Preuss U, Schuler F, Peter-Katalinic J, Gunawan J, Egge H
Institut für Physiologische Chemie, Universität Bonn, Federal Republic of Germany.
Arch Biochem Biophys. 1991 Nov 15;291(1):139-46. doi: 10.1016/0003-9861(91)90116-z.
The glycosylphosphatidylinositol anchor (GPI) from the membrane form variant surface glycoprotein (mfVSG) of Trypanosoma brucei brucei was isolated and identified after radioactive labeling with [3H]myristic acid, by immunostaining on HPTLC with a polyclonal antibody directed against mfVSG and by negative ion laser desorption and fast atom bombardment mass spectrometry of the GPI anchor before and after peracetylation. For the production of monoclonal antibodies the purified GPI molecule was incorporated into liposomes and injected intrasplenically in BALB/c mice. After fusion with the myeloma cell line X63-Ag 8.653 hybridoma cells were cloned by single cell cloning. The secreted antibodies were characterized by ELISA, Ouchterlony immunodiffusion, and Western blot and used in first immunofluorescent studies.
用[3H]肉豆蔻酸进行放射性标记后，通过用针对布氏布氏锥虫膜型可变表面糖蛋白（mfVSG）的多克隆抗体在高效薄层层析（HPTLC）上进行免疫染色，以及对过乙酰化前后的GPI锚进行负离子激光解吸和快原子轰击质谱分析，分离并鉴定了布氏布氏锥虫膜型可变表面糖蛋白（mfVSG）的糖基磷脂酰肌醇锚（GPI）。为了制备单克隆抗体，将纯化的GPI分子掺入脂质体中，并经脾内注射到BALB/c小鼠体内。与骨髓瘤细胞系X63-Ag 8.653融合后，通过单细胞克隆对杂交瘤细胞进行克隆。通过酶联免疫吸附测定（ELISA）、双向免疫扩散和蛋白质印迹法对分泌的抗体进行表征，并将其用于首次免疫荧光研究。
