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


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.
Pfleger
Christian
C
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
Schloot
Nanette
N
ter Veld
Frank
F
D001798:Blood Proteins / Q000737:chemistry*; D002138:Calibration; D016207:Cytokines / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005434:Flow Cytometry / Q000592:standards; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D015203:Reproducibility of Results
D001798:Blood Proteins; D016207:Cytokines; D007202:Indicators and Reagents