Ford C H, Osborne P O, Mathew A, Rego B G
Department of Surgery, Faculty of Medicine, Kuwait University, Safat.
J Chromatogr B Biomed Sci Appl. 2001 Apr 25;754(2):427-35. doi: 10.1016/s0378-4347(01)00026-3.
We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semipreparative hydroxyapatite affinity chromatography to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secreting antibodies recognising carcinoembryonic antigen (CEA) and the chemotherapeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60-360 mM phosphate buffer gradient was found to give better separation than elution with a 60-180 mM phosphate buffer gradient. Careful monitoring of HPLC fractions by enzyme linked immunosorbent assays for anti-CEA, anti-Dox and dual anti-CEA/anti-Dox activity, and pooling of fractions on the basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments.
我们开发了一种方法，该方法结合了蛋白A亲和色谱法以及HPLC分析型和半制备型羟基磷灰石亲和色谱法，用于从分泌识别癌胚抗原（CEA）和化疗药物阿霉素（Dox）的抗体的杂交杂交瘤中纯化双特异性抗体（BsMabs）。结果发现，用60 - 360 mM磷酸盐缓冲液梯度洗脱HPLC羟基磷灰石柱，比用60 - 180 mM磷酸盐缓冲液梯度洗脱能实现更好的分离。通过酶联免疫吸附测定法对抗CEA、抗Dox和双抗CEA/抗Dox活性进行仔细监测，并根据这些结果合并馏分，从而能够纯化新型BsMabs，用于体外和临床前体内实验。
