[Urinary albumin fragmentation and immunoreactivity].

Urinary albumin (ALB) has been measured as a marker for the early detection of diabetic nephropathy. In 2004, Comper et al. developed a gel-filtration high-performance liquid chromatography (HPLC) procedure for the determination of urinary ALB. They demonstrated the presence in its albumin fraction of non immunoreactive ALB with the total molecular weight of a monomeric ALB that was non-reactive with the existing anti-ALB antibody, and reported that the level of urinary non-immunoreactive ALB was higher in diabetic patients than in normal subjects. In this study, we isolated urinary ALB from diabetic patients using an anti-ALB antibody-coupled affinity column to test its immunoreactivity. In some diabetic patients, the results of HPLC and turbidimetric immunoassay for urinary ALB were discrepant. Western blot analysis showed that ALB samples from such patients were contaminated with proteins other than ALB, and contained ALB, whose molecular weight became lower using a reductive procedure. In addition, the reactivity of ALB with anti-ALB antibody differed depending on whether it was in a reduced or non-reduced state. These results indicate that ALB in such patients is susceptible to structural changes due to disease-induced urinary factors and, thus, their urine contains ALB with an altered reactivity to antibody.

[Construction of urinary Tamm-Horsfall protein assay by enzyme-linked immunosorbent assay and establishment of reference range of healthy subject].

Tamm-Horsfall protein (THP) is the most abundant protein in the urine. THP is expressed in the renal tubule as a precursor protein having 640 amino acid residues and anchored by GPI anchor in the cell membrane. Thereafter, THP is secreted as a glycoprotein is a molecular weight of about 95-100 kD. Despite several investigations from the perspective of renal failure, the physiological role of this protein is not yet clear. It has been reported that THP is also related to certain conditions, such as kidney stone formation and urinary tract infection. To examine the excretion of THP into urine, we constructed an enzyme linked immunosorbent assay(ELISA) for the measurement of THP in the urine. THP was purified from healthy human urine using Diatomaceous Earth. Then we obtained an antibody to purified THP and constructed a sandwich ELISA assay system to test urine samples. The sensitivity of measurement was 0.78 ng/ml. In this assay, the concentration of THP in spot urine can be linearly measured from 0.78 ng/ml to 50 ng/ml. The CV of assay was 2.4 to 4.1%. The measurement was not disrupted by urinary albumin (approximately 15 mg/ml), hemoglobin (approximately 15 microg/ml), glucose (approximately 30 mg/ml) and ascorbic acid (approximately 10 mg/ml). Pretreatment by centrifugation or filtration of urine affected the concentration of THP because of the agglutinated form of THP. We showed that the urinary excretion rate remained almost constant in our test population, 1.00-1.65 mg/hr (average +/- 1SD 1.30 +/- 0.25) for healthy men and 0.61-1.51 mg/hr (0.90 +/- 0.30) for healthy women and 1.10 +/- 0.34 mg/hr overall.