应用FITC系统建立雌二醇直接竞争化学发光免疫分析方法
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摘要
目的利用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)系统建立人血清雌二醇(estradiol,E_2)直接竞争化学发光免疫分析方法。方法采用兔抗-FITC抗体、FITC-抗-E_2两步包被制备预包被板,采用直接竞争法原理建立E_2化学发光免疫分析检测体系,并对FITC-anti-E_2包被浓度、酶标抗原稀释比例、缓冲体系、反应时间等各种影响因素进行优化。结果本系统线性范围为30～3000 pg/mL,空白检测限4.8pg/mL,定量检测限30 pg/mL,分析内精密度5.6%,批间精密性8.7%,平均回收率95.9%,与雌三醇、睾酮、孕酮均无明显交叉反应,与贝克曼全自动化学发光试剂同时测定164份样本,二者测定结果的相关系数r为0.979。结论成功建立了雌二醇直接竞争化学发光免疫分析方法 ,该方法性能指标优秀,具有一定的临床应用价值。
Objective A direct competitive chemiluminescence immunoassay for human serum estradiol(E_2)was established using the fluorescein isothiocyanate(FITC) system. Methods Two-step coating of rabbit antiFITC antibody and FITC-anti-E_2 were used to prepare the pre-clad plate. The direct competitive method was applied, investigated and optimized, to establish the new CLIA detection system for E2. The E_2 chemiluminescence immunoassay detection system was established by the principle of direct competition,considering a variety of factors such as dilution ratio of enzyme-labeled antigen, the concentration of FITCanti-E_2 coating,buffer system and incubate time. Results The linear detection range was 30 ～3000 pg/mL in this system,with the parameters as:the limit of the detection 4.8 pg/mL,the limit of quantitative detection 30 pg/mL,the precision within the analysis 5.6%,the inter-assay precision 8.7%,the recovery rate for accuracy95. 9%. There were no cross reactions with estriol, testosterone and progesterone. The simultaneous determination of 164 samples with Beckman's automated chemiluminescence reagents yielded a correlation coefficient of 0. 979. Conclusion The CLIA system for detecting E2 has been established, which has an excellent performance indicators and can be widely used in the clinical testing.
Objective A direct competitive chemiluminescence immunoassay for human serum estradiol(E_2)was established using the fluorescein isothiocyanate(FITC) system. Methods Two-step coating of rabbit antiFITC antibody and FITC-anti-E_2 were used to prepare the pre-clad plate. The direct competitive method was applied, investigated and optimized, to establish the new CLIA detection system for E2. The E_2 chemiluminescence immunoassay detection system was established by the principle of direct competition,considering a variety of factors such as dilution ratio of enzyme-labeled antigen, the concentration of FITCanti-E_2 coating,buffer system and incubate time. Results The linear detection range was 30 ～3000 pg/mL in this system,with the parameters as:the limit of the detection 4.8 pg/mL,the limit of quantitative detection 30 pg/mL,the precision within the analysis 5.6%,the inter-assay precision 8.7%,the recovery rate for accuracy95. 9%. There were no cross reactions with estriol, testosterone and progesterone. The simultaneous determination of 164 samples with Beckman's automated chemiluminescence reagents yielded a correlation coefficient of 0. 979. Conclusion The CLIA system for detecting E2 has been established, which has an excellent performance indicators and can be widely used in the clinical testing.
引文
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[9]李琦,朱勇,贾卫,等.抗FTTC单克隆抗体的制备及初步应用[J].细胞与分子免疫学杂志,2003,19(2):180.
[10]陈茶,孔海霞,雷鹏,等.应用FITC系统建立非均衡竞争雌二醇(E2)化学发光法[J].国际检验医学杂,2012,33(6):676-678.
[2]贺敏.孕酮化学发光免疫分析方法的建立[J].标记免疫分析与临床,2017,24(7):812-815.
[3] CARON E,SHEEDY C,FARENHORST A,et al. Development of competitive ELISAs for 17β-estradiol and 17β-estradiol+estrone+estriol using rabbit polyclonal antibodies[J]. Journal of EnvironmentalScienceand Health:Part B,2010,45(2):145-151.
[4]詹屋强,王弘.小分子物质酶联免疫分析方法的研究进展[J].食品安全质量检测学报,2015,6(3):857-861.
[5]王克珍.雌二醇化学发光免疫测定法的建立[J].临床检验杂志,2004,22(4):252-253.
[6]赵丹慧,李成文,薛延等.高价特异半抗原雌二醇抗体及酶标雌二醇复合物的研制[J].卫生研究1998,27(增刊):191-193.
[7]金声,周浣芳,常文保,等.酶联免疫吸附法直接测定血清雌二醇[J].分析化学,1994,22(2):115-119.
[8]刘珒,雷红涛,孙远明,等,雌二醇间接竞争酶联免疫吸附方法的建立[J].食品科学,2012,33(8):146-150.
[9]李琦,朱勇,贾卫,等.抗FTTC单克隆抗体的制备及初步应用[J].细胞与分子免疫学杂志,2003,19(2):180.
[10]陈茶,孔海霞,雷鹏,等.应用FITC系统建立非均衡竞争雌二醇(E2)化学发光法[J].国际检验医学杂,2012,33(6):676-678.