A pilot study to evaluate the application of a generic protein standard panel for quality control of biomarker detection technologies

Abstract

Background: Protein biomarker studies are currently hampered by a lack of measurement standards to demonstrate quality, reliability and comparability across multiple assay platforms. This is especially pertinent for immunoassays where multiple formats for detecting target analytes are commonly used.

Findings: In this pilot study a generic panel of six non-human protein standards (50 - 10^7 pg/mL) of varying abundance was prepared as a quality control (QC) material. Simulated "normal" and "diseased" panels of proteins were prepared in pooled human plasma and incorporated into immunoassays using the Meso Scale Discovery® (MSD®) platform to illustrate reliable detection of the component proteins. The protein panel was also evaluated as a spike-in material for a model immunoassay involving detection of ovarian cancer biomarkers within individual human plasma samples. Our selected platform could discriminate between two panels of the proteins exhibiting small differences in abundance. Across distinct experiments, all component proteins exhibited reproducible signal outputs in pooled human plasma. When individual donor samples were used, half the proteins produced signals independent of matrix effects. These proteins may serve as a generic indicator of platform reliability.Each of the remaining proteins exhibit differential signals across the distinct samples, indicative of sample matrix effects, with the three proteins following the same trend. This subset of proteins may be useful for characterising the degree of matrix effects associated with the sample which may impact on the reliability of quantifying target diagnostic biomarkers.

Conclusions: We have demonstrated the potential utility of this panel of standards to act as a generic QC tool for evaluating the reproducibility of the platform for protein biomarker detection independent of serum matrix effects.

Figure 1

Evaluation of cross-reactivity associated with the panel of protein standards. Uniplexed assays for each analyte: (A) CCL6, (B) lungkine, (C) caronte, (D) soggy, (E) luciferase and (F) lysozyme were performed, incorporating the omission of a single protein from the complete mixture of six protein QC material, to evaluate the contribution of each analyte to the overall mean signal output. The data points denote the mean values for each experiment, for day 1 (blue rhombuses), day 2 (pink squares) and day 3 (green triangles), and the error bars represent the SD from triplicate determinants.

Figure 2

Evaluation of the utility of each spike protein in single donor plasma samples. The mean signal output of (A) CCL6, (B) lungkine, (C) caronte, (D) soggy, (E) luciferase and (F) lysozyme from twelve single donor plasma samples supplemented to comprise the 1× stock of the QC material were determined from three separate experiments with n = 3. The data points denote the mean values for experiment 1 (blue rhombuses), experiment 2 (pink squares) and experiment 3 (green triangles), and the error bars represent the SD.

Figure 3

Evaluation of the reproducibility of IL-8 detection. IL-8 detection of the twelve single donor plasma samples, are displayed as (A) the mean signal output and as (B) interpolated concentrations of IL-8 from the internal standard curves. These data points (blue rhombuses) are the mean values of triplicate experiments, incorporating triplicate replicate determinants. The error bars denote the SD of the three separate experiments.