High‐sensitivity dye binding assay for albumin in urine

Abstract

We developed a dye‐binding method for albumin in urine based on bis (3′,3″‐diiodo‐4′4″‐dihydroxy‐5′5″‐dinitrophenyl)‐3,4,5,6‐tetrabromosulfonphthalein (DIDNTB), a dye that has a higher chemical sensitivity and specificity for albumin when compared to two other commonly used dyes. We prepared urine dipsticks with DIDNTB and certain other compounds to prevent "nonspecific" binding to the dipstick matrix. The detection limit for albumin with DIDNTB as the dye is about 10 mg/L. The extent of dye binding to proteins and other compounds was studied using ultracentrifugation and a selectively permeable membrane that permitted the passage of free but not bound dye; we believe this method is superior to photometric titration. The affinity of the dyes for albumin was found to be pH dependent with stronger binding at pH 1.8 than at pH 7.0. At pH 1.8, DIDNTB had a ca.10‐fold greater binding coefficient to albumin when compared to the widely used dyes, tetrabromophenol blue (CI 4430‐25‐5) or bromophenol blue (CI 115‐39‐9). We developed a system that minimized nonspecific binding by the dye through the use of polymethyl vinyl ethers and bis‐(heptapropylene glycol) carbonate. DIDNTB showed a greater chemical specificity for albumin when compared to most other proteins. The new albumin dipsticks are resistant to many potential interferences at substantial concentrations, making the dipsticks suitable to screen for albuminuria. J. Clin. Lab. Anal. 13:180–187, 1999.© 1999 Wiley‐Liss, Inc.