New sandwich ELISA for human urinary N-acetyl-β-D-glucosaminidase isoenzyme B as a useful clinical test

Abstract

We have developed a new ELISA for quantifying N-acetyl-β-D- glucosaminidase (NAG) isoenzyme B in human urine after raising monoclonal antibodies against the isoenzyme from human placenta. Though the obtained antibodies reacted not only to isoenzyme B but also to A, we could detect isoenzyme B selectively by a two-step sandwich ELISA with a pair of selected antibodies at low pH in the first reaction. The detected limit was 0.5 μg/L for a sample volume of 25 μL. Within-run CVs ranged from 2.5% to 5.4% and between run CVs ranged from 6.2% to 9.1%. Recoveries of NAG isoenzyme B added to each of three urine samples ranged from 91% to 114%. The dilution curves of urine samples showed good linearity. The cross-reactivity of NAG isoenzyme A was practically negligible (2-3%). The mean value for NAG isoenzyme B in spot urines from healthy adults was 2.9μg/g creatinine. This ELISA method is rapid and precise enough for routine determination of NAG isoenzyme B in human urine.