ELISA定量检测抗dsDNA抗体试剂盒性能验证方法的探讨

Verification for performance of anti-dsDNA antibody quantitative ELISA kit

目的探讨ELISA定量检测抗双链DNA抗体(下简称抗ds DNA抗体)试剂盒的性能验证方法。方法依据美国临床和实验室标准化协会(Clinical and Laboratory Standards Institute,CLSI)批准的EP15-A2文件方法验证精密度。通过检测过往室间质量评价(external quality assessment,EQA)质评物、与比较试剂进行比较、回收试验等3种方法验证准确度。通过检测空白样本验证检测低限(lower limit of detection,LLD)。通过《CNAS-CL39:医学实验室质量和能力认可准则在临床免疫学检验领域的应用说明》、CLSI C28-A3C文件提供的方法验证临界值。通过改良的Doumas法验证线性范围。结果实测批内、批间变异系数均小于厂家声称的变异系数或按EP15-A2公式计算的验证值。检测EQA质评物阴性符合率为98.4%(63/64),阳性符合率为100%(20/20),总符合率为98.8%(83/84)。与比较试剂的阴性符合率为91.2%(52/57),阳性符合率为87.0%(40/46),总符合率为89.3%(92/103),Kappa值为0.783(P=0.062),两种试剂具有优秀的一致性。平均回收率为99.65%。3种方法验证准确性均通过。实测LLD为0.5 IU/m L,低于厂家声称的1 IU/m L。按CNAS-CL39方法,实测的临界值为18.51IU/m L,与说明书建议的20 IU/m L接近。按C28-A3C方法,临界值验证也通过。线性回归方程为Y=0.978 8X-3.125 4,r^2为0.996 1,截距项-3.125 4与0差异无统计学意义(t=-0.772,P=0.483)。线性范围是1.6~212.5 IU/m L,与厂家声明一致,线性验证通过。结论候选抗ds-DNA抗体试剂盒的精密度、准确度、LLD、临界值、线性范围与厂家的声明一致,能满足临床诊断与治疗的需要。本研究采用的一系列验证方法为ELISA定量检测试剂的性能验证提供了新思路。

Objective To explore the verification methods for the performance of quantitative detection kit of anti-ds DNA antibodiy with enzyme-linked immunosorbent assay（ ELISA）. Methods The precision was verified according to the EP15-A2 document approved by the American Clinical and Laboratory Standards Institute（ CLSI）. The accuracy was verified by detecting the samples of previous external quality evaluation（ EQA）,compared with the comparative kits and recovery test. The lower limit of detection（ LLD） was calculated by the results of blank samples. The cut-off value was verified according to the C28-A3C document approved by CLSI and ＂ CNAS-CL39： Guidance on the Application of Accreditation Criteria for the Medical Laboratory Quality and Competence in the Field of Clinical Qualitative Immunology＂ respectively. The improved Doumas method was used to verify the range of linearity. Results The measured intra-assay and inter-assay coefficients of variation were lower than those announced by the manufacturer or the calculated values according to the EP15-A2 document. The coincidence rates for negative and positive EQA samples between detected and expected values were 98. 4%（ 63/64） and 100%（ 20/20） respectively. The total coincidence rate was 98. 8%（ 83/84）. The coincidence rate for negative and positive samples between the results from candidate and comparative kits were 91. 2%（ 52/57） and 87. 0%（ 40/46）respectively. The total coincidence rate was 89. 3%（ 92/103） and the Kappa value was 0. 783（ P = 0. 062）,which implied excellent consistency between the two kits. The mean recovery rate was 99. 65%. The measured LLD was 0. 5 IU/m L which was lower than 1IU/m L as claimed by the manufacturer. The measured cut-off value according to the CNAS-CL39 document was 18. 51 IU/m L,which was close to 20 IU/m L announced by the manufacturer. Based on the C28-A3 C method,the cut-off value could be approved. The linear regression equation was Y = 0. 978 8X-3. 125 4,r2= 0. 996 1. There was no statistical difference between the intercept（-3. 125 4） and 0（ t =-0. 772,P = 0. 483）. The range of linearity was from 1. 6 to 212. 5 IU/m L,which was consistent with the values declared by the manufacturer. All the verifications of the five performances above-mentioned could be passed. Conclusion The precision,accuracy,LLD,cut-off value and range of linearity of the candidate quantitative ELISA kit for anti-ds DNA antibody were consistent with the statement of the manufacturer,which indicated the performance of the kits may meet the requirements of clinic diagnosis and treatment. A series of methods used in this study provided a simple protocol for verifying the performance of quantitative ELISA kits.