Nice E, Dempsey P, Layton J, Morstyn G, Cui D F, Simpson R, Fabri L, Burgess A
Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Victoria, Australia.
Growth Factors. 1990;3(2):159-69. doi: 10.3109/08977199009108278.
One approach to the localization of functionally active regions of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is to map the epitopes recognized by neutralizing anti-hGM-CSF monoclonal antibodies. We have defined the epitope recognized by one neutralizing antibody (LMM102) using proteolytic fragments obtained by enzymic digestion of bacterially synthesized hGM-CSF. RP-HPLC fractionation of a tryptic digest resulted in the identification of an immunoreactive "tryptic core" peptide containing 66 amino acids (52% of the protein). Further digestion of this "tryptic core" with S. aureus V8 protease produced a unique immunoreactive hGM-CSF product comprising two peptides, residues 86-93 and 112-127, linked by a disulfide bond between residues 88 and 121. The individual peptides, generated by reduction with dithiothreitol, were not recognized by the antibody. An analog of this peptide has been synthesized chemically and shown to have similar immunoreactivity to the epitope obtained by enzymic digestion. A series of modified peptides has also been synthesized to identify further the region required for antibody recognition.
一种定位人类粒细胞 - 巨噬细胞集落刺激因子（hGM - CSF）功能活性区域的方法是绘制可被中和性抗hGM - CSF单克隆抗体识别的表位图谱。我们利用通过对细菌合成的hGM - CSF进行酶切获得的蛋白水解片段，确定了一种中和抗体（LMM102）所识别的表位。对胰蛋白酶消化产物进行反相高效液相色谱（RP - HPLC）分级分离，鉴定出一种含66个氨基酸（占该蛋白的52%）的免疫反应性“胰蛋白酶核心”肽。用金黄色葡萄球菌V8蛋白酶对该“胰蛋白酶核心”进行进一步消化，产生了一种独特的免疫反应性hGM - CSF产物，由两个肽段（残基86 - 93和112 - 127）组成，它们通过残基88和121之间的二硫键相连。用二硫苏糖醇还原产生的单个肽段不能被该抗体识别。已化学合成了该肽的类似物，并显示其与通过酶切获得的表位具有相似的免疫反应性。还合成了一系列修饰肽，以进一步确定抗体识别所需的区域。
