竞争性ELISA法测定软骨细胞蛋白聚糖代谢片段的可行性分析

Feasibility of ELISA for the Measurement of 5D4 Fragments in Culturedarticular Chondrocytes

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.

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 .