title: Optimization of pH values to formulate the bireagent kit for serum uric acid assay.
abstract: A new formulation of the bireagent kit for serum uric acid assay was developed based on the effects of pH on enzyme stability. At 4 °C, half-lives of uricases from Bacillus fastidious and Arthrobacter globiforms were longer than 15 months at pH 9.2, but became shorter at pH below 8.0; half-lives of ascorbate oxidase and peroxidase were comparable at pH 6.5 and 7.0, but became much shorter at pH higher than 7.4. In the new formulation of the bireagent kit, Reagent A contained peroxidase, 4-aminoantipyrine, and ascorbate oxidase in 50 mM phosphate buffer at pH 6.5; Reagent B contained B. fastidious or A. globiforms uricase in 50 mM sodium borate buffer at pH 9.2; Reagents A and B were mixed at 4:1 to produce a final pH from 7.2 to 7.6 for developing a stable color. The new bireagent kit consumed smaller quantities of three enzymes for the same shelf life. With the new bireagent kit, there were linear responses of absorbance at 546 nm to uric acid up to 34 mM in reaction mixtures and a good correlation of uric acid levels in clinical sera with those by a commercial kit, but stronger resistance to ascorbate. Therefore, the new formulation was advantageous.
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, People's Republic of China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Huang
authors.forename: Yuanxiang
authors.initials: Y
authors.lastname: Chen
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.forename: Hua
authors.initials: H
authors.lastname: Zhao
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Hu
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.forename: Juan
authors.initials: J
authors.lastname: Liao
authors.forename: Gaobo
authors.initials: G
authors.lastname: Long
authors.forename: Fei
authors.initials: F
authors.lastname: Liao
mesh_terms: D000675:Ampyrone / Q000737:chemistry; D001774:Blood Chemical Analysis / Q000379:methods*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D012449:Safety; D014527:Uric Acid / Q000097:blood*
chemical_list: D007202:Indicators and Reagents; D000675:Ampyrone; D014527:Uric Acid; D010088:Oxidoreductases
keywords: ascorbate oxidase; bireagent kit; serum uric acid; stability; uricase