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Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting
Published on: December 12, 2011
Accuracy and reproducibility of a multiplex immunoassay platform: a validation study
Julie A Bastarache1, Tatsuki Koyama, Nancy E Wickersham
1Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, United States. Julie.bastarache@vanderbilt.edu
Journal of Immunological Methods
|February 1, 2011
Summary
The Searchlight® multiplex immunoassay showed high inter-assay variability and poor recovery, indicating it needs optimization before clinical research. Singleplex assays performed better.
Area of Science:
- Biotechnology
- Immunology
- Assay Development
Background:
- Multiplex immunoassays enable simultaneous analysis of multiple analytes, offering advantages over singleplex assays.
- Validation of a specific multiplex cytokine immunoassay (Human 9-plex cytokine array on the Searchlight® platform) was performed before its use in a large clinical study.
Purpose of the Study:
- To validate the performance of the Searchlight® multiplex cytokine immunoassay.
- To compare its performance against traditional singleplex immunoassays.
Main Methods:
- Spike and recovery of recombinant proteins were compared between the Searchlight® platform and R&D Systems singleplex immunoassays.
- Identical patient samples were measured on both platforms and on different days to assess intra- and inter-assay variability.
Main Results:
- The Searchlight® platform exhibited inefficient recovery of spiked proteins compared to R&D Systems singleplex assays.
- While intra-assay variability on the Searchlight® platform was acceptable (CV% 9.1-13.7), inter-assay variability was unacceptably high (CV% 16.7-119.3), suggesting plate-to-plate inconsistencies.
- R&D Systems singleplex assays demonstrated significantly lower intra-assay (CV% 1.6-6.4) and inter-assay (CV% 3.8-7.1) variability.
Conclusions:
- The Searchlight® multiplex immunoassay platform demonstrated significant variability and recovery issues.
- Extensive assay optimization is required before the Searchlight® platform can be reliably used in widespread clinical research.
