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

J Immunol Methods. 2008 Jun 1;335(1-2):79-89. doi: 10.1016/j.jim.2008.02.018. Epub 2008 Mar 26.


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. Serum IgG antibodies to B. pertussis, Diphtheria and Tetanus can be measured easily, specific and reproducible using the pentaplex MIA. The pentaplex MIA shares features of the ELISA with the additional advantages of high sample throughput and small sample volumes and antigen required.

Publication types

  • Comparative Study
  • Validation Study

MeSH terms

  • Adhesins, Bacterial / immunology
  • Antibodies, Bacterial / blood*
  • Bacterial Outer Membrane Proteins / immunology
  • Bordetella pertussis / immunology*
  • Diphtheria Toxin / immunology*
  • Diphtheria-Tetanus-Pertussis Vaccine / immunology*
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Immunoassay / methods*
  • Immunoassay / standards
  • Microspheres
  • Pertussis Toxin / immunology
  • Reproducibility of Results
  • Tetanus Toxin / immunology*
  • Virulence Factors, Bordetella / immunology*


  • Adhesins, Bacterial
  • Antibodies, Bacterial
  • Bacterial Outer Membrane Proteins
  • Diphtheria Toxin
  • Diphtheria-Tetanus-Pertussis Vaccine
  • Tetanus Toxin
  • Virulence Factors, Bordetella
  • filamentous hemagglutinin adhesin, Bordetella pertussis
  • pertactin
  • Pertussis Toxin