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

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.

Determination of DTaP vaccine potency by multiplex immunogenicity testing using electrochemiluminescence

“…Precision of results was in the range or better than those published for singleplex ELISAs (Coombes et al 2012;Peng et al 2014;Reizenstein et al 1995) and bead-based multiplex assays (Bandiera et al 2019;Kadam et al 2019;Rajam et al 2019;Stenger et al 2011;van Gageldonk et al 2008;McAlister et al 2021). Furthermore, the enormous dynamic range of antibody quantitation over up to 4 logs scale using ECL detection represented a great advantage, because every single serum could be tested at the same starting dilution (1:400), thus avoiding repeat tests of sera with very low or very high IgG concentrations.…”

“…For many decades, enzyme-linked immunosorbent assay (ELISA) represented the standard readout method to quantify disease-or antigen-specific serum antibodies. Meanwhile, various multiplex testing platforms, such as bead-based flow cytometric assay and electrochemiluminescence (ECL) immunoassay (ECLIA) have been developed which have led not only to decreased sample and reagent volume needed but also to an increased testing throughput (Bandiera et al 2019;Bolton et al 2020;Bykonia et al 2023;Kadam et al 2019;McAlister et al 2021;Rajam et al 2019;Stenger et al 2011;Thomas et al 2022). Furthermore, multiplex assays were shown to perform at least similarly or even better with regards to sensitivity and reproducibility of antibody quantitation (Bolton et al 2020;Caboré et al 2016;Pickering et al 2002;Reder et al 2008;van Gageldonk et al 2008).…”

Statistical analysis plan for the OPTIMUM study: optimising immunisation using mixed schedules, an adaptive randomised controlled trial of a mixed whole-cell/acellular pertussis vaccine schedule

“… Compare vaccine antibody responses in each study group: vaccine antigen-specific IgG concentrations/titres will be measured at approximately ages 6, 7, 18, and 19 months using a multiplex fluorescent bead assay [ 4 , 5 ], for the following during stage 1 only (150 participants). Pertussis toxin (PT) Filamentous haemagglutinin (FHA) Pertactin (PRN) Tetanus toxoid (TT) Polyribosylribitol phosphate (Hib-PRP) 13-valent pneumococcal vaccine (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) Hepatitis B surface antigen (HBsAg) Diphtheria toxoid (DT) The seropositive thresholds for pertussis antigens are: ≥5 IU/mL for PT, FHA, and PRN The seroprotective thresholds other antigens are: ≥0.1 IU/mL for TT [ 6 ] and DT ≥10 mIU/mL for HBsAg ≥1 μ g/mL for Hib-PRP [ 7 ] ≥0.35 μ g/mL for pneumococcal serotypes [ 8 , 9 ] For each antigen, we will assess the ratio of the geometric mean titres (GMT) or concentrations (GMC) across study groups at each time point, the fold-rise in concentration from 18 (immediately before boosting with a dose of DTaP with inactivated polio vaccine) to 19 months (21-35 days after vaccination), and proportions of seroprotection or seropositivity.…”

“…For pneumococcal IgG responses, the lower limit of quantification ranges from 0.3 to 1.9 ng/mL [ 10 ]. For the other antigen-specific IgG, the lower limits of quantification range from 0.01 mIU/mL (DT) to 0.43 mIU/mL (FHA) [ 5 ].…”

“…Compare vaccine antibody responses in each study group: vaccine antigen-specific IgG concentrations/titres will be measured at approximately ages 6, 7, 18, and 19 months using a multiplex fluorescent bead assay [ 4 , 5 ], for the following during stage 1 only (150 participants). Pertussis toxin (PT) Filamentous haemagglutinin (FHA) Pertactin (PRN) Tetanus toxoid (TT) Polyribosylribitol phosphate (Hib-PRP) 13-valent pneumococcal vaccine (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) Hepatitis B surface antigen (HBsAg) Diphtheria toxoid (DT) …”