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. 2024 Jul 25;26(5):87.
doi: 10.1208/s12248-024-00946-2.

Recommendations for Development and Validation of a Fit-For-Purpose Biomarker Assays Using Western Blotting; An-AAPS Sponsored Initiative to Harmonize Industry Practices

Arvind Kinhikar  1 , Mohamed Hassanein  2 , Jake Harman  3 , Catherine Soderstrom  4 , Kimberly Honrine  5 , Amy Lavelle  6 , Marie-Anne Valentin  7 , Joel Mathews  8
Affiliations

Recommendations for Development and Validation of a Fit-For-Purpose Biomarker Assays Using Western Blotting; An-AAPS Sponsored Initiative to Harmonize Industry Practices

Arvind Kinhikar et al. AAPS J. .

Abstract

Western blot (WB) assays are routinely used for detection and quantification of biomarkers. Although assay validation to measure biomarkers in complex matrices has become a mainstay process for ligand binding assays (LBA) and mass spectrometry (MS), no guidelines exist yet validate biomarker methods using WB techniques. In this cross-industry white paper, we outlined in detail the key steps for development and for validation of WB assays for protein biomarkers under different contexts of use (COU). In addition, we described how to determine the level of assay validation needed for biomarker assays using Western blotting. For simplicity, we described two paths of WB assay validation. The first path (Path 1) is for biomarkers being analyzed for exploratory research or for internal go- or no/go- decision making. The second path (Path 2) is for clinical decision making such as dose determination or drug response that need to be run in a regulated environment. This work is supported through AAPS Biomarkers and Precision Medicine subteam and represents AAPS members opinion.

Keywords: Antibodies; Assay; Biomarker; Validation; Western blot.

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References

    1. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76(9):4350–4. - PubMed - PMC - DOI
    1. Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, Farquharson C, Gillingwater TH, et al. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PLoS ONE. 2013;8(8):e72457. - PubMed - PMC - DOI
    1. Ni D, Xu P, Gallagher S. Immunoblotting and immunodetection. Curr Protoc Cell Biol. 2017;74(6 2 1–6 2):37.
    1. Taylor SC, Berkelman T, Yadav G, Hammond M. A defined methodology for reliable quantification of Western blot data. Mol Biotechnol. 2013;55(3):217–26. - PubMed - PMC - DOI
    1. Chen GF, Zhang CY, Wang YY, Chen W. Application of reverse dot blot hybridization to simultaneous detection and identification of harmful algae. Environ Sci Pollut Res Int. 2015;22(14):10516–28. - PubMed - DOI

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