Vilím Vladimír, Vobůrka Zdenek, Vytásek Richard, Senolt Ladislav, Tchetverikov Ilja, Kraus Virginia B, Pavelka Karel
Institute of Rheumatology, Na Slupi 4, 128 50 Prague 2, Czech Republic.
Clin Chim Acta. 2003 Feb;328(1-2):59-69. doi: 10.1016/s0009-8981(02)00375-3.
Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP 5) is one of the most promising serologic markers with regard to an ability to prognose development of osteoarthritis (OA). Our aim was to map the epitopes of three monoclonal antibodies (mAb) to COMP and to develop and characterize a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring COMP levels in human body fluids.
COMP was digested with trypsin and the NH(2)-terminal sequence of the fragments recognized by each of the mAbs was determined. Steric competition among the mAbs was tested with an antibody capture assay. A sandwich ELISA was developed using unlabeled mAb 16-F12 as a capture antibody, and mAb 17-C10 labeled with biotin as the second antibody.
Epitopes of the three mAbs were mapped to three different domains within the COMP subunit (16-F12, NH(2)-terminal domain; 17-C10, EGF-like domain; 12-C4, COOH-terminal domain). These epitopes did not overlap. mAbs 17-C10 and 12-C4 yielded similar serum COMP results when used as the secondary antibodies. Serum COMP levels measured with the new sandwich ELISA using mAbs 16-F12 and 17-C10 correlated strongly with results based on an inhibition ELISA with mAb 17-C10 alone (r(2) = 0.836; P < 0.0001). We characterized the new sandwich ELISA with regards to inter- and intra-assay variability, the range of COMP levels that can be expected in human synovial fluids (SF) and sera (controls and OA and rheumatoid arthritis (RA) patients), and the day-to-day and diurnal variability of COMP levels in sera.
We have developed and characterized a sandwich ELISA for COMP that is sensitive and yields highly reproducible COMP results upon analysis of human sera and synovial fluids.
软骨寡聚基质蛋白/血小板反应蛋白5（COMP/TSP 5）是预测骨关节炎（OA）发展能力方面最有前景的血清学标志物之一。我们的目的是确定三种单克隆抗体（mAb）针对COMP的表位，并开发和鉴定一种用于测量人体体液中COMP水平的夹心酶联免疫吸附测定（ELISA）。
用胰蛋白酶消化COMP，并确定每种mAb识别的片段的NH(2)末端序列。用抗体捕获试验检测mAb之间的空间竞争。使用未标记的mAb 16-F12作为捕获抗体和生物素标记的mAb 17-C10作为第二抗体开发夹心ELISA。
三种mAb的表位定位于COMP亚基内的三个不同结构域（16-F12，NH(2)末端结构域；17-C10，表皮生长因子样结构域；12-C4，COOH末端结构域）。这些表位不重叠。当用作第二抗体时，mAb 17-C10和12-C4产生相似的血清COMP结果。使用mAb 16-F12和17-C10的新型夹心ELISA测量的血清COMP水平与仅基于mAb 17-C10的抑制ELISA结果密切相关（r(2)=0.836；P<0.0001）。我们对新型夹心ELISA的批间和批内变异性、人滑液（SF）和血清（对照、OA和类风湿关节炎（RA）患者）中预期的COMP水平范围以及血清中COMP水平的日间和昼夜变异性进行了表征。
我们已经开发并鉴定了一种用于COMP的夹心ELISA，该方法灵敏，在分析人血清和滑液时能产生高度可重复的COMP结果。
