Well-developed ligand-binding assays demonstrate robust performance using singlet analysis

Area of Science:

• Bioanalytical Chemistry
• Pharmacokinetics
• Assay Development

Background:

• Historically, replicate testing was essential for bioanalytical laboratory testing, particularly for ligand-binding assays (LBAs), due to the imprecision of older assay technologies.
• While regulatory guidelines permit singlet testing with demonstrated assay robustness, duplicate testing remains a common practice in many laboratories.

Purpose of the Study:

• To re-evaluate the necessity of replicate sample testing in modern bioanalytical assays, specifically ligand-binding assays (LBAs).
• To determine if singlet analysis yields comparable results to duplicate analysis in LBAs, even without automation.

Main Methods:

• Re-analysis of data from five pharmacokinetic assay validations and five clinical/preclinical studies.
• Original assays were performed in duplicate; re-analysis was conducted using singlet testing methodology.

Main Results:

• Data re-evaluated using singlet testing showed results nearly identical to the original duplicate testing results.
• The findings indicate that well-developed LBAs produce comparable data regardless of whether singlet or duplicate testing is employed.

Conclusions:

• The practice of replicate testing for LBAs should be re-evaluated in light of modern assay precision and accuracy.
• Automation is not a prerequisite for successful singlet testing in LBAs, challenging the notion that it is required.