Related Experiment Video
Updated: Jul 10, 2026
Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens
Published on: September 12, 2016
Effect of serum content and diluent selection on assay sensitivity and signal intensity in multiplex bead-based
Christian Pfleger1, Nanette Schloot, Frank ter Veld
1Institute for Clinical Diabetes Research, German Diabetes Center, Leibniz Institute at the Heinrich-Heine-University, Auf'm Hennekamp 65, D-40225 Duesseldorf, Germany. ch.pfleger@web.de
Insights
Choosing the right diluent is crucial for Luminex multiplex immunoassays. RD6 demonstrated superior performance over DY997, showing less signal reduction with increased human serum concentrations in cytokine determination.
Area of Science:
- Biochemistry
- Immunology
- Analytical Chemistry
Background:
- Complex biological samples like human serum require dilution in multiplexed immunoassays to mitigate matrix effects.
- Diluents must mimic the sample matrix for accurate interpolation and ensure optimal binding for all target analytes.
- The choice of diluent significantly impacts multiplex immunoassay results, necessitating quality assessment methods.
Purpose of the Study:
- To evaluate and compare the performance of two commercial diluents (DY997 and RD6) in a Luminex 19-plex immunoassay.
- To assess the impact of human serum concentration on signal intensity and assay sensitivity using different diluents.
- To determine the most appropriate diluent for cytokine analysis in human serum samples using the Luminex system.
Main Methods:
- A 19-plex immunoassay was performed using the Luminex system.
- Two commercial diluents, DY997 and RD6, were tested.
- Spiked samples with varying concentrations of human serum (25% and 50% v/v) and protein were analyzed.
Main Results:
- Diluent DY997 showed a 55% reduction in signal intensity with 50% human serum compared to 25% serum.
- Diluent RD6 exhibited only a 20% signal intensity reduction with 50% human serum compared to 25% serum.
- DY997 demonstrated decreased assay sensitivity with increasing protein concentrations, more so than RD6.
Conclusions:
- Diluent RD6 is more suitable than DY997 for cytokine determination in human serum using the Luminex multiplexed immunoassay system.
- The study highlights the importance of diluent selection for maintaining signal intensity and assay sensitivity in complex matrices.
- Performance metrics including signal intensity, assay sensitivity, and background levels are influenced by serum volume-fraction and protein concentration.
Introduction:
In the Luminex multiplexed immunoassay system, complex samples such as human serum are diluted to minimize disturbing matrix effects with a specific diluent. This diluent has to imitate the sample matrix to allow interpolation and has to provide optimal cytokine-antibody binding for all cytokines. Because diluents influence multiplex immunoassay results, this paper explores several methods to determine the quality of a chosen diluent.
Materials And Methods:
Two commercially available diluents, DY997 and RD6 from R&D Systems, were compared in a 19-plex immunoassay setup from Luminex.
Results:
Using diluent DY997, multiplex signal intensity was reduced by 55% when spiked samples (chemokines and cytokines at 100 pg/mL) contained 50% v/v human serum, compared to samples containing 25% v/v. When using diluent RD6, signal intensity was reduced by 20% when samples contained 50% v/v human serum, compared to 25% v/v human serum. Diluent DY997 showed decreasing multiplex assay sensitivity with increasing protein concentrations, but not as low as in the presence of 50% v/v human serum.
Conclusions:
In a 19-plex setup, this paper describes signal intensity, assay sensitivity and background signal levels in relation to the total volume-fraction of serum and protein concentration. For the determination of cytokines in serum samples with the multiplexed system Luminex the diluent RD6 seems more appropriate than the diluent DY997.
