Abstract

FAJARDO, Esther María et al. Validation of an inhibition-type ELISA for quantifying Vi polysaccharide content in the Cuban Typhoid Vaccine vax-TyViä. Vaccimonitor [online]. 2006, vol.15, n.2, pp. 5-12. ISSN 1025-0298.

Validation of an inhibition-type ELISA intended for quantifying a vaccine antigen, Vi polysaccharide from Salmonella typhi, is reported here for the first time in a scientific paper. It is devoted to the quality control of the Cuban typhoid vaccine vax-TyViä. The test requires six steps: (1) Coating the plate with poly-L-lysine and then Vi polysaccharide; (2) Blocking with skimmed milk; (3) Inhibition (or neutralization) in tubes of rabbit Vi antiserum with Vi polysaccharide contained in the Standard Curve (concentrations from 1 - 32 μg/mL), Positive Control and vaccine samples (3 dilutions) respectively; (4) Neutralization of free Vi antibodies existing in the previous mixtures by the coating polysaccharide; (5) Recognition of Vi antibodies bonded to the plate (Anti-rabbit IgG-alkaline phosphatase) and (6) Revealing by means of the enzyme-substrate reaction. The studied validation parameters and their results were: (1) Precision, expressed as the coefficient of variation, at three different polysaccharide concentration levels comprising the vaccine specification range (35, 50 and 70 μg/mL) and evaluated in terms of Repeatability, Intermediate Precision (four analysts) and Reproducibility (six analysts): £ 20%; (2) Linearity (100*R2): 99.68%; (3) Detection Limit: 0.5 μg/mL;(4) Accuracy (recovery at three different sample dilutions): between 100 y 118%, and (5) Robustness: neither 1.5 h blocking nor ± 5 min for plate reading influences the test (p = 0.52 and 0.56 respectively); however, water quality greatly influences the results (p = 0.026); water for injection showed the best results. The test is appropriate for the purpose it was validated; it also allows evaluating in vitro immunogenicity of Vi polysaccharide.

Keywords : ELISA; Vi polysaccharide; Salmonella typhi; Typhoid Vaccine.

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