Optimising a 6-plex tetanus-diphtheria-pertussis fluorescent bead-based immunoassay

Sonia M McAlister; Anita H J van den Biggelaar; Ruth B Thornton; Peter C Richmond

doi:10.1016/j.mex.2021.101360

Optimising a 6-plex tetanus-diphtheria-pertussis fluorescent bead-based immunoassay

MethodsX. 2021 Apr 22:8:101360. doi: 10.1016/j.mex.2021.101360. eCollection 2021.

Authors

Sonia M McAlister^{1

2}, Anita H J van den Biggelaar^{1

2}, Ruth B Thornton^{1

2}, Peter C Richmond^{1

2

3}

Affiliations

¹ Vaccine Trials Group, Wesfarmers Centre of Vaccines & Infectious Disease, Telethon Kids Institute, Perth, Western Australia, Australia.
² Division of Paediatrics, School of Medicine, The University of Western Australia, Perth, Western Australia, Australia.
³ Departments of Immunology and General Paediatrics, Perth Children's Hospital, Perth, Western Australia, Australia.

Abstract

Small volume assays are required for large-scale research studies and in particular paediatric trials, where multiple measures are required from a single sample. Fluorescent bead-based technology (Bioplex/Luminex) allows high through-put and simultaneous quantification of multiple analytes in a single test. This technology uses sets of microspheres, each with a unique spectral address that can be coated with a different antigen of interest. Following the addition of a detector antibody, specific for the isotype of interest and labelled with R-Phycoerythrin, the bioplex reader determines the amounts of antigen-specific antibodies in each test sample relative to a reference standard. Here we outline the optimisations undertaken to establish a 6-plex fluorescent bead-based immunoassay that can accurately measure human IgG to individual tetanus-diphtheria-acellular pertussis (Tdap) antigens from 2 to 4 ul of human serum/ plasma. This protocol was adapted from previously published methods and aligns with current recommendations for developing pertussis-serological assays. To our knowledge, this is the first Tdap-specific multiplex immunoassay (MIA) established in Australia. All components were optimised and validated in-house including: microsphere preparation conditions, reference serum and QC development, and assay running.•Determining optimal antigen coating dose and conjugation method.•Optimising an in-house reference serum with clinically relevant titres.•Determining assay specificity and reproducibility.

Keywords: Antibody; Bioplex; Conjugation; FMIA; IgG; Luminex; MIA; Microsphere; Multiplex; Whooping cough.