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J Immunol Methods. 2008 Jan 1;329(1-2):214-8. Epub 2007 Oct 22.

Effect of serum content and diluent selection on assay sensitivity and signal intensity in multiplex bead-based immunoassays.

Author information

1
Institute 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

Abstract

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.

PMID:
17964594
DOI:
10.1016/j.jim.2007.09.011
[Indexed for MEDLINE]

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