RESUMEN
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.
Asunto(s)
Biomarcadores , Western Blotting , Biomarcadores/análisis , Humanos , Western Blotting/normas , Industria Farmacéutica/normas , Reproducibilidad de los ResultadosRESUMEN
Aim: Globally, neurodegeneration accounts for significant morbidity and mortality among the elderly. Millions of people are afflicted with neurodegenerative diseases, with the most notable cases attributed to Alzheimer's, Huntington's, amyotrophic lateral sclerosis and Parkinson's diseases. Sensitive assays that can detect proteopathic anomalies indicative of early neurodegeneration have remained elusive. Therefore, there is an urgent need for sensitive diagnostic and prognostic biomarker assays that can guide the therapeutic regimen in the clinic. Materials & methods: Single molecule array digital immunoassay platform has sensitivity about 1000-fold higher than traditional ligand binding assays. Consequently, we are now beginning to implement ultrasensitive techniques in bioanalysis. Conclusion: In the current study, we evaluated single molecule array technology and report specifications to quantitate neurofilament light chain, a bona-fide biomarker for neurodegeneration. Preliminary neurofilament light screening results from 100 human geriatric cerebrospinal fluid samples displayed huge biological variation and warrants further investigation.
Asunto(s)
Inmunoensayo/métodos , Pruebas Inmunológicas/métodos , Proteínas de Neurofilamentos/metabolismo , HumanosRESUMEN
The 5th GCC in Barcelona (Spain) and 6th GCC in San Antonio (TX, USA) events provided a unique opportunity for CRO leaders to openly share opinions and perspectives, and to agree upon recommendations on biomarker bioanalytical method validation.