CA19-9时间分辨荧光免疫层析检测方法的建立

Establishment of time-resolved fluorescence immunochromatographic assay for detection of carbohydrate antigen 19-9

摘要:

本研究旨在建立一种定量检测血清中CA19-9含量的时间分辨荧光免疫层析检测方法。采用双抗体夹心法与荧光免疫层析技术，以羧基荧光微球和NC膜为载体将CA19-9配对抗体进行标记和包被，制备CA19-9检测试纸条。通过标记、包被抗体量对工艺进行优化，并通过线性范围、最低检出限、精密性等性能指标对CA19-9时间分辨荧光层析检测方法进行评价。最终确定20 μL荧光微球的标记抗体量为80 μg，检测线包被抗体浓度为1.5 mg/mL时，检测时间为15 min，线性范围为12.5–800 U/mL，最低检出限为6.32 U/mL，批内精密性与批间精密性均小于15%，平均回收率为101%，与罗氏电化学发光检测试剂盒平行检测50份临床样本，两者相关系数为0.980 6。初步建立了定量检测血清中CA19-9的荧光免疫层析检测方法，有较好的临床应用前景。

关键词:CA19-9，荧光免疫层析，定量检测，快检

Abstract:

To establish a time-resolved fluorescence immunochromatographic assay for quantitative determination of carbohydrate antigen 19-9 (CA19-9) in serum, we prepared CA19-9 test strips by integrating double-antibody sandwich method and fluorescence immunochromatography technique. Carboxy fluorescent microspheres and nitrocellulose membrane were used as carriers for labeling and coating CA19-9 pairing antibodies. We optimized the process by adjusting the amount of labeling and coating antibody. According to the linear range, lowest detection limit and precision, We evaluated the time-resolved fluorescence immunochromatographic assay of CA19-9. When the amount of labeled antibody was 80 μg for 20 μL fluorescent microspheres, and the concentration of coated antibody on the test line was 1.5 mg/mL, the optimal reaction time was 15 minutes. Assay linear range was 12.5 to 800 U/mL and the minimum detection limit was 6.32 U/mL. The Within-run and between-run coefficient of variation were less than 15%. Average recovery rate was 101%. By detecting 50 clinical samples in parallel with Roche electrochemical luminescence detection kit, correlation coefficient was 0.980 6. The experiment, initially established a fluorescence immunochromatographic detection method to quantitative detection of serum CA19-9, which has a good clinical application prospect.

Key words:carbohydrate antigen 19-9, fluorescence immunochromatography, quantitative detection, rapid test