A NOVEL METHOD FOR CONCENTRATING URINARY TYPE-IV COLLAGEN-BASED ON PRECIPITATION WITH POLYETHYLENE-GLYCOL - APPLICATION TO ITS MEASUREMENT BY ENZYME-IMMUNOASSAY

We investigated the efficacy of polyethylene glycol (PEG) for effectiv e and reproducible concentration of urinary type IV collagen prior to measurement by enzyme immunoassay (EIA). Human placental type IV colla gen at low concentrations (5 and 10 mu g/L) and urinary type IV collag en were readily precipitated by PEG-4000 added at a concentration of a bout 150 g/L in the presence of 0.5 g/L gamma-globulin. Type IV collag en measurement by EIA from PEG-concentrated urine samples showed compl ete recovery and good reproducibility. Analysis of size distribution b y Sephacryl S-300HR gel chromatography and Western blotting following polyacrylamide gel electrophoresis confirmed that type IV collagen in PEG-concentrated urine samples was of high molecular weight comparable to that of human placental type IV collagen. After PEG concentration, type IV collagen was detectable by EIA even in the urine of healthy s ubjects. Significantly higher concentrations of urinary type IV collag en were found in 30 diabetic patients with nephropathy than in 20 heal thy subjects [99.5 (8.9) mu g/L, mean (SEM) versus 21.4 (2.6) mu g/L, P < 0.0001]. Thus, urinary type IV collagen can be measured effectivel y by EIA following concentration with PEG. This method has potential f or the assessment of the progression of diabetic nephropathy.