国产血清人附睾蛋白4检测化学发光法的建立及初步性能评价

Establishment and performance evaluation of a domestic chemiluminescence immunoassay of serum HE4

目的 建立一种基于化学发光免疫分析法（CLIA）原理检测血清人附睾蛋白4（HE4）的方法 ,并进行方法性能验证.方法 利用一对HE4单克隆抗体分别偶联异硫氰酸荧光素（FITC）和碱性磷酸酶（ALP）,建立一个测定血清HE4的双抗体夹心的化学发光法.通过磁珠结合抗FITC抗体进行分离,加入ALP底物后光信号值与HE4浓度成正比.然后对方法的精密度、准确度、线性范围、特异性和稳定性等进行初步评价;并与市场上已有的国外进口HE4测定试剂盒比较.结果 方法的重复性精密度和日间精密度分别小于4.0%和5.0%;回收实验在90.0%~110.0%范围内;最低检出限和功能灵敏度分别为5.0 pmol/L和为15.0 pmol/L;线性范围是15.0~1500.0 pmol/L;试剂可以稳定12个月;与市场上已有进口试剂盒比对,其相关系数均大于0.99;本研究方法曲线下面积为0.896其他两种方法分别为为0.895和0.900.结论 本研究建立了一种敏感、重复性好和线性范围宽的血清HE4化学发光法.

Objective To establish and evaluate a domestic Chemiluminescence immunoassay （CLIA） for detecting serum human epididymis protein4 （HE4）. Methods To establish a double antibody sandwich CLIA for the determination of serum HFA. Fluoreseein isothioeyanate （FITC） and alkaline phosphatase （ALP） were used to label two different monoclonal antibodies of HE4. The separation could be realized by the magnetic particles coated with anti-FITC antibodies. Measure the relative light unit （RLU） after adding substrate solution, the RLU is proportional to the concentration of HFA. After evaluating the analytical performance including sensitivity, precision, accuracy, linearity, specificity under the optimized condition, this study compared the method with the commerical HFA kits as well. Results The precision of with-in lot , and with-out lot are less than 4. 0% and 5.0% respectively; Recovery is within 90. 0% - 110. 0% ; LoB is 5.0 pmol/L; functional sensitivity is 15.0 pmol/L; measure range is 15.0 - 1500. 0 pmol/L; the Correlation coefficient is bigger than 0. 99 comparing with commercial kits. The AUC （area under curve） of three methods is 0. 895,0. 900 and 0. 896 respectively. Conclusions This study established a sensitive CLIA method with high repeatability and wide measure range; it has good correlation with commercial HFA test kits.