level.
CaptiRec experiment with anti-EGFR antibody (mouse mAb, clone 225, SIGMA-ALDRICH #E2156) and Insulin on MDA-MB-231 human breast cancer cells in triplicates. The receptor candidate space is defined by an enrichment factor of > 4 fold and statistical significance (FDR-adjusted p-value<0.01). [/av_textblock] [av_image src='http://www.dualsystems.com/wp-content/uploads/2014/09/Biaffin-anti-EGFR-300x219.jpg' attachment='938' attachment_size='medium' align='center' styling='' hover='' link='' target='' caption='' font_size='' appearance='' overlay_opacity='0.4' overlay_color='#000000' overlay_text_color='#ffffff' animation='no-animation'][/av_image] [av_textblock size='' font_color='' color=''] Figure 5:

Surface Plasmon Resonance (SPR, BiacoreTM) analysis performed by Biaffin GmbH & Co KG to determine the affinity of the anti EGFR-antibody used in the above experiment. Human EGFR monomers were immobilized on the chip and the anti EGFR-antibody was used at a concentration range of 46pM bis 100nM. Affinity (KD) of the antibody to the EGFR is 0.2nM.

Conclusion:

In process positive control: Insulin Receptor (INSR) and Insulin-like growthfactor 1 receptor (IGF-1R) were detected as the target of insulin on the living human breast cancer cells (MDA-MB-231) on the peptide level. INSR was identified as the target receptor of insulin on the protein level on MDA-MB-231 cells. EGF-Receptor was identified as the target of the Anti-EGFR receptor Antibody on the living human breast cancer cells (MDA-MB-231). As expected, the ligand itself IgG (Immunglobulin G) is also in the receptor space since anti-EGFR antibody is glycosylated.