Radiopeptide internalisation and externalization assays: cell viability and radioligand integrity

Area of Science:

• Pharmacology
• Cell Biology
• Radiopharmaceutical Science

Background:

• Radiopeptide receptor-mediated assays are crucial for evaluating targeted therapies.
• Understanding ligand-receptor interactions and cell processing is key to assay development.
• Optimizing conditions for removing unbound ligands without compromising cell integrity is essential.

Purpose of the Study:

• To assess the integrity of externalized peptides in radiopeptide assays.
• To investigate the impact of pH and incubation time of acid wash steps on assay efficacy.
• To determine the optimal conditions for acid washing to remove surface-bound ligands while maintaining cell viability.

Main Methods:

• Radiopeptide receptor-mediated cell internalization and externalization assays were performed.
• The integrity of externalized peptides was analyzed.
• Varying pH levels (e.g., ≤ 2.8) and incubation times for acid wash steps were tested.
• Cell viability and integrity were assessed post-treatment.

Main Results:

• The intact proportion of externalized peptides was observed to be between 5-10%.
• Acid wash buffers with a pH of 2.8 or lower were detrimental to cell viability and integrity.
• Prolonged incubation times, especially with low pH buffers, exacerbated negative effects on cells.

Conclusions:

• Acid wash steps in radiopeptide assays must be carefully optimized to balance ligand removal and cell health.
• A pH of 2.8 or below is not recommended for acid washing due to significant damage to cell viability and integrity.
• Maintaining peptide integrity and cell viability requires cautious selection of acid wash parameters, with higher pH and shorter incubation times being preferable.