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.