Validation of the modified hemagglutination inhibition assay (mHAI), a robust and sensitive serological test for analysis of influenza virus-specific immune response

• Published in: Journal of clinical virology Vol. 56; no. 4; pp. 323 - 330
• Main Authors: Morokutti, A, Redlberger-Fritz, M, Nakowitsch, S, Krenn, B.M, Wressnigg, N, Jungbauer, A, Romanova, J, Muster, T, Popow-Kraupp, T, Ferko, B
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2013
• Subjects: Allergy and Immunology; Animals; Antibodies, Viral - blood; Bioassay validation; Erythrocytes - immunology; Ferrets - immunology; Ferrets - virology; GxP analysis; Hemagglutination Inhibition Tests - methods; Hemagglutinins, Viral - analysis; Humans; ICH guideline; Immunogenicity; Infectious Disease; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza serology; Influenza virus; Orthomyxoviridae Infections - immunology; Reproducibility of Results; Sensitivity and Specificity; Serum Albumin, Bovine; Vaccine efficacy/effectiveness; Viral Load; GCLP; IVR; good clinical laboratory practise; NYMC; receptor destroying enzyme; hemagglutination assay; hemagglutination inhibition assay; SPF; World Health Organisation; GLP; good clinical practise; red blood cell; RBC; RDE; Bioassay validation; hemagglutination unit; good laboratory practise; Influenza serology; geometric mean titre; GxP analysis; modified; NIBSC; ICH guideline; GxP; WHO; cytopathic effect; Vaccine efficacy/effectiveness; m; influenza virus resource; GMT; ICH; HAI; GCP/GLP/GCLP; Immunogenicity; GCP; International Conference on Harmonisation; National Institute of Biological Standards and Controls; CPE; HA; New York Medical College; specific pathogen free; HAU
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/j.jcv.2012.12.002

Abstract Background The hemagglutination inhibition assay (HAI) is universally regarded as the gold standard in influenza virus serology. Nevertheless, difficulties in titre readouts are common and interlaboratory variations are frequently reported. Objective We developed and validated the modified HAI to facilitate reliable, accurate and reproducible analysis of sera derived from influenza vaccination studies. Study design Clinical and preclinical serum samples, NIBSC reference sera and seasonal influenza virus type A (H1N1 and H3N2) and type B antigens were employed to validate the mHAI. Moreover, pandemic virus strains (H5N1 and H1N1pdm09) were used to prove assay robustness. Results Utilisation of a 0.08% solution of stabilised human erythrocytes, assay buffer containing bovine serum albumin and microscopical plate readout are the major differences between the modified and standard HAI assay protocols. Validation experiments revealed that the mHAI is linear, specific and up to eightfold more sensitive than the standard HAI. In 95.6% of all measurements mHAI titres were precisely measured irrespective of the assay day, run or operator. Moreover, 96.4% (H1N1) or 95.2% (H3N2 and B), respectively, of all serum samples were determined within one dilution step of the nominal values for spiked samples. Finally, the mHAI results remained unaffected by variations in virus antigens, erythrocytes, reagents, laboratory location, sample storage conditions or matrix components. Conclusion The modified HAI is easy to analyse, requires only a single source of erythrocytes and allows utilisation of numerous influenza virus antigens, also including virus strains which are difficult to handle by the standard HAI (e.g. H3N2, H5N1 and H1N1pdm09).