Kanakura Y, Cannistra S A, Brown C B, Nakamura M, Seelig G F, Prosise W W, Hawkins J C, Kaushansky K, Griffin J D
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115.
Blood. 1991 Mar 1;77(5):1033-43.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40-77, 78-94, or 110-127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.
粒细胞-巨噬细胞集落刺激因子（GM-CSF）是一种糖蛋白，造血祖细胞的存活、生长和分化都需要它。虽然通过cDNA克隆已了解GM-CSF的一级结构，但GM-CSF结构与功能之间的关系尚未完全明确。制备了15种针对人GM-CSF的不同单克隆抗体（MoAb），以在免疫学上定位该分子不同的区域。每种MoAb都进行了生物素化，并通过酶联免疫吸附测定法显示其能与固定在固相上的重组GM-CSF结合。然后使用15种未偶联的MoAb中的每一种与每种生物素化的MoAb竞争结合GM-CSF。这些交叉阻断研究确定了天然GM-CSF的8个不同表位。通过蛋白质印迹法发现，其中7个表位也存在于变性的GM-CSF中，并且4个表位在经β-巯基乙醇还原的GM-CSF上至少部分保守。针对8个表位中4个表位的MoAb在GM集落形成单位（CFU-GM）测定中中和了重组（糖基化和非糖基化）及天然人GM-CSF，并阻断了GM-CSF诱导的中性粒细胞活化。对于大多数抗体，中和活性与阻断125I-GM-CSF与中性粒细胞或母细胞结合的能力之间存在良好的相关性。针对一个表位的非中和抗体部分阻断了125I-GM-CSF与中性粒细胞的结合。没有一种MoAb能中和白细胞介素-3、粒细胞集落刺激因子（G-CSF）或巨噬细胞集落刺激因子（M-CSF）。通过确定对GM-CSF合成肽或人-鼠嵌合GM-CSF的反应性，可部分定位7个表位相对于氨基酸结构的位置。发现中和抗体定位于氨基酸40-77、78-94或110-127。因此，这些MoAb可用于识别GM-CSF的功能域以及可能参与受体相互作用的区域。
