The development and validation of a competitive, microtiter plate enzymeimmunoassay for human albumin in urine

We have described a fast, simple and sensitive microtiter scale, solid phase, competitive enzymeimmunoassay (EIA) for the determination of urinary albumin. The albumin used in the test system was purified by the combination of PEG precipitation and DEAE-cellulose column chromatography. In this EIA, microtiter plates were coated with rabbit antihuman albumin IgG, and incubated with HRP-albumin conjugate with either sample or standards. O-phenylenediamine (OPD) and H2O2 solution was used as substrate for HRP. Results obtained correlate well (r = 0.994) with those of an in-house RIA in which same antibody and standards were used as in EIA. The present assay covers the range of 0.5 to 10 mg/L and can be performed in 2 hours. The detection limit was 0.15 mg/L of albumin. Within-assay coefficient of variation was 8.1% and 6.6% and between-assay variation was 10.6% and 8.6% at 1.25 and 2.5 mg/L respectively.