Llewellyn David, Miura Kazutoyo, Fay Michael P, Williams Andrew R, 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
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK.
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, 20852, USA.
Sci Rep. 2015 Sep 16;5:14081. doi: 10.1038/srep14081.
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.
对自然获得性和疫苗诱导的针对恶性疟原虫血液期疟疾免疫力的评估一直备受关注。然而，该领域一直缺乏能够可靠地测量抗体与免疫细胞共同诱导的抗寄生虫活性的体外检测方法。在此，我们优化了抗体依赖性呼吸爆发（ADRB）检测方法，该方法可评估抗体激活人类中性粒细胞释放活性氧以应对恶性疟原虫血液期寄生虫的能力。我们特别关注影响血清制备和浓度的检测参数，并重要地评估了可重复性。我们的标准化方案包括用三名独立的中性粒细胞供体对每个血清样本进行单份检测，并以肯尼亚混合超免疫血清的标准阳性对照为参照对反应进行指数化。该方案可用于快速筛选免疫流行病学研究或临床疫苗试验中入组个体的大量样本队列，且每个样本仅需6微升血清。通过对86个样本的队列研究，我们发现接触过疟疾的个体比未接触过疟疾的个体诱导出更高的ADRB活性。ADRB检测方法的开发补充了血液期疟疾中与细胞无关的检测方法（如生长抑制活性检测）的应用，并在该领域提供了一种重要的基于细胞的标准化检测方法。
