title: Effect of serum content and diluent selection on assay sensitivity and signal intensity in multiplex bead-based immunoassays.
abstract: 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.
authors.affiliation: 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
authors.forename: Christian
authors.initials: C
authors.lastname: Pfleger
authors.forename: Nanette
authors.initials: N
authors.lastname: Schloot
authors.forename: Frank
authors.initials: F
authors.lastname: ter Veld
mesh_terms: 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
chemical_list: D001798:Blood Proteins; D016207:Cytokines; D007202:Indicators and Reagents