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 effective and reproducible concentration of urinary type IV collagen prior to measurement by enzyme immunoassay (EIA). Human placental type IV collagen at low concentrations (5 and 10 micrograms/L) and urinary type IV collagen were readily precipitated by PEG-4000 added at a concentration of about 150 g/L in the presence of 0.5 g/L gamma-globulin. Type IV collagen measurement by EIA from PEG-concentrated urine samples showed complete recovery and good reproducibility. Analysis of size distribution by 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 subjects. Significantly higher concentrations of urinary type IV collagen were found in 30 diabetic patients with nephropathy than in 20 healthy subjects [99.5 (8.9) micrograms/L, mean (SEM) versus 21.4 (2.6) micrograms/L, P < 0.0001]. Thus, urinary type IV collagen can be measured effectively by EIA following concentration with PEG. This method has potential for the assessment of the progression of diabetic nephropathy.
Banu
N
N
Second Department of Internal Medicine, Hiroshima University School of Medicine, Japan.
Hara
H
H
Kataoka
S
S
Egusa
G
G
Yamakido
M
M
D000328:Adult; D015153:Blotting, Western; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003094:Collagen / Q000737:chemistry / Q000652:urine*; D003924:Diabetes Mellitus, Type 2 / Q000652:urine; D003928:Diabetic Nephropathies / Q000652:urine; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D011233:Precipitin Tests; D015203:Reproducibility of Results; D005719:gamma-Globulins / Q000737:chemistry
D005719:gamma-Globulins; D011092:Polyethylene Glycols; D003094:Collagen