benzidine, which is prepared by dis- solving 1 mg of tetramethylbenzidine in 1 mL of dimethyl sulfoxide and adding it slowly to 100 mL of 0.1 mmol/L sodium ...
neonates during the first two posthatal weeks (1-3). These studies, involving immunoassay kits having strong reactivity to DLIS, showed that the initial concentration of DLIS detected was 0.3 ,ug/L in patients whose DLIS concentration increased temporally. Similar studies, such as reported recently (7), need to be performed with kits having weak reactivity to DLIS, to confirm the validity of this cutoff-value approach. I thank the manufacturers of the immunoassay kits for generously providing the kits for this study and the medical technologists of the chemistry laboratory for performing the analysis. References 1. Valdes R, Graves SW, Brown BA, Landt M. Endogenoussubstance in newborn infants causing false positive digoxin measurements J Pediatr 102,947-950(1983). 2. Pudek MB, Seccombe DW, Jacobson BE, Whitfleld MF. Seven different digoxin inmunoassay kits compared with respect to interference by a digoxin-like immunoreactive substance in serum from premature and full-term infants. Clin Chem 29,19721974 (1983). 3. Seccombe DW, Pudek MB, Whitfleld MF, et al. Perinatal changes in a cligoxinlike immunoreactive substance. PediatrRes 18, 1097-1099 (1984). 4 Hicks JM, Brett EM. Falsely increased digoxin concentrations in samples from neonates and infants. TherDrugMonit 6,461464(1984). & Boink ABTJ, Kruyswijk HI!, Willebrands .AF, Mass AHJ. Some factors affecting a commercial kit for radioimmunoassay of digoxin using tritiated digoxin. J Clin Chem Clin Biochem 15, 261-266 (1977). 6. Yatscoff RW, Deajardins PRE, Dalton JG. Digoxin-like inununoreactivity in the serum of neonates and uremic patients, as measured in the Abbott TDx. Clin Chem 30, 588 (1984). Letter. 7 Gortner L, Hellenbrecht D. An RIA kit that is more specific for digoxin in serum of newborns. Clin Chem 31, 155-156 (1985). Letter. 8 Soldin SJ, Papanastasiou-Dismandi A, Lingwood C, Kuksis A. Extraction and purification of digoxin iminunoreactive materials from urine of normal adults by liquid chromatography. Clin Chem 30, 1013 (1984). Abstract. Robert
C. McCarthy
Dept. of Pathol. The Children’s Hospital Denver, CO 80218
SimplerMeasurementof AlbuminIn UrIneor Plasma To the Editor: Fielding et al. (1) recently published an enzyme immunoassay method for measuring human albumin in concen-
trations of 3 to 1000 4ug/L. We have developed a simpler “single-sandwich” technique, in which antibody of the same species is used both for binding and for detection of the albumin. The following procedure was adopted: 1. Incubate microtiter plates overnight at 4#{176}C with each well containing 100 pL of goat antiserum to human albumin (Cappel Laboratories, Cochranville, PA) diluted 1:1000 in coating buffer (0.5 mmol/L carbonate/bicarbonate buffer, pH 9.6). 2. Empty the wells and wash three times with diluent buffer (50 mmol/L Tris HC1, pH 8.0, containing 500 iL of Tween 20 surfactant per liter). 3. Incubate the plates for 1 h at room temperature with each well containing 100 pL of albumin standard or sample (urine or plasma) in diluent buffer, then empty and wash as in step 2. 4. Incubate the plates for 1 h at room temperature with each well containing 100 zL of goat antihuman albumin IgG conjugated with peroxidase (Cappel Laboratories) diluted 1:2000 in diluent buffer; empty and wash as in step 2. 5. Incubate the plates with each well containing 100 pL of the peroxidase substrate 3,3’,5,5’-tetramethylbenzidine, which is prepared by dissolving 1 mg of tetramethylbenzidine in 1 mL of dimethyl sulfoxide and adding it slowly to 100 mL of 0.1 mmol/L sodium acetate/citric acid buffer, pH 6.0, then immediately before use, adding sufficient H202 to give a final concentration of 1.3 mmol/L. In contrast to o-phenylenediamine, tetramethylbenzidine need not be incubated in the dark. 6. Stop the peroxidase reaction after exactly 10 mm by adding 50 &L of 2 mol/L H2SO4; the blue color changes to yellow. 7. Measure the absorbance of well contents at 450 nm in a microtiter plate reader. We assessed the characteristics of the assay with standard curves of 10 albumin concentrations, measured in triplicate. The lowest concentration that differed from zero readings by more than 2 SD was 5 zg/L. Nonspecific binding was always
