Dempsey P J, Layton J E, Duhrsen U, Nicola N A, Cebon J, Burgess A W, Morstyn G
Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Australia.
Hybridoma. 1990 Dec;9(6):545-58. doi: 10.1089/hyb.1990.9.545.
We have produced monoclonal antibodies to bacterially synthesized, human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and have studied in detail the characteristics of three strongly neutralizing antibodies. The antibodies reacted with GM-CSF at high dilution (EC50 = 0.1-1.7 nM) in an indirect ELISA but did not react with murine GM-CSF or other cytokines. They also recognized glycosylated hGM-CSF produced by human lymphocytes. The antibodies were able to immunoprecipitate rhGM-CSF, but only reacted weakly with rhGM-CSF on Western blots, indicating that they recognized a conformation-dependent epitope. Cross-blocking studies showed that the three antibodies recognized overlapping epitopes. The antibodies inhibited binding of 125I-labeled rhGM-CSF to HL-60 cells at nanomolar concentrations and neutralized GM-CSF activity in two different bioassays. These antibodies thus provide a useful tool for analyzing the specificity of bioassays and for further studies of the production and function of GM-CSF in vitro and in vivo.
我们制备了针对细菌合成的人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）的单克隆抗体，并详细研究了三种强中和抗体的特性。这些抗体在间接ELISA中以高稀释度（EC50 = 0.1 - 1.7 nM）与GM-CSF反应，但不与鼠GM-CSF或其他细胞因子反应。它们还能识别由人淋巴细胞产生的糖基化hGM-CSF。这些抗体能够免疫沉淀rhGM-CSF，但在Western印迹上与rhGM-CSF的反应较弱，表明它们识别的是构象依赖性表位。交叉阻断研究表明，这三种抗体识别重叠表位。这些抗体在纳摩尔浓度下可抑制125I标记的rhGM-CSF与HL-60细胞的结合，并在两种不同的生物测定中中和GM-CSF活性。因此，这些抗体为分析生物测定的特异性以及进一步研究GM-CSF在体外和体内的产生和功能提供了有用的工具。
