title: Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria.
abstract: The assessment of naturally-acquired and vaccine-induced immunity to blood-stage Plasmodium falciparum malaria is of long-standing interest. However, the field has suffered from a paucity of in vitro assays that reproducibly measure the anti-parasitic activity induced by antibodies in conjunction with immune cells. Here we optimize the antibody-dependent respiratory burst (ADRB) assay, which assesses the ability of antibodies to activate the release of reactive oxygen species from human neutrophils in response to P. falciparum blood-stage parasites. We focus particularly on assay parameters affecting serum preparation and concentration, and importantly assess reproducibility. Our standardized protocol involves testing each serum sample in singlicate with three independent neutrophil donors, and indexing responses against a standard positive control of pooled hyper-immune Kenyan sera. The protocol can be used to quickly screen large cohorts of samples from individuals enrolled in immuno-epidemiological studies or clinical vaccine trials, and requires only 6 μL of serum per sample. Using a cohort of 86 samples, we show that malaria-exposed individuals induce higher ADRB activity than malaria-naïve individuals. The development of the ADRB assay complements the use of cell-independent assays in blood-stage malaria, such as the assay of growth inhibitory activity, and provides an important standardized cell-based assay in the field.
authors.affiliation: The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
authors.forename: David
authors.initials: D
authors.lastname: Llewellyn
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, 20852, USA.
authors.forename: Kazutoyo
authors.initials: K
authors.lastname: Miura
authors.affiliation: Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Fay
authors.affiliation: The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Williams
authors.affiliation: The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.|KEMRI Centre for Geographic Medicine Research, Coast, P.O. Box 230-80108, Kilifi, Kenya.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Murungi
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Shi
authors.affiliation: The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
authors.forename: Susanne H
authors.initials: SH
authors.lastname: Hodgson
authors.affiliation: The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Douglas
authors.affiliation: KEMRI Centre for Geographic Medicine Research, Coast, P.O. Box 230-80108, Kilifi, Kenya.
authors.forename: Faith H
authors.initials: FH
authors.lastname: Osier
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, 20852, USA.
authors.forename: Rick M
authors.initials: RM
authors.lastname: Fairhurst
authors.affiliation: Malaria Research and Training Centre, Faculty of Medicine, Pharmacy and Odonto-stomatology, University of Bamako, Bamako, Mali.
authors.forename: Mahamadou
authors.initials: M
authors.lastname: Diakite
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Pleass
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, 20852, USA.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Long
authors.affiliation: The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Draper
mesh_terms: D000328:Adult; D000913:Antibodies, Protozoan / Q000276:immunology*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D017780:Malaria Vaccines / Q000276:immunology; D016778:Malaria, Falciparum / Q000276:immunology / Q000469:parasitology; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D010963:Plasmodium falciparum / Q000276:immunology*; D011961:Receptors, Fc / Q000378:metabolism; D015203:Reproducibility of Results; D016897:Respiratory Burst / Q000276:immunology*
chemical_list: D000913:Antibodies, Protozoan; D017780:Malaria Vaccines; D011961:Receptors, Fc