Houranieh A, Statland B E, Davis G L, Ito R K, Relias V, Kudryk B, Yunis E J
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Hybridoma. 1988 Feb;7(1):55-68. doi: 10.1089/hyb.1988.7.55.
Cross-linked Fibrin II was prepared using Kabi grade (L) fibrinogen. Fibrin plasmic digest was separated on Sepharose CL-6B. Fragments Mr 135-300 kDa were used to immunize 6-9 weeks old female BALB-c mice. A stable hybridoma secreting monoclonal antibody (MAb) TD-1 (IgG 2a, Kappa) was prepared by fusion using myeloma cells (P3-NS1/1-Ag4-1) and immunized cells. Fibrinogen and plasmin digest of fibrinogen in serial dilutions did not compete with the immunizing antigen. To prove that TD-1 binds specifically to cross-linked fibrin, immunoprecipitation with S. aureus and affinity chromatography were performed. In both experiments, we demonstrated that TD-1 binds specifically to a protein Mr greater than 200 kDa which is found in XL-fibrin and not fibrinogen. Reduced samples showed the antibody bands (heavy and light chains) and three protein bands, Mr greater than 80 kDa (gamma-gamma dimer), Mr greater than 45 kDa (beta chain of fragment D) and Mr greater than 16 kDa (alpha chain from fragment D) were present. TD-1 reacted strongly with HPLC fraction of the immunizing antigen Mr 220 kDa (probably DD/E complex). Affinity binding constants (Scatchard Plot Analysis) were determined. The highest affinity was obtained with XL-fibrin fraction Mr 220 kDa, KD = 1.39 X 10(-8) and high molecular weight XL-fibrin fragments, KD = 1.6 X 10(-7). Fragment DD had KD of 2.8 X 10(-6). These results suggest that TD-1 is specific for the DD region of human cross-linked Fibrin II.
交联纤维蛋白II是使用卡比级（L）纤维蛋白原制备的。纤维蛋白血浆消化产物在琼脂糖凝胶CL-6B上进行分离。使用分子量为135 - 300 kDa的片段免疫6 - 9周龄的雌性BALB - c小鼠。通过骨髓瘤细胞（P3 - NS1/1 - Ag4 - 1）与免疫细胞融合制备出分泌单克隆抗体（MAb）TD - 1（IgG 2a，κ链）的稳定杂交瘤。纤维蛋白原以及系列稀释的纤维蛋白原纤溶酶消化产物不与免疫抗原竞争。为证明TD - 1特异性结合交联纤维蛋白，进行了金黄色葡萄球菌免疫沉淀和亲和层析实验。在这两个实验中，我们都证明TD - 1特异性结合一种分子量大于200 kDa的蛋白质，该蛋白质存在于交联纤维蛋白中而非纤维蛋白原中。还原后的样品显示出抗体条带（重链和轻链）以及三条蛋白条带，分子量大于80 kDa（γ - γ二聚体）、分子量大于45 kDa（片段D的β链）和分子量大于16 kDa（片段D的α链）。TD - 1与免疫抗原分子量为220 kDa的高效液相色谱馏分（可能是DD/E复合物）强烈反应。测定了亲和结合常数（Scatchard图分析）。与分子量为220 kDa的交联纤维蛋白馏分结合时亲和力最高，解离常数KD = 1.39×10⁻⁸，与高分子量交联纤维蛋白片段结合时KD = 1.6×10⁻⁷。片段DD的KD为2.8×10⁻⁶。这些结果表明TD - 1对人交联纤维蛋白II的DD区域具有特异性。
