MONOCLONAL-ANTIBODIES TO HUMAN ESTROGEN-RECEPTOR
MONOCLONAL-ANTIBODIES TO HUMAN ESTROGEN-RECEPTOR
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DOI：
10.1073/pnas.77.9.5115
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
JENSEN, EV
中科院分区：
文献类型：
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作者：
GREENE, GL;NOLAN, C;JENSEN, EV
MCF-7人乳腺癌细胞的核外雌激素受体蛋白（嗜雌激素）通过细胞匀浆的胞质溶胶部分通过连接到琼脂糖的雌二醇亲和柱来纯化。用在10%（体积/体积）二甲基甲酰胺/0.5M硫氰酸钠中的50 μ M [3 H]雌二醇洗脱，得到40%的[3 H]雌二醇-亲雌激素的回收率，显示纯复合物预期的比放射性的14%。用这种部分纯化的雌二醇受体复合物免疫刘易斯大鼠，其血清中含有抗雌激素抗体，该抗体不仅与MCF-7细胞的核和胞核雌二醇受体复合物反应，而且与大鼠、小牛和猴子宫、母鸡输卵管和人乳腺癌中的雌激素反应。将来自免疫大鼠的脾淋巴细胞与2种不同的小鼠骨髓瘤系（P3-X63-Ag 8和Sp2/0-Ag 14）的细胞融合以产生杂交瘤培养物，其中2%产生抗雌激素的抗体。通过有限稀释克隆后，3杂交瘤细胞株分泌antiestrophilin扩大悬浮培养和腹水肿瘤在无胸腺小鼠，以提供大量的单克隆抗体，这些抗体识别哺乳动物，但不是禽雌激素，并表现出不同程度的反应与受体从nonprimate来源。通过在[35 S]甲硫氨酸存在下生长来自Sp2/0的克隆，制备放射性标记的单克隆IgG。这些单克隆抗体可用于研究人类生殖组织的雌激素受体，特别是用于乳腺癌中受体的放射免疫化学测定和免疫细胞化学定位。[This将在指导治疗选择方面发挥重要作用。
Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to sepharose. Elution with 50 .mu.M [3H]estradiol in 10% (vol/vol) dimethyl formamide/0.5 M sodium thiocyanate gave a 40% recovery of [3H]estradiol-estrophilin, showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells, but also with estrophilin from rat, calf and monkey uterus, hen oviduct and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of 2 different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, 3 hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies; these antibodies recognize mammalian, but not avian estrophilin, and show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2/0 in the presence of [35S]methionine, radiolabeled monoclonal IgG was prepared. These monoclonal antibodies may be useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers. [This will be important in guiding therapy selection.].