Interference in microsphere flow cytometric multiplexed immunoassays for human cytokine estimation.

Area of Science:

• Immunology
• Biochemistry
• Assay Development

Background:

• Multiplexed immunoassays are valuable for simultaneous measurement of multiple cytokines.
• Accurate cytokine quantification is essential for understanding biological processes and disease states.

Purpose of the Study:

• To investigate positive and negative interference in human cytokine measurements using multiplexed bead-based immunoassays.
• To identify factors affecting the accuracy and precision of IL-6 and TNF-alpha quantification.

Main Methods:

• Analysis of 30 normal human plasma samples using 2-plex and multiplexed assays from three vendors.
• Evaluation of sample diluents and assessment of interference from endogenous factors in spiked samples.

Main Results:

• Significant differences in IL-6 and TNF-alpha measurements were observed based on assay multiplexity and vendor.
• Proprietary blocking ingredients in sample diluents impacted measured cytokine values.
• Endogenous antibodies, soluble receptors, or anti-cytokine antibodies caused negative interference in 10-26% of samples.

Conclusions:

• Multiplexed immunoassay performance for cytokine profiling is affected by various interference factors.
• Further research is needed for universally optimized sample diluents, calibrated standards, and internal assay controls.
• Standardization is key to improving inter- and intralaboratory accuracy and precision in cytokine measurements.