Anti-lubricin monoclonal antibodies created using lubricin-knockout mice immunodetect lubricin in several species and in patients with healthy and diseased joints.
Anti-lubricin monoclonal antibodies created using lubricin-knockout mice immunodetect lubricin in several species and in patients with healthy and diseased joints.
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DOI：
10.1371/journal.pone.0116237
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Warman ML
中科院分区：
文献类型：
--
作者：
Ai M;Cui Y;Sy MS;Lee DM;Zhang LX;Larson KM;Kurek KC;Jay GD;Warman ML
由PRG 4基因编码的润滑素是关节中的主要润滑剂。我们用纯化的人润滑素免疫遗传缺陷的小鼠（Prg 4-/-），并产生了几个单克隆抗体。我们使用生物样品和重组润滑素亚结构域确定每个mAb的结合表位、灵敏度和特异性，并且我们还开发了竞争ELISA测定来测量滑液和血液中的润滑素。我们发现这些单克隆抗体都能识别含有O-连接寡糖的表位，这些寡糖与肽基序KEPAPTTT偶联。通过western blot，mAb在1 μl来自几种动物物种（包括人）的滑液中检测到润滑素。这些单克隆抗体对润滑素是特异性的，因为它们不与来自患有弯曲指关节病-髋内翻-心包炎综合征（CACP）的患者的其他滑液成分发生交叉反应，CACP患者在遗传上缺乏这种蛋白质。竞争ELISA检测润滑素的血液样品中的健康人，但没有从患者与CACP，表明血液可用于诊断测试怀疑有CACP的患者。血液中的润滑素表位不代表滑液的降解片段。因此，尽管血液润滑素水平不能区分炎性关节疾病患者与健康对照，但表位特异性抗润滑素mAb可用于监测滑液中的疾病活动。
Lubricin, encoded by the gene PRG4, is the principal lubricant in articulating joints. We immunized mice genetically deficient for lubricin (Prg4-/-) with purified human lubricin, and generated several mAbs. We determined each mAb’s binding epitope, sensitivity, and specificity using biologic samples and recombinant lubricin sub-domains, and we also developed a competition ELISA assay to measure lubricin in synovial fluid and blood. We found the mAbs all recognized epitopes containing O-linked oligosaccharides conjugated to the peptide motif KEPAPTTT. By western blot, the mAbs detected lubricin in 1 μl of synovial fluid from several animal species, including human. The mAbs were specific for lubricin since they did not cross-react with other synovial fluid constituents from patients with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP), who genetically lack this protein. The competition ELISA detected lubricin in blood samples from healthy individuals but not from patients with CACP, indicating blood can be used in a diagnostic test for patients suspected of having CACP. Lubricin epitopes in blood do not represent degradation fragments from synovial fluid. Therefore, although blood lubricin levels did not differentiate patients with inflammatory joint disease from healthy controls, epitope-specific anti-lubricin mAbs could be useful for monitoring disease activity in synovial fluid.
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