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Updated: May 12, 2026
Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test
Published on: July 14, 2023
Comparative evaluation of performance and precision of multiplex immunoassays measuring human IgG to Streptococcus
Michael Morici1,2, Jill Gilmour3,4, Omar Rossi5
1The Australian Strep A Vaccine Initiative (ASAVI), Australia.
Insights
Two qualified multiplex immunoassays for Streptococcus pyogenes (Strep A) vaccine development show comparable precision and repeatability. These validated assays are suitable for clinical trials and seroepidemiological studies of Strep A immunity.
Area of Science:
- * Infectious Disease Immunology
- * Vaccinology
- * Clinical Assays
Background:
- * *Streptococcus pyogenes* (Group A Streptococcus, Strep A) is a major global pathogen with significant disease burden and sequelae.
- * No licensed Strep A vaccines are available, and correlates of protection are not established.
- * Standardized assays are crucial for measuring vaccine-induced immune responses to support Strep A vaccine development and licensure.
Purpose of the Study:
- * To directly compare two qualified multiplex immunoassays for detecting human IgG antibodies against Strep A vaccine antigens.
- * To evaluate the precision and repeatability of MSD and Luminex platforms for Strep A serology.
Main Methods:
- * Comparison of MSD (electrochemiluminescence) and Luminex (fluorescent) multiplex immunoassay platforms.
- * Use of standardized intravenous immunoglobulin dilutions as reference standards.
- * Evaluation of assay precision using samples from human volunteers targeting four Strep A antigens: Streptolysin O, *S. pyogenes* cell envelope protein, *S. pyogenes* adhesion and division protein, and group A carbohydrate.
Main Results:
- * Both MSD and Luminex platforms demonstrated comparable intermediate precision and repeatability.
- * Variance increased in samples with very low antibody concentrations.
- * Operator variability and day-to-day factors did not significantly impact assay results, with inter-assay CVs for standards below 15%.
Conclusions:
- * Both multiplex immunoassay platforms are suitable for quantifying human IgG to Strep A vaccine antigens.
- * These validated assays can support early-phase clinical trials and seroepidemiological studies for Strep A vaccines.
Introduction:
Streptococcus pyogenes (Group A Streptococcus, Strep A) is a Gram-positive bacterium and one of the most significant global bacterial pathogens, with disproportionate disease burden affecting low and middle-income countries. Despite substantial mortality and immune sequelae caused by Strep A, no licensed vaccines exist, and correlates of protection remain unestablished. Advancing Strep A vaccine development requires standardized and qualified assays to accurately measure vaccine-induced immune responses to support vaccine development and licensure/post-licensure monitoring. This study reports a direct comparison of two qualified multiplex immunoassays designed to detect human IgG antibodies targeting four distinct Strep A vaccine antigens.
Methods:
The MSD assay platform employs positional multiplexing with electrochemiluminescent detection, while the Luminex system uses bead-based multiplexing with fluorescent detection. Both assays used standardized intravenous immunoglobulin dilutions as reference standards. Assay precision was evaluated by analyzing eight samples from human volunteers selected to span the dynamic range for the four antigens Streptolysin O, S. pyogenes cell envelope protein, S. pyogenes adhesion and division protein, and group A carbohydrate.
Results:
Both platforms demonstrated comparable intermediate precision and repeatability profiles. Increased variance was observed in samples with very low antibody concentrations. Neither operator variability nor day-to-day factors significantly impacted assay results. Additionally, inter-assay coefficients of variation for standards remained below 15% within the pre-established quantitative ranges for both platforms.
Discussion:
The findings demonstrate that both multiplex immunoassays show equivalent precision and repeatability for quantifying human IgG to four Strep A vaccine antigens, making both platforms suitable for supporting early clinical trials and seroepidemiological studies.
