双波长微板赖氏法检测丙氨酸氨基转移酶方法的优化

Optimization of two-wavelength microplate Reitman-Frankel assay for ALT testing

目的对检测丙氨酸氨基转移酶(alanine aminotransferase,ALT)的双波长微板赖氏法进行优化。方法采用正交试验优化双波长微板赖氏法的空白试剂(磷酸缓冲液或纯化水)和Na OH浓度(0.5、1.0、1.5、2.0 mol/L),考察方法的稳定性、线性拟合度、精密性和准确性。结果以纯化水作空白,1.0 mol/L Na OH作终止溶液时,该方法具有很好的稳定性;线性拟合度好,R2=0.998 3>0.990 0;检测38和57卡门单位的丙酮酸标准溶液的板内A492/630值的变异系数均<10%,100%的样本检测结果在可接受范围内。结论优化的检测ALT的双波长微板赖氏法具有很好的稳定性、线性、精密性和准确性,且通量高,适用于大规模检测。

Objective To optimize the two-wavelength microplate Reitman-Frankel assay for ALT testing. Methods The blank reagent（PBS or purified water） and concentration of sodium hydroxide for two-wavelength microplate ReitmanFrankel assay were optimized by orthogonal test, and the optimized method was evaluated for stability, linearity, precision and accuracy. Results The optimized method showed good stability and linearity when purified water was used as bland reagent and 1. 0 mol / L sodium hydroxide as stop solution（R2 = 0. 998 3 0. 990 0）. Both the CVs of A492 / 630 values in intra-assay on 38 and 57 Karmen units of standard pyroracemic acid were less than 10%, and all the determination results of the samples were within the acceptable range. Conclusion The optimized two-wavelength microplate Reitman-Frankel assay showed good stability, linearity, precision and accuracy and high flux, which was suitable for large-scale determination of ALT.