Yamamoto H, Murphy L J
Department of Internal Medicine, University of Manitoba, Winnipeg, Canada.
J Clin Endocrinol Metab. 1995 Apr;80(4):1179-83. doi: 10.1210/jcem.80.4.7536201.
Urinary insulin-like growth factor-I (IGF-I) from healthy human subjects was examined using two antisera directed toward the whole molecule (WM) and the N-terminal of IGF-I. Pooled urine samples from normal adults were dialyzed, lyophilized, then subjected to Sephacryl S-200 chromatography. The gel filtration profile of immunoreactive IGF-I measured by RIA using WM antiserum showed two peaks. Of the total IGF-I, approximately 40% was free, and the rest was present as a 50-kilodalton complex. To characterize the IGF-I forms present in those two peaks, antibody capture enzyme-linked immunoassays (EIA) using the two antisera were established for detection of intact IGF-I and N-terminal-truncated IGF-I variants. The WM antibody recognizes intact IGF-I and des(1-3)-IGF-I, an N-terminal-truncated variant, equally well, whereas the N-terminal IGF-I antibody recognizes intact IGF-I, but not des(1-3)-IGF-I (< 1% cross-reactivity). As both antibodies show similar cross-reactions with IGF-II, the difference between IGF-I levels recognized by the two antisera was considered to indicate the presence of N-terminal-truncated IGF-I variants. Of the free immunoreactive IGF-I in the urine, 64% was not recognized by N-terminal IGF-I antiserum and was considered to represent N-terminal-truncated IGF-I. In contrast, only 6% of the IGF-I present in the 50-kilodalton fraction was truncated. Urine samples from normal human subjects were analyzed by RIA with WM antiserum and EIA with both WM and N-terminal IGF-I antisera after extraction of IGF-I from binding proteins. IGF-I values measured by EIA with the WM antiserum correlated well with those values obtained by RIA using WM antiserum (r = 0.98; P < 0.001). The total urinary IGF-I level measured by EIA with the WM antiserum was 216.0 +/- 41.1 ng/L (mean +/- SEM), and 35.2 +/- 6.1% of this was considered to represent N-terminal-truncated IGF-I. Using an immobilized biotinylated peptide corresponding to the N-terminal six amino acids of IGF-I, we detected proteolytic activity toward the N-terminal of IGF-I in all four human serum samples tested. In contrast, only two of seven urine samples had detectable protease activity, and in these samples, activity was very low compared to that in serum.(ABSTRACT TRUNCATED AT 400 WORDS)
使用两种分别针对胰岛素样生长因子-I（IGF-I）全分子（WM）和N端的抗血清，对健康人类受试者的尿IGF-I进行了检测。收集正常成年人的尿液样本，透析、冻干后，进行Sephacryl S - 200柱层析。用WM抗血清通过放射免疫分析（RIA）测定免疫反应性IGF-I的凝胶过滤图谱显示有两个峰。在总的IGF-I中，约40%是游离的，其余以50千道尔顿的复合物形式存在。为了鉴定这两个峰中存在的IGF-I形式，建立了使用这两种抗血清的抗体捕获酶联免疫分析（EIA），用于检测完整的IGF-I和N端截短的IGF-I变体。WM抗体对完整的IGF-I和N端截短变体des(1 - 3)-IGF-I的识别能力相同，而N端IGF-I抗体能识别完整的IGF-I，但不能识别des(1 - 3)-IGF-I（交叉反应性<1%）。由于两种抗体与IGF-II的交叉反应相似，两种抗血清识别的IGF-I水平差异被认为表明存在N端截短的IGF-I变体。尿液中游离的免疫反应性IGF-I中，64%不被N端IGF-I抗血清识别，被认为代表N端截短的IGF-I。相比之下，50千道尔顿组分中的IGF-I只有6%是截短的。从结合蛋白中提取IGF-I后，用WM抗血清通过RIA和用WM及N端IGF-I抗血清通过EIA对正常人类受试者的尿液样本进行分析。用WM抗血清通过EIA测定的IGF-I值与用WM抗血清通过RIA获得的值相关性良好（r = 0.98；P < 0.001）。用WM抗血清通过EIA测定的总尿IGF-I水平为216.0±41.1 ng/L（平均值±标准误），其中35.2±6.1%被认为代表N端截短的IGF-I。使用与IGF-I N端六个氨基酸对应的固定化生物素化肽，我们在所有检测的四个人类血清样本中检测到了针对IGF-I N端的蛋白水解活性。相比之下，七个尿液样本中只有两个具有可检测到的蛋白酶活性，而且在这些样本中，活性与血清中的相比非常低。（摘要截断于400字）
