Objective Monoclonal antibodies ( Mab) of 5D4 which specifically against structural carbohydrate epitopes on the KS of aggrecan have been produced and used to detect the fragment of aggrecan which represents the abnormal metabolism of articular cartilage . Methods Articular chondrocytes were cultured with stimulated subject of TNF -αfor up to 8 days.Aggrecan catabolic fragments in me-dium were measured by ELISA using Mab -5D4.The inter -run variability and intra -run variability were calculated .The GAG was measured by using a modification of a 1,9-dimethylmethylene blue spectrophotometric assay (DMMB) and the correlations with the re-sults of ELISA were detected .Results The coefficient of variance of inter -run and intra-run of ELISA were10.38%and 3.91%, re-spectively.Fragments of 5D4 in medium of TNF-αgroups significantly increase than control (328.22ng/ml vs 184.61ng/ml,t=5.67, P=0.001).There were good correlations between the results of ELISA and GAG detected by DMMB assay ( r=0.453 -0.579,P=0.001).Conclusion The consistency and repeatability of ELISA to detect the fragments of 5D4 released by chondrocytes is acceptable . The method can be used in the study of metabolism of aggrecan .%目的 本研究探讨竞争性ELISA法测定软骨细胞蛋白聚糖代谢片段的可行性,为蛋白聚糖相关研究提供一个新的方法. 方法 利用单克隆抗体(Mab-5D4)通过竞争性ELISA法检测体外培养的兔关节软骨细胞蛋白聚糖的代谢片段,摸索最佳实验条件,计算本方法的板间差异和板内差异,同时通过DMMB分光光度法测定蛋白聚糖的代谢物糖胺聚糖(GAG),并比较它们的相关性. 结果 竞争性ELISA法测定蛋白聚糖5D4片段方法的板间差异为10.38%,板内差异为3.91%,实验组培养上清液5D4片段的浓度明显高于对照组(328.22ng/ml vs 184.61ng/ml,t=5.67,P=0.001). 与常规分光光度法测定蛋白聚糖的代谢物GAG有较好的相关性(r=0.453~0.579,P=0.001). 结论 竞争性ELISA法测定蛋白聚糖代谢产物方法简便,重复性好,可应用于蛋白聚糖代谢的相关研究.
展开▼
机译:6.一种化合物,其包含具有根据20的EP-B1-2563920(SEQ ID NO:80)的具有由20个连接的核苷组成的核碱基序列的修饰的寡核苷酸,其中所述修饰的寡核苷酸包含:由十个连接的脱氧核苷组成的缺口片段;和由五个连接的核苷组成的5'翼区段; 3'翼段由五个连接的核苷组成;其中所述间隙片段位于5'侧翼片段和3'侧翼片段之间,其中每个侧翼片段的每个核苷包含2'-O-甲氧基乙基糖;其中修饰的寡核苷酸的每个胞嘧啶是5-甲基胞嘧啶,并且其中修饰的寡核苷酸的每个核苷间键是硫代磷酸酯键;特别是inotersen;及其衍生物,例如由EP-B1-2563920保护的其盐,包括钠盐。
机译:6.一种化合物,其包含具有根据20的EP-B1-2563920(SEQ ID NO:80)的具有由20个连接的核苷组成的核碱基序列的修饰的寡核苷酸,其中所述修饰的寡核苷酸包含:由十个连接的脱氧核苷组成的缺口片段;和由五个连接的核苷组成的5'翼区段; 3'翼段由五个连接的核苷组成;其中所述间隙片段位于5'侧翼片段和3'侧翼片段之间,其中每个侧翼片段的每个核苷包含2'-O-甲氧基乙基糖;其中修饰的寡核苷酸的每个胞嘧啶是5-甲基胞嘧啶,并且其中修饰的寡核苷酸的每个核苷间键是硫代磷酸酯键;特别是inotersen;及其衍生物,例如由EP-B1-2563920保护的其盐,包括钠盐。