Accuracy and reproducibility of a multiplex immunoassay platform: a validation study

Abstract

Background: Multiplex immunoassays offer many advantages over singleplex assays for the analysis of multiple analytes in a single sample. We sought to validate a specific multiplex cytokine immunoassay (Human 9-plex cytokine array on the Searchlight® platform by Thermoscientific) prior to use in a large clinical study.

Methods: We compared spike and recovery of recombinant proteins on the Searchlight® platform to singleplex immunoassays purchased from R&D Systems, measured identical patient samples on the two different platforms, and measured identical patient samples on different days to measure intra- and inter-assay variability.

Results: Assays using the Searchlight® platform had inefficient recovery of spiked recombinant proteins compared to R&D Systems singleplex assays. Assaying identical patients samples on different days on the Searchlight platform had acceptable intra-assay variability (intra-assay coefficient of variation (CV%) range for all analytes of 9.1-13.7) but unacceptably high inter-assay variability (CV% range for all analytes 16.7-119.3) suggesting plate-to plate variability. Similar assays for individual cytokines on the R&D platform had an intra-assay CV% range of 1.6-6.4 and an inter-assay CV% range of 3.8-7.1. Some deficiencies in Searchlight® assay performance may be due to irregularities in spotting of capture antibodies during manufacturing.

Conclusions: We conclude that the Searchlight® multiplex immunoassay platform would require extensive additional assay optimization prior to widespread clinical research use.

Figure 1. Intra-well spot irregularities

Digital images of representative duplicate wells in a Searchlight® human 9-plex cytokine kit. Panel A shows an ideal well with each spot visible and no overlap of spots. Panel B shows intense signal of one analyte spot (arrow) with the luminescence of that spot bleeding over into an adjacent spot. Panel C shows the comet effect (arrow) with the signal from one spot interfering with an adjacent spot. Panel D shows capture antibody drop-out (arrow) in a pair of duplicate wells. Panel E shows halos around the edge of wells affecting a large portion of the plate (circled area).

Figure 2. Variability in analyte values in control human plasma

Identical aliquots of pooled control human plasma with the same number of freeze thaw cycles were run on seven lot matched Searchlight® plates. The individual biomarker and the corresponding CV% are noted on the x-axis with the value of each of the seven individual marker measurements displayed on the y-axis.

Figure 3. Spike and recovery of R & D proteins on the R & D platform and Searchlight proteins on Searchlight platform

Average values of recombinant proteins spiked into control human plasma for all 9 analytes. Each graph shows the expected concentration of the sample (spike, x-axis) and the measured concentration of the sample (recovery, y-axis). R&D data is plotted on the left and Searchlight data is plotted on the left.

Figure 4. Patient samples assayed on the two platforms

Average values for 5 analytes measured in 10 patient samples on both platforms. R&D values are shown on the x-axis, Searchlight values are shown on the y-axis. Diagonal line represents 100% agreement between the two platforms.