Highly Specific and Sensitive Measurements of Human and Monkey Interleukin 21 Using Sequential Protein and Tryptic Peptide Immunoaffinity LC-MS/MS
Highly Specific and Sensitive Measurements of Human and Monkey Interleukin 21 Using Sequential Protein and Tryptic Peptide Immunoaffinity LC-MS/MS
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DOI：
10.1021/ac4006765
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发表时间：
2013-06-04
影响因子：
7.4
通讯作者：
Neubert, Hendrik
中科院分区：
文献类型：
--
作者：
Palandra, Joe;Finelli, Alyce;Neubert, Hendrik
开发、确认并实施了一种用于定量人和食蟹猴白细胞介素21（IL-21）的高特异性和灵敏度免疫亲和LC-MS/MS测定法。工作流程包括使用抗IL-21捕获抗体离线富集IL-21，然后使用磁珠分离，胰蛋白酶消化，使用抗肽抗体在线富集IL-21衍生的胰蛋白酶肽，在人和食蟹猴血清和组织中开发并确认了该测定法，定量下限为0.78 pg/ml。mL基于完整细胞因子。发现批内和批间精密度和准确度以及稳定性和回收率均可接受。自身免疫性疾病患者血清中未检测到IL-21。然而，在食蟹猴中定量IL-21水平。脾和结肠组织以及正常和炎性肠病（IBD）人结肠组织以及增生的人扁桃体。
A highly specific and sensitive immunoaffinity LC-MS/MS assay for quantification of human and cynomolgus monkey interleukin 21 (IL-21) was developed, qualified, and implemented. The workflow includes offline enrichment of IL-21 using an anti-IL-21 capture antibody, followed by isolation using magnetic beads, trypsin digestion, online enrichment of IL-21 derived tryptic peptides using antipeptide antibodies, and quantification using nanoflow LC-MS/MS. This assay was developed and qualified in human and cynomolgus monkey serum and tissues with a lower limit of quantitation of 0.78 pg/mL based on the intact cytokine. Both intra- and interbatch precision and accuracy, as well as stability and recovery, found to be acceptable. IL-21 was not detected in serum from normal healthy from autoimmune disease patients. However, IL-21 levels were quantified in cynomolgus monkey., spleen and colon tissue and normal and inflammatory bowel disease (IBD) human colon tissue as well as hyperplasia human tonsils.