Bessos H, Prowse C V
Thromb Haemost. 1986 Aug 20;56(1):86-9.
This study examines the suitability of four recently characterised monoclonal antibodies (MAbs) for the immunoaffinity purification of human coagulation factor IX (FIX) from plasma concentrates. Initial studies using 125I-FIX indicated that appreciable amounts of bound FIX could be eluted from immobilised MAbs with 0.2 M glycine, 50% ethanediol pH 10 (buffer N). Further studies with FIX concentrates showed that buffer N eluted FIX without compromising the activity of the zymogen. Although FIX was eluted from all four MAbs with this buffer, the best yield (82%) was obtained with MAb ESN-3. ESN-3 bound 40 to 60 iu FIX per mg MAb when immobilised on Sepharose 4B. After washing, column elution with buffer N yielded FIX at 100-200 iu/mg. The purity of the product was confirmed by sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting. The product contained no detectable mouse IgG (less than 3%) and less than 1% FII, X, or protein C.
本研究考察了四种最近鉴定出的单克隆抗体（MAb）用于从血浆浓缩物中免疫亲和纯化人凝血因子IX（FIX）的适用性。使用¹²⁵I-FIX的初步研究表明，用0.2M甘氨酸、50%乙二醇，pH10（缓冲液N）可从固定化的MAb上洗脱可观量的结合FIX。对FIX浓缩物的进一步研究表明，缓冲液N能洗脱FIX且不损害酶原的活性。虽然用该缓冲液可从所有四种MAb上洗脱FIX，但使用MAb ESN-3时获得了最佳产率（82%）。当固定在琼脂糖4B上时，ESN-3每毫克MAb结合40至60国际单位（iu）的FIX。洗涤后，用缓冲液N进行柱洗脱得到的FIX为100 - 200 iu/mg。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹法证实了产物的纯度。该产物未检测到小鼠IgG（小于3%），且FII、X或蛋白C含量小于1%。
