The xMAP™ technique can be used for detection of the inflammatory cytokines IL-1β, IL-6 and TNF-α in bovine samples

Abstract

Infectious diseases can cause large health problems in cattle. The infections cause an acute inflammatory response, mediated by pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. By mapping the pattern of cytokines during inflammations, valuable information about the course of an infection is gained. The aim of the present study was to evaluate a particle-based flow cytometric method, the xMAP TM technique, using ovine/bovine reagents, for quantification of IL-1β, IL-6 and TNF-α, for application in studies on ruminant infectious diseases with emphasis on bovine milk and plasma samples. Singleplex, duplex and triplex xMAP™ assays were evaluated, and limits of detection (LODs) as well as intra- and inter-assay variabilities were determined for each assay. Cross-reactivity between reagents in multiplex assays was also tested. In addition, presence of cytokines in milk and plasma samples from healthy and mastitic cows was studied. The LODs were significantly lower for singleplex xMAP™ assays than for duplex and triplex assays. In singleplex assays, the LODs were 0.08, 0.2 and 0.5 ng/ml, for IL-1β, IL-6 and TNF-α, respectively. Corresponding LODs in triplex assays were 2.0, 6.5 and 3.5 ng/ml. Data indicate that the linear ranges of the multiplex assays were narrower than in singleplex assays. The intra-assay coefficients of variation were ≤ 10.7% for singleplex assays, while they ranged from 6.2 to 23.2% in the triplex assay. The inter-assay variance ranged from 5.1 to 35.8% in singleplex assays, and from 8.8 to 78.4% in triplex assays. Cross-reactivity between reagents was not observed, and all three cytokines were detected in bovine milk and plasma samples collected from cows with clinical mastitis. In conclusion, our results show that the xMAP™ technique can be used for quantification of IL-1β, IL-6 and TNF-α in bovine samples, and that further work is required to optimize the multiplex assays.