TY - JOUR

T1 - Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology

AU - Kofoed, Kristian

AU - Schneider, Uffe Vest

AU - Scheel, Troels

AU - Andersen, Ove

AU - Eugen-Olsen, Jesper

PY - 2006/7

Y1 - 2006/7

N2 - BACKGROUND: Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently used markers.METHODS: We used a combination of in-house and commercially available multiplex immunoassays based on Luminex xMAP technology to assay biomarkers of potential interest in EDTA-plasma samples.RESULTS: A 3-plex assay for soluble urokinase plasminogen activator receptor (suPAR), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1beta (IL-1beta), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-alpha human cytokine panel. No cross-reactivity was observed when the assays were combined. Correlation between values obtained with the 8-plex, the 5-cytokine panel, the 3 in-house 1-plex assays, and a suPAR ELISA ranged from 0.86 to 0.99. Mean within- and between-run CVs were 8.0% and 11%, respectively. Recoveries of suPAR, sTREM-1, and MIF calibrators were 108%, 88%, and 51%, respectively. In plasma collected from 10 patients with bacterial sepsis confirmed by blood culture, the assay detected significantly increased concentrations of all 8 analytes compared with healthy controls.CONCLUSIONS: A commercially available xMAP panel can be expanded with markers of interest. The combined multiplex assay can measure the 8 analytes with high reproducibility. The xMAP technology is an appealing tool for assaying conventional cytokines in combination with new markers.

AB - BACKGROUND: Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently used markers.METHODS: We used a combination of in-house and commercially available multiplex immunoassays based on Luminex xMAP technology to assay biomarkers of potential interest in EDTA-plasma samples.RESULTS: A 3-plex assay for soluble urokinase plasminogen activator receptor (suPAR), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1beta (IL-1beta), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-alpha human cytokine panel. No cross-reactivity was observed when the assays were combined. Correlation between values obtained with the 8-plex, the 5-cytokine panel, the 3 in-house 1-plex assays, and a suPAR ELISA ranged from 0.86 to 0.99. Mean within- and between-run CVs were 8.0% and 11%, respectively. Recoveries of suPAR, sTREM-1, and MIF calibrators were 108%, 88%, and 51%, respectively. In plasma collected from 10 patients with bacterial sepsis confirmed by blood culture, the assay detected significantly increased concentrations of all 8 analytes compared with healthy controls.CONCLUSIONS: A commercially available xMAP panel can be expanded with markers of interest. The combined multiplex assay can measure the 8 analytes with high reproducibility. The xMAP technology is an appealing tool for assaying conventional cytokines in combination with new markers.

KW - Biological Markers

KW - Chemotactic Factors

KW - Cross Reactions

KW - Granulocyte-Macrophage Colony-Stimulating Factor

KW - Humans

KW - Immunoassay

KW - Interleukin-1

KW - Interleukin-6

KW - Interleukin-8

KW - Macrophages

KW - Membrane Glycoproteins

KW - Receptors, Cell Surface

KW - Receptors, Immunologic

KW - Receptors, Urokinase Plasminogen Activator

KW - Sepsis

KW - Tumor Necrosis Factor-alpha

U2 - 10.1373/clinchem.2006.067595

DO - 10.1373/clinchem.2006.067595

M3 - Journal article

C2 - 16690735

VL - 52

SP - 1284

EP - 1293

JO - Clinical Chemistry

JF - Clinical Chemistry

SN - 0009-9147

IS - 7

ER -