We designed a semiquantitative analysis of urinary low protein levels using silver dot blot assay. In this method, 3 microl of urine are blotted to one dot onto a cellulose acetate membrane, which is stained by a colloidal silver staining reagent, and the optical density of the silver stained urinary protein is measured at 500 nm using a densitometer. There was a good linearity between 2.5 mg/L and 100 mg/L and a gentle linearity between 100 mg/L and 200 mg/L, and the minimum sensitivity was 2.5 mg/L. This method is suitable for semiquantitative analysis of urinary protein levels less than 300 mg/L, which can not be determined precisely by dipstick.
Cellulose acetate membrane electrophoresis with colloidal silver stain re-vealed that the width of the albumin fraction in IgA nephropathy (IgAN) urine before treatment was significantly expanded. This phenomenon was not shown in IgAN urine after treatment or in non-IgAN urine. There was a reverse correlation between the width of the albumin fraction and the albumin con-centration in IgAN urine. By immuno-fixation, Tamm-Horsfall protein (THP) was located in the same position as the albumin band in IgAN urine before treatment; however, in the urine of a healthy subject it was located in the same position as alpha(1)-globulin. By ELISA, the THP-albumin complex concentration in IgAN urine before treatment was significantly higher than in the other two diseases. The width of the albumin fraction and the sodium ion concentra-tion of the urine were significantly correlated. The THP/albumin ratio in IgAN urine before treatment was significantly higher than in the other two groups. This suggests that the characteristic expanded width of albumin found by immunofixation indicates a THP-albumin complex, and that the sodium concentration of urine is involved in the formation of this complex.
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