Khosravi M J, Diamandi A, Mistry J, Krishna R G, Khare A
Diagnostic Systems Laboratories Canada, Inc., Toronto, Ontario, Canada.
J Clin Endocrinol Metab. 1997 Dec;82(12):3944-51. doi: 10.1210/jcem.82.12.4415.
Although the acid-labile subunit (ALS) of the approximately 150-kDa insulin-like growth factor (IGF)-binding protein (IGFBP) complex was described over a decade ago, details of ALS physiology have remained largely uncertain. We evaluated antibodies to synthetic human ALS and constructed a noncompetitive ALS enzyme-linked immunosorbent assay. Whereas uncomplexed ALS is directly measured, determination of total levels required sample pretreatment with SDS, which was found to optimally dissociate complexed ALS and significantly enhance ALS immunoreactivity. ALS in random adult sera was approximately 50% uncomplexed, and samples devoid of complexed ALS by immunoaffinity separation contained about 54% of the total levels. Serum ALS fractionated by gel filtration high performance liquid chromatography eluted in a single peak at approximately 150 kDa with IGF-I and IGFBP-3, but appeared at about 400-500 kDa after sample acidification and fractionation under acidic condition. The unexpected shift in ALS immunoreactivity remained unchanged when acid-neutralized or SDS-treated samples were fractionated under neutral pH and was reproducible when the 150-kDa complex was isolated, treated with acid or SDS, and rechromatographed. ALS in adult sera more tightly correlated with IGFBP-3 than IGF-I or IGF-II. The total levels (mean +/- SD) were 16.7 +/- 3.7 mg/L in 22 normal subjects, 28.3 +/- 8.1 mg/L in 20 acromegalic patients, and 9.5 +/- 3.8 in 32 GH-deficient adults. Little or no ALS was detectable in amniotic fluid, cerebrospinal fluid, seminal plasma, or milk, whereas high levels were present in synovial fluid. The development of ALS enzyme-linked immunosorbent assay should greatly facilitate further investigations of this unique glycoprotein.
尽管大约150 kDa的胰岛素样生长因子（IGF）结合蛋白（IGFBP）复合物的酸不稳定亚基（ALS）在十多年前就已被描述，但ALS生理学的细节在很大程度上仍不确定。我们评估了针对合成人ALS的抗体，并构建了一种非竞争性ALS酶联免疫吸附测定法。未结合的ALS可直接测量，而总水平的测定需要用SDS对样品进行预处理，发现SDS能最佳地解离结合的ALS并显著增强ALS免疫反应性。随机抽取的成人血清中约50%的ALS未结合，通过免疫亲和分离去除结合的ALS的样品中含有约54%的总水平。通过凝胶过滤高效液相色谱法分离的血清ALS与IGF-I和IGFBP-3在约150 kDa处单峰洗脱，但在样品酸化并在酸性条件下分级后出现在约400 - 500 kDa处。当酸中和或SDS处理的样品在中性pH下分级时，ALS免疫反应性的意外变化保持不变，并且当分离出150 kDa的复合物、用酸或SDS处理并重新色谱分析时可重现。成人血清中的ALS与IGFBP-3的相关性比与IGF-I或IGF-II更紧密。22名正常受试者的总水平（平均值±标准差）为16.7±3.7 mg/L，20名肢端肥大症患者为28.3±8.1 mg/L，32名生长激素缺乏的成年人中为9.5±3.8 mg/L。羊水、脑脊液、精浆或乳汁中几乎检测不到或检测不到ALS，而滑液中含量很高。ALS酶联免疫吸附测定法的开发应极大地促进对这种独特糖蛋白的进一步研究。
