Neurofilament ELISA validation

Abstract

This multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online.

Multicenter Immunoassay Validation of Cerebrospinal Fluid Neurofilament Light: A Biomarker for Neurodegeneration

“…In agreement with our observations, Petzold and colleagues also observed variations in the concentration of standards and their associated optical densities (OD) 26 . They noted that nonsystematic error due to variations in the preparation of standards could be responsible for up to twofold differences in sample concentration, and identified differences in the accuracy and types of pipettes used to make the standards and dispense samples as possible sources of variation.…”

“…Nonetheless, despite the variation the data do point to the positive effects of implementation of a standardised protocol in that no one laboratory reported consistently high or low values and discrepancies seem to be spread out among the laboratories. In support of the positive effect of implementation of validation protocols a previous multi-centre study, albeit with greater numbers of participating laboratories, which did not use standardised protocols in the validation of the UmanDiagnostics NfL ELISA reported a significantly higher inter-laboratory precision CV of 59% 26 .…”

“…Alternatively, failure to observe parallelism, and indeed some of the other validation parameters, could be in part due to some of the less practiced operators experiencing difficulty with interpretation or complexity of the plate plans and SOPs. It has been argued that as NfL has the tendency to re-aggregate following suspension [39][40][41][42] that delays in sample and standard incubation could account for the failure to satisfactorily measure NfL in many experiments 26 . The data show that between 15 and 60 minutes were taken, from the time standards were reconstituted, to incubate the standards and samples on the ELISA plates.…”

“…To date attempts at investigating the performance of assays for amyloid and neurodegenerative biomarkers of AD have proven to be problematic with unacceptable levels of inter-laboratory variation being reported 24,25 . More recently, a multicentre NfL ELISA assay validation experiment, which examined whether parameters such as pipette calibration, duration of colorimetric development or delay in analysis could contribute to the observed high levels of inter-laboratory variation reported that disparity in the accuracy of standard preparation, contributed most significantly to the observed large inter-laboratory coefficient of variation (59%) in that particular experiment 26 .…”

Proteome signatures—how are they obtained and what do they teach us?

“…Traditionally, ELISA has been the most used method for targeted protein quantification and remains the gold standard to date (Pan et al 2009), providing good specificity and sensitivity. If high-quality antibodies exist, the validation of these unique patterns of protein expression can be a relatively straightforward process (Köhler and Seitz 2012;Petzold et al 2010), although it remains, to this day, the bottleneck in biomarker research (Diamandis 2012a). However, for many of the most recently discovered candidates in proteomic studies, ELISA is still limited by the lack of availability of highly specific antibodies.…”