Uto I, Ishimatsu T, Hirayama H, Ueda S, Tsuruta J, Kambara T
Department of Urology, Kumamoto University Medical School, Japan.
J Immunol Methods. 1991 Apr 8;138(1):87-94. doi: 10.1016/0022-1759(91)90067-p.
A method for enzyme-antibody conjugation using a new maleimide derivative as coupling reagent has been developed. Since a monomeric conjugate of horseradish peroxidase and Fab' antibody could be readily prepared with high efficiency and reproducibility, the enzyme activity and antigen-binding activity were well preserved and nonspecific staining was greatly reduced. The conjugate is suitable for use in both ELISA procedures and immunohistochemistry. Using both methods we examined the pathophysiological significance of Tamm-Horsfall protein (THP) and the present study describes the ELISA method to quantify urinary THP using the new method with rabbit anti-THP antibody. A low concentration (0.04 M) of urea added to the urine samples increased the linearity of the standard curve and the sensitivity of the assay, permitting the detection of as little as 20 ng/ml THP. Freezing and thawing the urine resulted in variable or lower values of THP concentration. THP concentrations in urine as determined by ELISA were stable for at least one month after -70 degrees C storage, but not after -30 degrees C storage. There was no correlation between THP concentrations in 24 h urine samples and the morning urine of the same patient. These results suggest that it is essential to use fresh or -70 degrees C stored 24 h urine samples with added urea (0.04 M) for the determination of THP concentrations in urine by the present enzyme-antibody conjugation method. The THP concentration in normal 24 h urine of young children was found to be less than 51.8 mg/g Cr.
已开发出一种使用新型马来酰亚胺衍生物作为偶联试剂进行酶 - 抗体偶联的方法。由于辣根过氧化物酶与Fab'抗体的单体偶联物能够以高效率和可重复性轻松制备，酶活性和抗原结合活性得到了很好的保留，并且非特异性染色大大减少。该偶联物适用于ELISA程序和免疫组织化学。我们使用这两种方法研究了Tamm - Horsfall蛋白（THP）的病理生理意义，本研究描述了使用兔抗THP抗体的新方法定量尿THP的ELISA方法。向尿液样本中添加低浓度（0.04 M）的尿素可提高标准曲线的线性和检测灵敏度，能够检测低至20 ng/ml的THP。尿液的冻融导致THP浓度值可变或降低。通过ELISA测定的尿液中THP浓度在-70℃储存后至少一个月稳定，但在-30℃储存后不稳定。同一患者24小时尿液样本中的THP浓度与晨尿之间没有相关性。这些结果表明，使用添加了尿素（0.04 M）的新鲜或-70℃储存的24小时尿液样本对于通过本酶 - 抗体偶联方法测定尿液中的THP浓度至关重要。发现幼儿正常24小时尿液中的THP浓度低于51.8 mg/g Cr。
