Tharakan J, Strickland D, Burgess W, Drohan W N, Clark D B
American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Md.
Vox Sang. 1990;58(1):21-9. doi: 10.1111/j.1423-0410.1990.tb02050.x.
An immunoaffinity process based on monoclonal antibody (MAb) to factor IX (FIX) has been developed. Initially, vitamin-K-dependent proteins from cryoprecipitate-poor plasma are isolated on DEAE-Sephadex. The eluate is applied to an immunoaffinity column that utilizes a divalent metal-ion-dependent MAb directed against FIX. After washing the column with high salt in the presence of magnesium ion, the FIX is eluted using a citrate- or EDTA-containing buffer. Coagulation assays and Western blots show no detectable amounts of any contaminating proteins. Purity of the FIX product is established using reduced and nonreduced Coomassie-stained SDS-PAGE and HPLC. The N-terminal 20 amino acids of the single peak of the HPLC were shown to be identical to those reported for FIX. The process shows no detectable leakage of monoclonal antibodies (MAb), efficient utilization of MAb, and provides yields greater than 95%. The use of solvent/detergent treatment as a potential viral inactivation methods is incorporated in the process. Studies with tritiated Triton X-100 indicate that the detergent can be washed out of the MAb column so that less than 1 ppm (total) Triton X-100 coelutes with the FIX.
已经开发出一种基于针对凝血因子IX（FIX）的单克隆抗体（MAb）的免疫亲和方法。首先，在DEAE-葡聚糖凝胶上分离来自少冷沉淀血浆的维生素K依赖性蛋白。将洗脱液应用于免疫亲和柱，该柱利用针对FIX的二价金属离子依赖性单克隆抗体。在镁离子存在下用高盐洗涤柱子后，使用含柠檬酸盐或乙二胺四乙酸（EDTA）的缓冲液洗脱FIX。凝血试验和蛋白质免疫印迹显示未检测到任何污染蛋白。使用还原和非还原考马斯亮蓝染色的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和高效液相色谱（HPLC）确定FIX产品的纯度。HPLC单峰的N端20个氨基酸显示与报道的FIX相同。该方法未检测到单克隆抗体（MAb）的泄漏，MAb利用效率高，产率大于95%。该方法采用溶剂/去污剂处理作为潜在的病毒灭活方法。用氚标记的 Triton X-100进行的研究表明，去污剂可以从MAb柱中洗出，因此与FIX共洗脱的 Triton X-100总量小于1 ppm。
