Development and optimization of an enzyme-linked immunosorbent assay employing two murine monoclonal antibodies for absolute quantitation of human beta-glucuronidase.
We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) for absolute quantitation of human beta-glucuronidase. This is a double antibody sandwich system employing two murine monoclonal antibodies specific for human beta-glucuronidase developed in our laboratories. The method involves (a) coating of the high binding polystyrene microtitration plate with the first antibody (7B6 IgG), (b) blocking of remaining active sites with 3% bovine serum albumin in phosphate-buffered saline, (c) application of samples, (d) addition of the biotinylated second antibody (6D2 IgG), (e) addition of streptavidin-horseradish peroxidase, and (f) development of color with o-phenylenediamine dihydrochloride-H2O2 and reading in a microplate reader at a wavelength of 490 nm. The method is highly sensitive with an optimal range of 10 to 100 ng/ml of the enzyme and is reproducible with intraday and interday precisions of 3.2 and 4.1%, respectively. The enzyme contents of 20 urine and 20 bile samples quantitated by this ELISA method were, respectively, 148 +/- 101 and 6380 +/- 3780 ng/ml (means +/- SD) which correlated well with their enzyme activities. Such a method for absolute quantitation of human beta-glucuronidase is essential for studying its pathophysiologic roles in cholelithiasis and carcinogenesis and can also be used clinically as an indicator for tissue damage or malignancy.
Ho
K J
KJ
Department of Pathology, University of Alabama, Birmingham.
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001646:Bile / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005966:Glucuronidase / Q000032:analysis* / Q000276:immunology / Q000652:urine; D006801:Humans; D051379:Mice; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D005966:Glucuronidase