Interference in microsphere flow cytometric multiplexed immunoassays for human cytokine estimation.
The present study describes positive and negative interference of human cytokine measurement in multiplexed bead-based immunoassays. Significant differences in measured IL-6 and TNF-alpha values in 30 normal human plasma samples were apparent depending on whether measurements were with a 2-plex assay or embedded in a multiplex of 8-or more cytokine antibody pairs, as well as among the kits of 3-different vendors. Sample diluents containing proprietary blocking ingredients were shown to greatly affect the outcome of measured cytokine values. Additionally, recovery of IL-6 and TNF-alpha from spiked samples suggests significant negative interference from either endogenous antibodies, soluble receptors or anti-cytokine antibodies in 10% and 26% of samples, respectively. While it is evident that multiplexed immunoassays hold great promise for cytokine profiling, there are still important issues needing further study. Especially needed are universally optimized sample diluents, uniformly calibrated standards with mass values, and internal assay controls, which should greatly facilitate intralaboratory accuracy and precision and interlaboratory comparisons of cytokine measurements. Possible causes of interference and remedies are discussed.
Phillips
Donald J
DJ
Centers for Disease Control and Prevention, National Center on Birth Defects and Development Disabilities, Mail Stop D02, 1600 Clifton Rd., NE, Atlanta, GA 30333, USA.
League
Stacy C
SC
Weinstein
Paula
P
Hooper
W Craig
WC
D017077:Culture Media, Conditioned / Q000737:chemistry; D016207:Cytokines / Q000032:analysis / Q000097:blood*; D005188:False Negative Reactions; D005189:False Positive Reactions; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods / Q000592:standards; D015850:Interleukin-6 / Q000032:analysis / Q000097:blood; D008863:Microspheres; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000097:blood; D020298:U937 Cells
D017077:Culture Media, Conditioned; D016207:Cytokines; D015850:Interleukin-6; D011933:Reagent Kits, Diagnostic; D014409:Tumor Necrosis Factor-alpha