影响α-淀粉酶参考方法准确测量因素的探讨

Exploration of the Influencing Factors on the Accuracy of Results for α-Amylase Reference Method

目的 通过对影响α-淀粉酶参考方法测量准确性的主要因素进行探讨,以帮助参考实验室更好地建立和运行该参考方法.方法 在其他因素不变的条件下,只改变实验中的一个因素,检测2009A,2009B（指2009年RELA样本,下同）各10次,计算其与原方法检测结果的相对偏倚及统计学意义（允许偏倚范围设定为参考方法等效限的1/4,即±1.25%）.主要探讨的影响因素有：a.反应液和样本加入步骤：a1.反应液和样本分步加入;a2.反应液和样本同时加入.b.加样方式：b1.手工加样,加样前润洗移液器,加样后不洗;b2.手工加样,加样前、后均不润洗移液器;b3.手工加样,加样前不洗,加样后回洗移液器;b4.稀释配液仪加样.c.混匀方式：c1.反应全程不搅拌;c2.搅拌至反应延迟时间（delay time）的2/3时停止;c3.反应全程搅拌.d.α-葡萄糖苷酶浓度：反应液中溶液2和α-葡萄糖苷酶（活性为1 014 ku/L）的比例为d1 100：1,d2 100：0.5,d3 100：0.8和d4 100：1.2.e.调节起始试剂pH方式：e1.静止条件下达到方法要求;e2.搅拌条件下达到方法要求.结果 ①a2,b1,c1,d4方法检测样本（2009A,2009B,下同）结果,相对偏倚为-0.44%～0.40%（t=-1.00～4.45,P值均＞0.05,差异均无统计学显著性意义）.②b2,b3和c3方法检测结果的相对偏倚为-2.14%～17.0%（t=-37.21～26.50,P值均＜0.05,差异均有统计学显著性意义）.③d2,d3方法检测两样本结果的相对偏倚为-0.47%～-0.04%（t=1.191～12.92,2009A样本结果P值均＞0.05,差异无统计学显著性意义,2009B样本结果P均＜0.05,差异有统计学显著性意义）.④e2方法检测两样本结果的相对偏倚为-0.48%和0.11%（t=2.523,1.728,2009A样本P值＜0.05,差异有统计学显著性意义,2009B样本P值＞0.05,差异无统计学显著性意义）.结论 通过对实验中常见影响因素的分析,优化了方法步骤,对参考方法的建立和运用具有一定的实用价值.

Objective To discuss the reference method for a-amylase much better, the main influencing factors was evaluated. Methods Changed one factor while the others kept invariant,and measured the samples of 2009 RELA A,B for 10 times, then computed the bias compared to the standard one （the allowed bias was 1/4 of the equivalent limit, that was ±1.25 %）. The Influencing factors were as follows ： a. The process of adding the reaction reagent and samples ： al. adding the reaction re-agent first,then the samples;a2, adding the reaction reagent and samples at the same time. b. The way of adding the sam-pies： bl. by the way of manual,rinsing the pipet before use with the sample; b2. by the way of manual,no rinsing; b3. by the way of manual,rinsing the pipet after adding samples;b4, adding samples with Microlab500C. c. The way of mixing the reac- tion reagent and samples：c1, no stir during reaction; c2. stir to the 2/3 delay time; c3：stir until the end. d. The concentration of a-glucosidase：the ratio of the reaction reagent 2 and a-glucosidase was dl 100 ： 1,d2 100 ： 0. 5,d3 100 ： 0.8 and d4 100 ： 1.2. e. The way of adjusting the pH of start reagent：el, to the required condition without stirring; e2. to the requirement with stirring. Results ① .the bias for method a2,bl,cl,d4 measuring samples （2009A,2009B,next was the same） were -0. 44%-0. 40% （t=-1.00-4. 45 ,all the P〉0.05） and there was not statistically significant for all, ②the bias for meth-od b2 ,b3 ,c3 were -2.14% -17.0% （t=-37.21-26.50 ,all the P〈0. 05） and there was statistical significance for all. ③ the bias for method d2,d3 were -0. 47%-0.04% （t=l. 191-12.92,P-value of 2009A〉0. 05） and there was not sta-tistically significant for 2009A, P-value of 2009B〈0.05 ,and there was statistical significance for 2009B. ④the bias for meth-od e2 were - 0.48%, 0.11% respectively （t = 2.523,1.728, P-value of 2009 A〈0.05 ） and there was statistically significant for 2009A,P-value of 2009B〉0.05,and there was not statistical significance for 2009B. Conclusion Through analyzing the main influencing factors of reference method for a-amylase,optimized the test process. It is useful for establishing and run- ning the method well.