Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria
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Standardization of the antibody-dependent respiratory burst assay with human neutrophils and Plasmodium falciparum malaria. / Llewellyn, David; Miura, Kazutoyo; Fay, Michael P.; Williams, Andrew Richard; Murungi, Linda M.; Shi, Jianguo; Hodgson, Susanne H.; Douglas, Alexander D.; Osier, Faith H.; Fairhurst, Rick M.; Diakite, Mahamadou; Pleass, Richard J.; Long, Carole A.; Draper, Simon J.
In: Scientific Reports, Vol. 5, 14081, 2015.Research output: Contribution to journal › Journal article › Research › peer-review
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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 -
ID: 145122946