Enjoh T, Hizuka N, Perdue J F, Takano K, Fujiwara H, Higashihashi N, Marumoto Y, Fukuda I, Sakano K
Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan.
J Clin Endocrinol Metab. 1993 Aug;77(2):510-7. doi: 10.1210/jcem.77.2.7688378.
Four immunoglobulin G1 class monoclonal antibodies (mAbs; 1D5, 1D9, 2B11, and 2H11) were produced against recombinant human insulin-like growth factor-II (rhIGF-II). Enzyme-linked immunosorbent assay established that these four mAbs specifically recognized rhIGF-II and hIGF-II, but not rhIGF-I. mAbs 1D5, 1D9, and 2H11 did not cross-react with mouse rIGF-II, although there are only six amino acid differences between mouse IGF-II and human IGF-II. The epitope for each mAb was partially defined by enzyme-linked immunosorbent assay using mouse-human chimera IGF-II mutants and other IGF-II mutants that were prepared by site-directed mutagenic procedures. These results indicated that the epitopes of mAbs 1D5, 1D9, and 2H11 are in the C-domain of hIGF-II around Ala32 to Ser36, and that of 2B11 is in the carboxyl-terminal end of the B- and A-domains of hIGF-II. A specific and sensitive RIA was developed using mAb 2H11. In this RIA, IGF-II variant ([RLPG/S29]IGF-II) and rhIGF-II competed equally with [125I]IGF-II for binding to mAb 2H11. Similar results were produced when mAbs 1D5, 1D9, and 2B11 substituted for 2H11. The potential usefulness of mAb 2H11 in an immunoblot procedure to characterize the heterogeneity of IGF-II in the sera and tumor tissues of patients with nonislet cell tumor hypoglycemia was evaluated. A procedure that combined acid-ethanol extraction of serum or tumor tissues and immunoaffinity concentration of the extracted IGF-II with mAb 2H11-immobilized resin was found to be an effective way to prepare the samples. In Western immunoblots, a quantity of rhIGF-II as low as 3 ng could be identified, whereas 200 ng rhIGF-I or rat IGF-II were not recognized. The levels of IGF-II in the sera of 12 patients with nonislet cell tumor hypoglycemia varied from normal to about twice normal. The mol wt (M(r)) of this IGF-II was between 10-17K. There was little of the processed 7.5K M(r) IGF-II in the sera of these patients. Finally, the source of the high M(r) forms of IGF-II was the tumor, because the ratios of high M(r) forms of IGF-II to 7.5K IGF-II changed dramatically from 99:1 and 91:9 to 4:96 and 32:68 in two patients after successful excision of their tumors.(ABSTRACT TRUNCATED AT 250 WORDS)
制备了四种抗重组人胰岛素样生长因子-II（rhIGF-II）的免疫球蛋白G1类单克隆抗体（mAb；1D5、1D9、2B11和2H11）。酶联免疫吸附测定表明，这四种单克隆抗体能特异性识别rhIGF-II和hIGF-II，但不能识别rhIGF-I。单克隆抗体1D5、1D9和2H11与小鼠rIGF-II无交叉反应，尽管小鼠IGF-II与人IGF-II之间只有六个氨基酸差异。通过使用小鼠-人嵌合IGF-II突变体和其他通过定点诱变程序制备的IGF-II突变体的酶联免疫吸附测定，部分确定了每种单克隆抗体的表位。这些结果表明，单克隆抗体1D5、1D9和2H11的表位在hIGF-II的C结构域中Ala32至Ser36附近，而2B11的表位在hIGF-II的B和A结构域的羧基末端。使用单克隆抗体2H11开发了一种特异性和灵敏的放射免疫分析方法。在这种放射免疫分析中，IGF-II变体（[RLPG/S29]IGF-II）和rhIGF-II与[125I]IGF-II竞争结合单克隆抗体2H11的能力相同。当用单克隆抗体1D5、1D9和2B11替代2H11时，产生了类似的结果。评估了单克隆抗体2H11在免疫印迹程序中用于表征非胰岛细胞瘤低血糖患者血清和肿瘤组织中IGF-II异质性的潜在用途。发现一种将血清或肿瘤组织的酸-乙醇提取与用固定有单克隆抗体2H11的树脂对提取的IGF-II进行免疫亲和浓缩相结合的程序是制备样品的有效方法。在Western免疫印迹中，低至3 ng的rhIGF-II量都可以被鉴定出来，而未识别出200 ng的rhIGF-I或大鼠IGF-II。12例非胰岛细胞瘤低血糖患者血清中IGF-II水平从正常到约为正常的两倍不等。这种IGF-II的分子量（M(r)）在
