Horton J K, Davies M, Woodhead J S, Weeks I
Department of Renal Medicine, University of Wales College of Medicine, Cardiff, UK.
Clin Chim Acta. 1988 May 31;174(2):225-37. doi: 10.1016/0009-8981(88)90389-0.
Tamm-Horsfall glycoprotein was purified to apparent homogeneity from human urine by repeated precipitation with 0.58 mol/l NaCl and gel permeation chromatography under dissociating conditions on Bio-Gel A1.5M. The protein was found to consist of a single polypeptide chain of Mr 100,000 under non-reducing conditions and Mr 75,000 under reducing conditions. Antibodies to Tamm-Horsfall glycoprotein were raised in rabbits and subsequently purified by affinity chromatography using the glycoprotein linked to Sepharose 4B. The specificity of these antibodies was confirmed by Western blotting and by indirect immunofluorescence staining of human kidney tissue. The purified antibodies were labelled with 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methyl-9-acridinium carboxylate fluorosulphonate, an acridinium ester, to a specific activity of 6 X 10(5) photon counts/ng of protein, and used to establish a two-site immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein in serum and urine. The bound and the free fractions were separated by a second antibody to Tamm-Horsfall glycoprotein linked to paramagnetic particles. The bound antibodies were quantified by chemiluminescence. The assay had a sensitivity of detection of 2 ng/ml and a working range, as determined by inter-assay precision profiles, of 30-500 ng/ml. The range in serum samples from volunteers with normal renal function (n = 92) was 74-520 ng/ml and the mean 24-h excretion rate in healthy subjects (n = 32) was 70 +/- 26 mg.
通过在0.58 mol/l氯化钠中反复沉淀以及在变性条件下于Bio-Gel A1.5M上进行凝胶渗透色谱法，从人尿中纯化出了具有明显均一性的Tamm-Horsfall糖蛋白。发现在非还原条件下该蛋白由一条分子量为100,000的单多肽链组成，在还原条件下分子量为75,000。用兔制备了针对Tamm-Horsfall糖蛋白的抗体，随后通过使用与琼脂糖4B偶联的糖蛋白进行亲和色谱法进行纯化。这些抗体的特异性通过蛋白质印迹法以及人肾组织的间接免疫荧光染色得以证实。将纯化的抗体用4-(2-琥珀酰亚胺氧基羰基乙基)苯基-10-甲基-9-吖啶鎓羧酸酯荧光磺酸盐（一种吖啶酯）标记，比活性达到6×10(5) 光子计数/ng蛋白，并用于建立一种双位点免疫化学发光测定法，以测量血清和尿液中的Tamm-Horsfall糖蛋白。结合部分和游离部分通过与顺磁性颗粒偶联的针对Tamm-Horsfall糖蛋白的第二抗体进行分离。通过化学发光对结合抗体进行定量。该测定法的检测灵敏度为2 ng/ml，根据批间精密度曲线确定的工作范围为30 - 500 ng/ml。肾功能正常的志愿者（n = 92）血清样本中的范围为74 - 520 ng/ml，健康受试者（n = 32）的平均24小时排泄率为70±26 mg。
