Development and validation of a multiplex immunoassay for the simultaneous determination of serum antibodies to Bordetella pertussis, diphtheria and tetanus

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• 作者：Pieter G.M. van Gageldonk ; Frank G. van Schaijk ; Fiona R. van der Klis ; Guy A.M. Berbers
• 关键词：Multiplex immunoassay ; ELISA ; Bordetella pertussis ; Diphtheria ; Tetanus ; Luminex
• 刊名：Journal of Immunological Methods
• 出版年：2008
• 出版时间：1 June 2008
• 年：2008
• 卷：335
• 期：1-2
• 页码：79-89
• 全文大小：1179 K

文摘

To increase testing of vaccine induced humoral immunity in immune surveillance studies and vaccine trials, a rapid and simple microsphere-based multiplex assay (pentaplex) was developed for the quantitation of IgG serum antibodies directed against the Bordetella pertussis antigens: Pertussis Toxin (Ptx), Filamentous hemagglutinin (FHA), Pertactin (Prn) and to Diphtheria toxin and Tetanus toxin. All individual antigens were covalently linked to carboxylated microspheres. The method was validated with different serum panels (n = 60–78 samples). With the Multiplex Immunoassay (MIA) no evidence for bead interference between monoplex and pentaplex was found. The specificity of the method was shown by a heterologous inhibition of < 16 % and homologous inhibition of > 92 % . The pentaplex MIA appeared sensitive with lower limits of quantitation (LLOQ) well below those for ELISA (enzyme-linked immuno-sorbant assay). Assay reproducibility was high with intra-assay variability less than 10 % and inter-assay variability below 14 % . The reproducibility of the bead conjugation was good and beads could be stored up to at least 6 months without quality reduction. Importantly, the correlation of the pentaplex MIA with the individual ELISAs was excellent, R > 0.98 for the Pertussis antigens and R = 0.95 for Diphtheria and R = 0.98 for Tetanus.