Inducible T-cell costimulator (ICOS), a homodimeric protein expressed on the surface of activated T-cells, is being investigated as a potential therapeutic target to treat various cancers. Recent studies have reported aberrant increases in the soluble form of ICOS (sICOS) in human serum in disease-state patients, primarily using commercial ELISA kits. However, results from our in-house immunoassay did not show these aberrant increases, leading us to speculate that commercial sICOS ELISAs may be prone to interference. We directly tested that hypothesis and found that one widely used commercial kit yields false-positives and is prone to human anti-mouse antibody interference. We then analyzed a panel of healthy, cancer, chronic hepatitis C virus, systemic lupus erythematosus, and diffuse cutaneous systemic sclerosis human serum using our in-house immunoassay and reported the measured sICOS concentrations in these populations. Since even well characterized immunoassay methods are prone to non-specific interference, we also developed a novel sICOS LC-MS/MS method to confirm the results. Using these orthogonal approaches, we show that sICOS is a low abundance soluble protein that cannot be measured above approximately 20 pg/mL in human serum.

诱导型 T 细胞共刺激物 (ICOS) 是一种在活化 T 细胞表面表达的同源二聚体蛋白，正在研究作为治疗各种癌症的潜在治疗靶点。最近的研究报告了疾病状态患者的人血清中可溶形式的 ICOS (sICOS) 异常增加，主要使用商业 ELISA 试剂盒。然而，我们内部免疫测定的结果并未显示这些异常增加，这使我们推测商业 sICOS ELISA 可能容易受到干扰。我们直接测试了这一假设，发现一种广泛使用的商业试剂盒会产生假阳性，并且容易受到人类抗小鼠抗体的干扰。然后我们分析了一组健康、癌症、慢性丙型肝炎病毒、系统性红斑狼疮、和弥漫性皮肤系统性硬化症人血清使用我们的内部免疫测定并报告了在这些人群中测量的 sICOS 浓度。由于即使是特征良好的免疫测定方法也容易受到非特异性干扰，我们还开发了一种新的 sICOS LC-MS/MS 方法来确认结果。使用这些正交方法，我们表明 sICOS 是一种低丰度可溶性蛋白，在人血清中无法测量到高于约 20 pg/mL。