This study introduces a new ELISA method for measuring anti-Vi IgG antibodies, which is cost-effective, precise, and reproducible. It uses Vi polysaccharide as the coating antigen, performs well across multiple laboratory settings, and ensures results can be standardized and compared globally. This method is compatible with both clinical and research applications, offering a reliable tool for assessing immune responses to typhoid vaccines.
Salmonella enterica serovar Typhi is the causative agent of typhoid fever, leading to approximately 9.2 million cases and 110,000 deaths annually worldwide, particularly among children and young adults in low- and middle-income countries. The Vi capsular polysaccharide, a virulence factor and the primary antigen for vaccines, has been targeted for vaccine development. Over the years, various vaccines have been created, including the oral live-attenuated Ty21a vaccine and injectable plain Vi polysaccharide vaccines. However, these vaccines are ineffective in children under two years of age, leaving this population underserved. Conjugate vaccines, such as Typhoid Conjugate Vaccines (TCVs), which link the Vi polysaccharide to carrier proteins, have been developed to protect infants and young children. Three TCVs have achieved prequalification status by the World Health Organization (WHO).
Recent efforts focus on developing multivalent vaccines targeting not only Salmonella Typhi but also Salmonella Paratyphi A and other invasive non-typhoidal Salmonella (iNTS) strains. This approach aims to broaden protection against multiple Salmonella diseases.
The development of an accurate, high-throughput, and cost-effective method to measure anti-Vi IgG levels is critical, especially as Vi is the target antigen for current monovalent TCVs and will be included in new multivalent combinations.
Historically, establishing an international anti-Vi reference standard serum and optimizing immunoassays for anti-Vi IgG measurement have been challenges. Initial efforts included freeze-dried standard sera, such as TYS, derived from horse immunization in 1935. Subsequent collaborative studies with sera from vaccinees led to the development of additional standards, including the 10/126 and Vi-IgGR1 standards. The 16/138 standard, a pooled serum from subjects immunized with Vi and Vi-TT vaccines, was later established as the WHO international standard with an assigned 100 IU/mL concentration.
This study introduces a new ELISA method designed to measure anti-Vi IgG antibodies, expressed in ELISA Units/mL (EU/mL). The assay was characterized for precision, dilution linearity, and specificity, and it was calibrated against the 16/138 international standard. Results demonstrated the assay’s suitability for clinical studies, allowing expression of anti-Vi IgG levels in EU/mL, µg/mL, or IU/mL for cross-study comparisons.
Key Steps in the ELISA Development:
- Antigen Preparation: Vi polysaccharide from Citrobacter freundii was used as the coating antigen.
- Assay Setup: Plates were coated with Vi, followed by blocking steps and sample addition. The assay incorporated primary antibodies, detection antibodies (alkaline phosphatase-conjugated goat anti-human IgG), and substrate.
- Calibration: The assay was calibrated against the 16/138 standard, establishing conversion factors between EU/mL, IU/mL, and µg/mL.
- Validation: The assay’s performance was compared to a commercial ELISA kit, demonstrating comparable precision and specificity.
This customized ELISA offers a cost-effective and reliable alternative to commercial kits, with the added advantage of flexibility and potential miniaturization for increased throughput. The assay’s ability to express results in multiple units facilitates cross-study comparison, enhancing its utility in clinical research and vaccine evaluation.
