Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Denmark.
PLoS One. 2012;7(6):e39228. doi: 10.1371/journal.pone.0039228. Epub 2012 Jun 27.
Antigen 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.
To 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.
We 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.
The 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 (r(2)>0.97).
This 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.
IP-10 的抗原特异性释放是感染结核分枝杆菌的既定标志物。与 IFN-γ 相比，IP-10 的释放浓度高出 100 倍，这使得开发新的检测方法成为可能。干血斑是高通量新生儿筛查的方便样本。
开发一种基于 ELISA 的灵敏且稳健的检测方法，用于检测血浆、干血斑（DBS）和干血浆斑（DPS）中的 IP-10；验证 ELISA 在临床相关样本中的性能；并评估该检测方法对巨细胞病毒（CMV）和结核分枝杆菌特异性免疫反应的检测性能。
我们用针对人 IP-10 的小鼠和大鼠单克隆抗体进行免疫，并开发了一种 ELISA。对该检测方法进行了验证，并应用于检测 18 例对结核分枝杆菌具有免疫反应性的患者和 32 例健康对照者的 CMV 和结核分枝杆菌特异性反应，其中 22 例对 CMV 具有免疫反应性，而对结核分枝杆菌均无免疫反应性。我们比较了该新检测方法与 IFN-γ 的性能。
该 ELISA 可可靠地检测血浆和滤纸片样本中的 IP-10。该 ELISA 的线性范围为 2.5-600pg/ml。DPS 样本中不易检测到 IFN-γ。在 37°C 下，IP-10 在滤纸片样本中至少稳定 4 周。从抗原刺激和非刺激的全血中，血浆、DPS 和 DBS 样本中检测到的 IP-10 之间的相关性非常好（r(2)>0.97）。
该新开发的检测方法可可靠地定量检测来自抗原刺激和非刺激全血的血浆、DBS 和 DPS 样本中的 IP-10。滤纸检测法可在环境温度下（例如通过邮寄系统）轻松获取和运输样本。该系统可能会简化结核分枝杆菌和 CMV 感染的诊断检测。
