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Updated: Jan 12, 2026
A High-throughput Shigella-specific Bactericidal Assay
Published on: February 27, 2019
Development and qualification of a multiplexed immunoassay to assess the immunogenicity of Shigella vaccines
Paul W Rhyne1, Justine Sunshine1, Wayne Hogrefe2
1Gates Medical Research Institute, One Kendall Square Building 600, Suite 6-301, Cambridge, MA 02139, United States.
Insights
A new multiplex immunoassay accurately measures antibody responses to Shigella vaccine candidates. This assay is crucial for evaluating multivalent Shigella vaccines, especially in resource-limited settings.
Area of Science:
- Immunology
- Vaccinology
- Microbiology
Background:
- Shigella causes significant diarrheal disease in children, particularly in low- and middle-income countries (LMICs).
- Rising antibiotic resistance and limited healthcare access necessitate an effective Shigella vaccine.
- Next-generation Shigella vaccines targeting lipopolysaccharide (LPS) O-antigen require robust immunogenicity assays.
Purpose of the Study:
- To develop and qualify a multiplex immunoassay for evaluating antibody responses to Shigella vaccine candidates.
- To assess the assay's performance across multiple Shigella serotypes and vaccine components.
Main Methods:
- A Luminex-based multiplex immunoassay was created using purified LPS from five Shigella serotypes and Shigella IpaB protein.
- Assay performance was evaluated using pooled post-vaccination sera, assessing linearity, accuracy, precision, and specificity.
Main Results:
- The assay demonstrated excellent linearity (R² ≥ 0.98) and accuracy across multiple Shigella antigens.
- High precision was observed, with some variability noted for specific serotypes at high dilutions.
- The assay showed specificity for homologous antigens, with cross-reactivity patterns aligning with known LPS structural similarities.
Conclusions:
- A qualified multiplex immunoassay accurately and efficiently measures Shigella vaccine-induced antibody responses.
- This assay provides a scalable solution for evaluating multivalent Shigella vaccines in clinical trials, especially in LMICs.
Background:
Shigella is a major cause of diarrheal disease in young children worldwide, particularly in low- and middle-income countries (LMICs). The emergence of antibiotic resistance and limited access to healthcare in LMICs underscores the urgent need for an effective Shigella vaccine. Several next-generation Shigella vaccines are under development, many of which target the O-antigen component of lipopolysaccharide (LPS) from multiple serotypes to achieve broad protective coverage. Reliable, multiplexed assays are needed to evaluate vaccine immunogenicity across Shigella serotypes and vaccine platforms.
Methods:
A Luminex-based multiplex immunoassay was developed incorporating purified LPS from five Shigella serotypes (S. sonnei, S. flexneri 1b, 2a, 3a, and 6) along with Shigella IpaB protein and common vaccine carrier proteins. Assay performance was evaluated using pooled post-vaccination serum samples with qualification parameters including linearity, accuracy, precision, and specificity.
Results:
Dilutional linearity was confirmed in all antigens across a broad dynamic range (R2 ≥ 0.98), with slopes ranging from 0.91 to 1.16. Within-run and between-run accuracy and intra-assay and inter-assay precision met predefined criteria for all antigens, with variability primarily observed at high dilutions for S. flexneri 1b and 3a. The assigned lower and upper limits of quantitation (LLOQ-ULOQ) spanned up to two orders of magnitude per antigen. The multiplex assay demonstrated specificity for homologous antigens, with some cross-reactivity patterns reflecting known structural similarities among LPS serotypes.
Conclusion:
This multiplex immunoassay enabled accurate and efficient measurement of vaccine-induced antibody responses across multiple Shigella antigens and carrier proteins. It therefore offers a qualified scalable solution for evaluating multivalent Shigella vaccines in clinical trials, particularly in resource-limited settings.
