Guglielmo-Viret V, Thullier P
Groupe de Biotechnologie des Anticorps, Département de Biologie des Agents Transmissibles, Centre de Recherche du Service de Santé des Armées, BP 87, 38702, La Tronche, France.
J Immunol Methods. 2007 Dec 1;328(1-2):70-8. doi: 10.1016/j.jim.2007.08.003. Epub 2007 Aug 29.
An electrochemiluminescence (ECL) assay for the detection of the B chain of ricin (RCA-B) in a 96-well plate format was developed in parallel with a colorimetric ELISA utilizing the same pair of antibodies. Sensitivity results were interpreted with the ANOVA and Tukey statistical tests, allowing a direct comparison between the two technologies, that can probably be extended to other protein antigens such as toxins. Reproducibility, repeatability and rapidity of the two techniques were also compared. The ELISA assay utilized an alkaline phosphatase conjugate for signal generation. After optimization, its limit of detection was 400 pg of RCA-B per ml buffer, with an intra-day standard deviation (SD) of 2.2% of the mean and an inter-day SD of 5.1%. The ECL assay utilized ruthenylated antibodies for detection. The ECL measurement was carried out using a Sector PR 400 plate reader. After optimization, its limit of detection was 50 pg of RCA-B per ml buffer, with an intra-day SD of 4.1% of the mean and an inter-day SD of 4.3%. Starting from a pre-coated plate, the ELISA assay was completed in 7 h and the ECL assay took 2.5 h. While reproducibility and repeatability of the two assays were equivalent, this ECL assay in plate format had an 8-fold better sensitivity for RCA-B detection than the colorimetric ELISA in buffer and in various matrices. The ECL assay was also three times faster, and retained the robustness and convenience of the 96-well plate format.
我们开发了一种用于在96孔板中检测蓖麻毒素B链（RCA-B）的电化学发光（ECL）检测方法，并同时开发了一种使用相同抗体对的比色酶联免疫吸附测定（ELISA）。使用方差分析（ANOVA）和Tukey统计检验来解释灵敏度结果，从而可以直接比较这两种技术，这种比较可能还可以扩展到其他蛋白质抗原，如毒素。我们还比较了这两种技术的重现性、重复性和速度。ELISA检测使用碱性磷酸酶偶联物来产生信号。优化后，其检测限为每毫升缓冲液中400 pg的RCA-B，日内标准偏差（SD）为平均值的2.2%，日间SD为5.1%。ECL检测使用钌标记抗体进行检测。使用Sector PR 400酶标仪进行ECL测量。优化后，其检测限为每毫升缓冲液中50 pg的RCA-B，日内SD为平均值的4.1%，日间SD为4.3%。从预包被板开始，ELISA检测在7小时内完成，ECL检测耗时2.5小时。虽然两种检测的重现性和重复性相当，但这种板格式的ECL检测对RCA-B检测的灵敏度比缓冲液和各种基质中的比色ELISA高8倍。ECL检测速度也快三倍，并且保留了96孔板格式的稳健性和便利性。
