Biswas T, Miller F W, Twitty S A, Plotz P H
J Immunol Methods. 1987 Apr 16;98(2):235-41. doi: 10.1016/0022-1759(87)90010-x.
A rapid method has been developed for enrichment of Jo-1 antigen (histidyl-tRNA synthetase) from HeLa cells. The enzyme has been prepared from post-ribosomal supernatant by successive chromatography with Blue Sepharose and Poly-U-Sepharose, followed by DEAE-high performance liquid chromatography (HPLC). By this method, enzyme could be obtained within 4 days of HeLa cell harvesting, with 40% recovery of the enzymatic activity. The apparent native molecular size of the enzyme as determined by HPLC-size exclusion column chromatography was approximately 120 kDa. Under denaturing conditions using SDS-polyacrylamide gel electrophoresis the enzyme subunit size was approximately 55 kDa. The antigen preparation, although not homogeneous, was found to react only with anti-Jo-1 positive antisera when tested by immunoblotting with many patient sera of defined autoantibody specificities, making the preparation useful for immunologic studies of anti-Jo-1 antibodies.
已开发出一种从HeLa细胞中富集Jo-1抗原（组氨酰-tRNA合成酶）的快速方法。该酶是通过用蓝色琼脂糖凝胶和聚尿嘧啶琼脂糖凝胶连续层析，然后进行DEAE-高效液相色谱（HPLC），从核糖体后上清液中制备的。通过这种方法，在收获HeLa细胞后的4天内即可获得该酶，酶活性回收率为40%。通过HPLC-尺寸排阻柱色谱法测定，该酶的表观天然分子大小约为120 kDa。在使用SDS-聚丙烯酰胺凝胶电泳的变性条件下，酶亚基大小约为55 kDa。通过用许多具有明确自身抗体特异性的患者血清进行免疫印迹检测发现，该抗原制剂虽然不均一，但仅与抗Jo-1阳性抗血清反应，这使得该制剂可用于抗Jo-1抗体的免疫学研究。
