Development of flow cytometric opsonophagocytosis and antibody-mediated complement deposition assays for non-typeable Haemophilus influenzae
Author(s): ,
Stephen Thomas
Affiliations:
Public Health England
,
Stephanie Leung
Affiliations:
Public Health England
,
Katy Knox
Affiliations:
Public Health England
,
Pascal Lestrate
Affiliations:
GlaxoSmithKline Biologicals SA
,
Dominique Wauters
Affiliations:
GlaxoSmithKline Biologicals SA
,
Andrew Gorringe
Affiliations:
Public Health England
Stephen Taylor
Affiliations:
Public Health England
PHE ePoster Library. Thomas S. Mar 20, 2018; 205933; 12611
Mr. Stephen Thomas
Mr. Stephen Thomas
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Abstract
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Abstract 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 response 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 functional immune assessment of NTHi candidate vaccines. Funding Funding was received from Department of Health Grant in Aid and grants from GlaxoSmithKline Biologicals SA.
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