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Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum.

Abstract：

:Quantification of biomarkers can provide important information about the safety and efficacy of candidate drugs. Unfortunately, limited sample volume and excess costs often limit analysis of multiple biomarkers. We developed, optimized, validated, and implemented a multiplex immunoassay for simultaneous measurement of multiple circulating cytokines: IL-1beta, TNFalpha, IL-6, IL-8, and IL-10. Multiplex immuoassays were performed using the Luminex LabMAP instrument. Capture antibodies for each cytokine were covalently bound to distinct microsphere subsets distinguished by differing dye ratios. The concentration of each individual cytokine determined by measuring orange fluorescence produced by a complex of a biotinylated cytokine-specific antibody and streptavidin-phycoerythrin. The lower limit of quantification for all assays was 20 pg/mL with the exception of IL-8 which was 100 pg/mL. The inter-assay precision was less than 25%CV for all analytes at all control levels both pre-study and in-study. The percent recovery ranged from 83 to 108% pre-study and 90 to 125% in-study. In a linearity assessment, a 15,000 pg/mL multi-analyte control could be diluted 1:50 and maintain expected accuracy. We measured the cytokine concentrations in more than 2000 serum samples from patients with sepsis. Multiplex results for IL-6 were compared to a conventional commercially available ELISA kit. The degree of agreement between the two methods as measured by the concordance correlation coefficient was 84.5%. Multiplex results were 2.36-fold higher than ELISA values on the average. After adjusting for this mean difference, the 95% empirical limits of agreement for the ratio of individual sample values were 0.33, 2.65. This multiplex immunoassay provided simultaneous measurement of circulating cytokines using 80% less patient specimen compared to traditional approaches and at a significantly decreased cost. Efficient use of this platform requires process improvements to fully maximize the positive impact of multiplex assays in clinical drug development.

journal_name

J Pharm Biomed Anal

journal_title

Journal of pharmaceutical and biomedical analysis

authors

Ray CA,Bowsher RR,Smith WC,Devanarayan V,Willey MB,Brandt JT,Dean RA

doi

10.1016/j.jpba.2004.05.024

subject

Has Abstract

pub_date

2005-01-04 00:00:00

pages

1037-44

issue

eissn

0731-7085

issn

1873-264X

pii

S0731708504002559

journal_volume

pub_type

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Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum.