Hemagglutination Inhibition Test
Overview
The hemagglutination inhibition (HI) test is a classical serological assay used to detect and quantify dengue-specific antibodies in patient plasma or serum. It exploits the ability of dengue virus to agglutinate red blood cells — antibodies against dengue antigens inhibit this agglutination in a titre-dependent manner. The HI test is one of the oldest dengue serological methods and was the primary tool for primary/secondary infection classification before IgG-capture ELISA became standard.
The standard method is Clarke and Casals (1958): acetone-extracted dengue antigens for each serotype (DENV-1, -2, -3, -4) are prepared at four haemagglutinating units. Plasma samples are treated to remove non-specific inhibitors and tested in serial dilutions. The HI titre is the reciprocal of the highest plasma dilution that inhibits haemagglutination.
Key Points from Literature
Primary vs. secondary infection classification
The HI assay is used to classify infection status:
- Secondary infection: HI titre ≥1:2,560 in a single acute-phase plasma sample (WHO 1997 criterion). The high titre reflects anamnestic recall of cross-reactive antibodies from prior heterotypic exposure.
- Primary infection: HI titre <1:1,280 or rising paired titres with lower peak.
- This classification underpins the secondary-infection-specific PAIgG mechanism described in Oishi2003 - PAIgG and Thrombocytopenia in Secondary Dengue.
Relationship to PAIgG formation
In Oishi2003, HI titre was used to confirm secondary infection in all 83 enrolled patients; the 53-patient study cohort were all acute-phase secondary infections. Notably, no correlation was found between the PAIgG level and plasma HI titre within secondary infection patients (r = −0.130, P = 0.361) — suggesting that the overall antibody burden (as reflected by HI titre) does not drive PAIgG formation. This supports the model that viral antigen on the platelet surface, rather than circulating antibody titre, is the rate-limiting factor in immune complex formation on platelets.
Contradictions & Debates
- HI titre-based classification is less specific than plaque reduction neutralisation tests (PRNT) and is used less frequently in modern research settings; its threshold (≥1:2,560) for secondary classification can misclassify some late primary responses.
- The IgG:IgM ratio from paired ELISA is now preferred for primary/secondary classification in many research settings (see IgM-IgG Serology ELISA).
Related Pages
Sources
- Oishi2003 - PAIgG and Thrombocytopenia in Secondary Dengue (HI titer ≥1:2,560 = secondary classification; Clarke & Casals 1958 method; all four DENV serotype antigens; no correlation of HI titre with PAIgG levels)