High-sensitivity dye binding assay for albumin in urine

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-tetrabromosulfonphthal ein (DIDNTB), a dye that has a higher chemical sensitivity and specificity for albumin when compared to two other commonly used dyes. We prepared urin e dipsticks with DIDNTB and certain other compounds to prevent "nonspecific " binding to the dipstick matrix. The detection limit for albumin with DIDN TB as the dye is about 10 mg/L. The extent of dye binding to proteins and o ther compounds was studied using ultracentrifugation and a selectively perm eable membrane that permitted the passage of free but not bound dye; we bel ieve 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 coef ficient 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 syste m that minimized nonspecific binding by the dye through the use of polymeth yl vinyl ethers and bis(heptapropylene glycol) carbonate. DIDNTB showed a g reater chemical specificity for albumin when compared to most other protein s. 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. (C) 1999 Wiley-Liss, Inc.