Development of flow cytometric opsonophagocytosis and antibody-mediated complement deposition assays for non-typeable Haemophilus influenzae

Stephen R. Thomas*, Stephanie Leung, Katy Knox, Tom M.A. Wilkinson, Karl J. Staples, Pascal Lestrate, Dominique Wauters, Andrew Gorringe, Stephen C. Taylor

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)


Background: Haemophilus influenzae is found in the nasopharynx of 80% of the human population. While colonisation with non-typeable Haemophilus influenzae (NTHi) is usually asymptomatic, it is capable of causing acute and chronic otitis media (OM) in infants, invasive disease in susceptible groups and is the leading cause of exacerbations of patients with chronic obstructive pulmonary disease (COPD). Current methods for assessing functional antibody immunity to NTHi are limited and labour intensive. Flow cytometric assays could provide an attractive alternative to evaluate immune responses to candidate vaccines in clinical trials. Results: We have developed a duplexed flow-cytometric uptake and oxidative burst opsonophagocytosis assay (fOPA). We have also developed a duplexed antibody-mediated complement C3b/iC3b and C5b-9 deposition assay (CDA). Antibody-mediated C3b/iC3b deposition correlated with opsonophagocytic uptake (r = 0.65) and with opsonophagocytic oxidative burst (r = 0.69). Both fOPA and CDA were reproducible, with the majority of samples giving a coefficient of variation (CV) of < 20% and overall assay CVs of 14% and 16% respectively. Conclusions: The high-throughput flow cytometric assays developed here were successfully optimised for use with NTHi. Assays proved to be sensitive and highly reproducible for the measurement of bacterial uptake and oxidative burst opsonophagocytosis and antibody-mediated deposition of C3b/iC3b and C5b-9. These assays are useful tools for use in large scale epidemiological studies and to assist in the assessment of functional antibody induced by NTHi candidate vaccines.

Original languageEnglish
Article number167
JournalBMC Microbiology
Issue number1
Publication statusPublished - 29 Oct 2018

Bibliographical note

Funding Information:
Funding was received from UK Department of Health and grants from GlaxoSmithKline Biologicals SA. UK department of health had no role in the design of the study or the collection, analysis and interpretation of data. GlaxoSmithKline Biologicals played a role in project initiation and reviewed and approved the final manuscript. The views expressed in this publication are those of the authors and not necessarily those of Public Health England or the Department of Health.

Publisher Copyright:
© 2018 The Author(s).

Copyright 2018 Elsevier B.V., All rights reserved.


  • Antibody
  • Complement
  • Flow cytometry
  • Non-typeable Haemophilus influenzae
  • Opsonophagocytosis
  • Vaccine


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