Diagnosis Performance Evaluation Of Restructuring Bacillus Fastidious Uricase

The uricase method determination uric acid levels can be divided into twotypes: indirect and direct uricase method. Indirect uricase method use themethod of uricase coupling with aldehyde dehydrogenase and peroxidasedynamics which has high efficiency, but the high cost, too much interferencefactors and bad repeatability make it difficult to promotion of clinicalapplication. Now the direct uricase method only has end balance methodwhich has been IFCC recommended reference method, but its precision is lowand can be interfered by0.40mmol/L of serum xanthine and a few othermaterial, the method also has resistance for most serum components. Theindirect balance method is peroxidase coupling uricase and differentcolour-display reagent, the characters of repeatability, simple operation andcost savings lead to be used widely on clinical laboratory. But the biggestinterference factors are the xanthine of serum.The bacillus fastidious intracellular uricase gene has been cloned andapplied for the invention patent （patent no.200910103741.1, accept Genbankregistration number FJ393559）, the uricase have resistance for1.50mmol/Lxanthine. his topic research aim is to examine optimal conditions of thisuricase in indirect end balance method, and evaluation diagnosisperformance in detail, Establish uric acid kit preliminary model.The optimum conditions of the final optimized reagent as follows:R1in duplicate （PBS6.0） R2, a copy （pH9.2borax）HRP2.7U/ml （1.8U/ml） uricase0.6U/ml （0.2U/ml）4-AAP0.45mmol/L （0.3mmol/L） Toos4.2mmol/L （1.4mmol/L）NaN₃,0.05%NaN₃,0.05%Accordance with the relevant requirements of the NCCLS, the diagnosticperformance of the independent research and the uric acid reagent wereevaluated in detail: detection limit of the LOD=LOB+1.65SD=14.4umol/L, uric acid within the scope of0-1800umol/L have good linearity, linearregression equation is: Y=0.0003X-0.0012, R²=0.9971, reportable range:14.4-3510umol/L and the intra-precision are2.2%and3.0%, theinter-precision are4.0%and4.8%, the stability of the reagents save up more than half a year, reference interval: Male207-428umol/L, female140-330umol/L,bilirubin concentration<320umol/L, ascorbicacidconcentration<0.4mmol/L, the concentration of hemoglobin concentration<4g/L can’t significantly interfere the determination of uric acid.The interference of anti-xanthine: Biosino uric acid kit, Chengdu Mikeuric acid kit, Metrobiological uric acid kit, homemade uric acid kit, there areno significant interference within xanthine concentration0-1.2mmol/L. Andthere is obvious negative interference within xanthine concentration>0.3mmol/L, and with increasing concentrations of xanthine the degree ofnegative interference on the determination results is also growing. Theabove experiments show that the laboratory-made prokaryotic expression ofeukaryotic expression of uricase has a distinct advantage than enkaryoticexpression of uricase in the anti-xanthine, the evaluation of other performanceindicators also reached the level of similar products in the market. Provides adetailed laboratory date for the declaration of recombinant uricase apply as adiagnostic tool enzyme in vitro diagnostics, also laid the foundation for thedevelopment of recombinant uricase pharmaceutical preparations.