Comparison of an electrochemiluminescence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain (RCA-B).
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.
Guglielmo-Viret
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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.
Thullier
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P
D000818:Animals; D003124:Colorimetry / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D015203:Reproducibility of Results; D012276:Ricin / Q000032:analysis*; D012680:Sensitivity and Specificity; D013995:Time
D012276:Ricin