Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples

Abstract

Background: Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance.

Methods: In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BAL) and plasma) samples.

Results: We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD were 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5 to 28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations.

Conclusions: In conclusion, we validated a multiplex electrochemiluminescent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISAs.

Keywords: Biomarkers; Bronchoalveolar lavage; ELISA; Immunoassay; Plasma.

Figure 1. Comparison of patient samples run on MSD and R&D platforms

Individual patient values are represented as open circles with the cytokine value from R&D plotted on the x axis and the value from MSD on the y axis. Diagonal line represents perfect correlation. R=0.97 for IL-6 and 0.97 for IL-8.

Figure 2

Intra-assay CV percent as a function of concentration. Mean IL-6 or IL-8 concentration from R&D (left panels) and MSD (right panels) is plotted on the x axis and CV% is plotted on the y axis. In general, the lowest protein concentrations have the highest CV%.

Figure 3. Spile and recovery for human validation

Recombinant protein standards form each platform were spiked into control plasma and percent recovery was measured. Black circles represent MSD proteins recovered on the MSD platform, red circles represent R&D proteins recovered on the R&D platform and blue circles represent R&D proteins recovered on MSD platform. Duplicates of each measurement are represented as circles connected by a vertical line.

Figure 4. Comparison of mouse plasma (red) and BAL (black) samples run on MSD and R&D platforms

Individual values are represented as dots with the cytokine value from R&D plotted on the x axis and the value from MSD on the y axis. Diagonal line represents perfect correlation.

Figure 5

Intra-assay CV percent as a function of concentration. Mean cytokine concentration from MSD is plotted on the x axis and CV% is plotted on the y axis. In general, the lowest protein concentrations have the highest CV%.

Figure 5

Intra-assay CV percent as a function of concentration. Mean cytokine concentration from MSD is plotted on the x axis and CV% is plotted on the y axis. In general, the lowest protein concentrations have the highest CV%.

Figure 6. Spike and recovery for mouse

Recombinant protein standards from MSD were spiked into control plasma (black circles) or BAL (red circles) and recovered values were measured. Diagonal line represents 100% recovery.

Figure 6. Spike and recovery for mouse

Recombinant protein standards from MSD were spiked into control plasma (black circles) or BAL (red circles) and recovered values were measured. Diagonal line represents 100% recovery.