TY - JOUR

T1 - Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria

AU - Llewellyn, David

AU - Miura, Kazutoyo

AU - Fay, Michael P.

AU - Williams, Andrew Richard

AU - Murungi, Linda M.

AU - Shi, Jianguo

AU - Hodgson, Susanne H.

AU - Douglas, Alexander D.

AU - Osier, Faith H.

AU - Fairhurst, Rick M.

AU - Diakite, Mahamadou

AU - Pleass, Richard J.

AU - Long, Carole A.

AU - Draper, Simon J.

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

U2 - 10.1038/srep14081

DO - 10.1038/srep14081

M3 - Journal article

C2 - 26373337

VL - 5

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 14081

ER -