Okret S, Wikström A C, Wrange O, Andersson B, Gustafsson J A
Proc Natl Acad Sci U S A. 1984 Mar;81(6):1609-13. doi: 10.1073/pnas.81.6.1609.
Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR.
用纯化的大鼠肝脏糖皮质激素受体（GR）免疫的一只BALB/c小鼠和一只C57/BL小鼠的脾细胞，与小鼠骨髓瘤细胞系Sp 2/0-Ag 14融合。用酶联免疫吸附测定法，以部分纯化的大鼠肝脏GR作为抗原，对杂交瘤产生抗GR抗体的情况进行筛选。进一步的筛选是通过第二抗体免疫沉淀测定法，使用来自大鼠肝脏胞质溶胶的[3H]曲安奈德-GR复合物作为示踪剂。在两种测定中均呈阳性的来自10个不同微孔板孔的杂交瘤，通过有限稀释法成功克隆为单克隆。通过等电聚焦分析时，单克隆抗体的不同等电点证实了它们的不同来源。四个单克隆杂交瘤细胞系分泌IgM抗体；两个分泌IgG1；三个分泌IgG2a；一个分泌IgG2b。当分别与单克隆IgG或IgM抗体孵育时，在甘油密度梯度中通过4S GR向8.5S或19S GR-抗体复合物的转变鉴定出GR-抗体复合物。这10种单克隆抗体识别GR上不同的决定簇，所有这些决定簇都位于受体与配体和DNA结合域分开的那个结构域上。此外，单克隆抗体对小鼠肝脏GR的交叉反应性也有所不同。未观察到与人淋巴细胞GR的交叉反应性。用0.5%纯GR制剂进行十二烷基硫酸钠电泳，然后用其中一种单克隆抗体进行免疫印迹，鉴定出一条分子量为94,000的单一肽段，与纯化的大鼠肝脏GR相同。
