Su J L, Schumacher B L, Lindley K M, Soloveychik V, Burkhart W, Triantafillou J A, Kuettner K, Schmid T
Department of Protein Sciences, GlaxoSmithKline, Research Triangle Park, NC 27709, USA.
Hybridoma. 2001 Jun;20(3):149-57. doi: 10.1089/027245701750293475.
In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.
在本报告中，我们描述了人浅表区蛋白（SZP）的纯化、跨物种单克隆抗体（MAb）的产生以及该蛋白在人和动物体液中的检测。用作免疫抗原的人SZP，要么从人软骨器官培养物的培养基中纯化，要么从人滑液中纯化。免疫抗原与RIBI佐剂以三种形式之一混合：未修饰的SZP、浅表区蛋白-钥孔戚血蓝蛋白缀合物（SZP-KLH）或浅表区蛋白与透明质酸的混合物（SZP-HA）。产生并鉴定了一组包括GW4.23、S6.79、S13.52、S13.233和S17.109的单克隆抗体。单克隆抗体（MAb）S6.79特别值得关注，它是一种来自SZP-KLH免疫的IgG2b，解离常数（K(D)）为3.14×10(-9) M。在酶联免疫吸附测定（ELISA）和蛋白质印迹法中，它都能与大分子形式的SZP发生强烈反应。在免疫组织化学中，它能使关节软骨的最表层染色，而软骨的中层和深层则不着色。当将MAb S6.79应用于人体体液的蛋白质印迹时，在滑液样本中检测到一条强的345 kDa条带，在血浆和血清样本中检测到大小相似的较弱条带。通过蛋白质印迹和抗体吸收实验证明，MAb S6.79在从牛、狗、豚鼠和兔子采集的滑液样本中也与SZP表现出跨物种免疫反应性。这种跨物种单克隆抗体将成为人和动物模型研究中监测SZP水平和组织分布的有用工具。它可能有助于确定SZP在正常关节中的作用，并且对于骨关节炎、类风湿性关节炎和先天性屈曲挛缩-关节病-髋内翻-心包炎等病理状况下SZP的测量可能具有诊断或预后价值。
