A Flow Cytometry-based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer...

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• Aptamers offer advantages over antibodies for cancer research.
• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is key for aptamer development.
• Efficient binding assays are crucial for confirming aptamer specificity post-SELEX.

Purpose of the Study:

• To detail a flow cytometry-based binding assay for evaluating aptamer selectivity and specificity.
• To demonstrate the assay's application using an aptamer targeting EpCAM, a protein overexpressed in carcinomas.
• To provide a protocol applicable to other aptamers targeting cell surface proteins.

Main Methods:

• Utilized flow cytometry to assess aptamer binding to EpCAM-positive and EpCAM-negative cells.
• Employed specific buffers and incubation steps for aptamer-cell interaction.
• Included a non-binding aptamer control for comparative analysis.

Main Results:

• The EpCAM-specific aptamer showed selective binding to EpCAM-positive cells.
• No significant binding was observed with EpCAM-negative cells.
• A distinct band shift in flow cytometry analysis confirmed aptamer specificity.

Conclusions:

• The described flow cytometry assay effectively demonstrates aptamer selectivity and specificity.
• This method is valuable for validating aptamers targeting cell surface proteins like EpCAM.
• The protocol's adaptability supports broader aptamer research in targeted drug delivery.