Critical Reagent Screening and Characterization: Benefits and Approaches for Protein Biomarker Assays by Hybrid LC–MS

Abstract

In recent years, hybrid ligand-binding assays (LBAs)/LC–MS assays have been increasingly used for quantitation of protein biomarkers in biological matrices. However, unlike in LBAs where the importance of critical reagent screening and characterization is well understood and widely reported, benefits of well-characterized hybrid LC–MS assay reagents are frequently underestimated. Two groups of analyte-specific reagents, binding reagents and assay calibrators, are considered the critical reagents for biomarker assays. In this article, we summarize the similarities and differences of critical reagents used in LBAs and hybrid LC–MS assays, overview the benefits and approaches of critical reagent screening, characterization, antibody conjugation and discuss bioanalytical considerations in hybrid LC–MS assay development for robust measurements of protein biomarkers in biological matrices.

Keywords:

• critical reagents
• endogenous biomarker
• hybrid LC–MS assay
• immunocapture (IC)
• ligand-binding assay (LBA)
• surrogate peptide

Financial & competing interests disclosure

The authors of this article are current employees of Bristol-Myers Squibb Company. 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.

Acknowledgment

The authors would like to thank RJ Neely and J Wang for the scientific review.

Author contributions

R Santockyte drafted this manuscript. N Zheng and J Zeng contributed to the conception of this work and revision for critical content.