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

Area of Science:

• Immunology
• Biotechnology
• Analytical Chemistry

Background:

• Small volume assays are crucial for large-scale studies, especially in pediatric trials requiring multiple measures from single samples.
• Fluorescent bead-based technology (Bioplex/Luminex) enables high-throughput, simultaneous quantification of multiple analytes.
• Accurate serological assays are needed to assess immune responses to vaccines like tetanus-diphtheria-acellular pertussis (Tdap).

Purpose of the Study:

• To establish and optimize a 6-plex fluorescent bead-based immunoassay for measuring human IgG to individual Tdap antigens.
• To validate the assay for use with small sample volumes (2-4 µL) of human serum or plasma.
• To develop the first Tdap-specific multiplex immunoassay (MIA) in Australia.

Main Methods:

• Optimized microsphere preparation, antigen coating dose, and conjugation methods.
• Developed and validated an in-house reference serum with clinically relevant titers.
• Established assay running conditions, including detector antibody selection and R-Phycoerythrin labeling.
• Determined assay specificity and reproducibility through rigorous validation.

Main Results:

• Successfully established a 6-plex fluorescent bead-based immunoassay for Tdap antigens.
• Validated the assay for accurate quantification of human IgG antibodies from 2-4 µL of serum/plasma.
• Demonstrated assay specificity and reproducibility, aligning with pertussis-serological assay recommendations.

Conclusions:

• The developed Tdap-specific MIA is suitable for large-scale research and pediatric trials requiring minimal sample volumes.
• This assay provides a valuable tool for assessing immune responses to Tdap vaccination in Australia.
• The optimized protocol advances multiplex immunoassay capabilities for vaccine serology.