Zahalka M A, Naor D
Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Clin Immunol Immunopathol. 1991 Apr;59(1):72-88. doi: 10.1016/0090-1229(91)90083-m.
An immunogenic protein with an identical Mr (64 kDa) was isolated from syngeneic concanavalin A-induced lymphoblasts (syn-Con A-blasts) and YAC lymphoma cells, both derived from A mice. The 64-kDa protein was purified by a sequence of biochemical steps: Sephadex G-100 gel filtration, ion-exchange chromatography in a fast protein liquid chromatography system, Con A-Sepharose affinity chromatography, and preparative gel electrophoresis. The immunogenic fraction isolated in each step was moved to the next one, and so on. The immunogenicity of the separated fractions was measured by a lymph node proliferation (LNP) assay, which is indicative of a delayed-type hypersensitivity response. For instance, the final 64-kDa isolated protein of the syn-Con A-blasts induced an efficient LNP response in A mice which was detected after challenge with the final 64-kDa isolated protein of YAC cells. In addition to their identical molecular weight, both proteins were eluted at the same ionic strength and both expressed affinity to Con A-Sepharose beads, suggesting that they were glycosilated. Similar 64-kDa proteins were isolated by a different purification procedure, which was performed in the presence of protease inhibitors, excluding the possibility that the final antigen was an autodigested product. As the 64-kDa protein is immunogenic in the syngeneic host, it may be employed as a immunotherapeutic reagent against the original tumor and perhaps against other tumors expressing the same antigen.
从同基因伴刀豆球蛋白A诱导的淋巴母细胞（同基因伴刀豆球蛋白A - 母细胞）和YAC淋巴瘤细胞（均源自A小鼠）中分离出一种具有相同分子量（64 kDa）的免疫原性蛋白。通过一系列生化步骤对64 kDa蛋白进行纯化：葡聚糖G - 100凝胶过滤、快速蛋白质液相色谱系统中的离子交换色谱、伴刀豆球蛋白A - 琼脂糖亲和色谱和制备性凝胶电泳。将每个步骤中分离出的免疫原性组分转移至下一步骤，依此类推。通过淋巴结增殖（LNP）试验测定分离组分的免疫原性，该试验可指示迟发型超敏反应。例如，同基因伴刀豆球蛋白A - 母细胞最终分离出的64 kDa蛋白在A小鼠中诱导了有效的LNP反应，在用YAC细胞最终分离出的64 kDa蛋白攻击后可检测到该反应。除分子量相同外，两种蛋白在相同离子强度下洗脱，且均表现出对伴刀豆球蛋白A - 琼脂糖珠的亲和力，表明它们被糖基化。通过不同的纯化程序分离出了类似的64 kDa蛋白，该程序在蛋白酶抑制剂存在的情况下进行，排除了最终抗原是自消化产物的可能性。由于64 kDa蛋白在同基因宿主中具有免疫原性，它可作为针对原始肿瘤以及可能针对表达相同抗原的其他肿瘤的免疫治疗试剂。
