Abstract
BackgroundAntigen specific release of IP-10 is an established marker for infection with M.tuberculosis. Compared to IFN-γ, IP-10 is released in 100-fold higher concentrations enabling the development of novel assays for detection. Dried blood spots are a convenient sample for high throughput newborn screening.AimTo develop a robust and sensitive ELISA-based assay for IP-10 detection in plasma, dried blood spots (DBS) and dried plasma spots (DPS); to validate the ELISA in clinically relevant samples; and to assess the performance of the assay for detection of Cytomegalovirus (CMV) and M.tuberculosis specific immune responses.MethodWe raised mice and rat monoclonal antibodies against human IP-10 and developed an ELISA. The assay was validated and applied to the detection of CMV and M.tuberculosis specific responses in 18 patients with immune reactivity towards M.tuberculosis and 32 healthy controls of which 22 had immune reactivity towards CMV and none towards M.tuberculosis. We compared the performance of this new assay to IFN-γ.ResultsThe ELISA was reliable for IP-10 detection in both plasma and filter paper samples. The linear range of the ELISA was 2.5–600 pg/ml. IFN-γ was not readily detectable in DPS samples. IP-10 was stabile in filter paper samples for at least 4 weeks at 37°C. The correlation between IP-10 detected in plasma, DPS and DBS samples was excellent (r2>0.97).ConclusionsThis newly developed assay is reliable for IP-10 quantification in plasma, DBS and DPS samples from antigen stimulated and non-stimulated whole blood. The filter paper assays enable easy sample acquisition and transport at ambient temperature e.g. via the postal system. The system can potentially simplify diagnostic assays for M.tuberculosis and CMV infection.
Highlights
The chemokine IP-10 (CXCL-10) is a small 7.2 Kd proinflammatory molecule, expressed by different cells including monocyte/macrophages, hepatocytes, endothelial cells, fibroblasts and astrocytes
The ELISA was reliable for IP-10 detection in both plasma and filter paper samples
IP-10 was stabile in filter paper samples for at least 4 weeks at 37uC
Summary
The chemokine IP-10 (CXCL-10) is a small 7.2 Kd proinflammatory molecule, expressed by different cells including monocyte/macrophages, hepatocytes, endothelial cells, fibroblasts and astrocytes. Elevated IP-10 levels have been associated with infectious diseases, immune dysfunction and tumour development [1,3,4,5], and can be used to monitor hepatitis C viral infection [6,7], tuberculosis [8,9,10,11] and malaria [12]. IP-10 is an inducible cytokine, and expression is stimulated at the transcription levels by multiple signals, mainly T cell derived IFN-c and TNF-a and IL-2, type II IFNs, IL-27, IL-17/IL-23 and IL-1b. IP-10 has emerged as a promising alternative read-out marker to IFN-c in cell mediated immune response (CMI) assays e.g. in the Quantiferon test (QFTTB, Qiagen, USA) used for diagnosis of infection with M.tuberculosis Dried blood spots are a convenient sample for high throughput newborn screening
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