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

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.

Introduction

There is increasing demand for multiplexed immunoassay systems that allow multiple cytokine determination by using a small amount of samples (Kurkjian et al., 2006, Martins et al., 2006, Nolan and Mandy, 2006, Skogstrand et al., 2005).

The Luminex-100 bead-based system combines the principle of ELISA and fluorescent bead-based flow cytometry and is theoretically capable of measuring up to 100 analytes simultaneously.

An important factor in multiplex assay optimization is the sample diluent; a protein mixture that minimizes disturbing influences such as heterophilic antibodies and unspecific binding. In addition, the diluent optimizes cytokine detection by sample dilution and, at the same time, mimics the sample matrix in the calibration curve (i.e. human serum) to allow accurate interpolation of unknown sample response in an external calibration curve. Diluents, of course, lack potentially disturbing factors such as endogenous cytokines and heterophilic antibodies (de Jager et al., 2005, Muller et al., 2002).

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. We present several methods to determine the quality of a diluent. As an example we used two widely used diluents from R&D Systems; DY997 and RD6. We demonstrate the importance of using a diluent that adequately imitates the sample matrix.