Kurkela R, Vuolas L, Vihko P
Biocenter, University of Oulu, Finland.
J Immunol Methods. 1988 Jun 13;110(2):229-36. doi: 10.1016/0022-1759(88)90108-1.
Monoclonal antibodies of IgG1 subclass raised against purified human prostate-specific acid phosphatase were subjected to different procedures to produce F(ab')2 fragments suitable for radioimaging of prostatic cancer, following derivatization and labeling with radionuclides. The main aim was to obtain highly purified fragments with preserved immunological activity. Optimized pepsin digestion led to the formation of mainly F(ab')2 and Fab fragments, and, following Sephacryl S-200 gel filtration, the yield of pure F(ab')2 fragments was 24 +/- 11% of the theoretical maximum. After papain digestion in the absence of thiols, no formation of Fab fragments was observed, and the F(ab')2 fragments formed could be efficiently separated from Fc fragments by chromatofocusing or ion exchange chromatography. The yield of F(ab')2 fragments from papain digestion was 50 +/- 5% of the theoretical maximum. Both the above procedures gave F(ab')2 fragments with immunoreactivity and affinity identical to those of the precursor IgG1, despite the fact that isoelectric focusing profiles of the two F(ab')2 preparations differed, suggesting different digestion sites.
以纯化的人前列腺特异性酸性磷酸酶为抗原制备的IgG1亚类单克隆抗体，经过不同处理以产生适用于前列腺癌放射性成像的F(ab')2片段，随后进行放射性核素衍生化和标记。主要目的是获得具有保留免疫活性的高度纯化片段。优化的胃蛋白酶消化主要产生F(ab')2和Fab片段，经Sephacryl S-200凝胶过滤后，纯F(ab')2片段的产量为理论最大值的24±11%。在无硫醇存在下进行木瓜蛋白酶消化时，未观察到Fab片段的形成，且通过色谱聚焦或离子交换色谱可有效地将形成的F(ab')2片段与Fc片段分离。木瓜蛋白酶消化产生的F(ab')2片段产量为理论最大值的50±5%。尽管两种F(ab')2制剂的等电聚焦图谱不同，表明消化位点不同，但上述两种方法产生的F(ab')2片段的免疫反应性和亲和力均与前体IgG1相同。
