Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Auckland, New Zealand.
Immunobiology, UCL Great Ormond Street Institute of Child Health, London, UK.
Methods Mol Biol. 2020;2136:323-335. doi: 10.1007/978-1-0716-0467-0_26.
Group A Streptococcus (GAS) is a major cause of global mortality, yet there are no licensed GAS vaccines. Vaccine progress has been hampered, in part, by a lack of standardized assays able to quantify antibody function in test antisera. The most widely used assay was developed over 50 years ago by Rebecca Lancefield and relies on human whole blood as a source of complement and neutrophils. Recently, an opsonophagocytic killing (OPK) assay has been developed for GAS by adapting the OPK methods utilized in Streptococcus pneumoniae vaccine testing. This assay uses dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60 cells) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in the Lancefield assays. This protocol outlines methods for performing a GAS OPK assay including titering test sera to generate an opsonic index. This in vitro assay could aid in selecting vaccine candidates by demonstrating whether candidate-induced antibodies lead to complement deposition and opsonophagocytic killing.
A 组链球菌（GAS）是导致全球死亡的主要原因，但目前尚无 GAS 疫苗获得许可。疫苗的研发进展受到阻碍，部分原因是缺乏能够定量检测抗血清中抗体功能的标准化检测方法。最广泛使用的检测方法是由丽贝卡·兰斯菲尔德（Rebecca Lancefield）在 50 多年前开发的，该方法依赖于人类全血作为补体和中性粒细胞的来源。最近，一种噬菌细胞杀伤（OPK）检测法已被开发用于 GAS，该方法通过改编用于肺炎链球菌疫苗检测的 OPK 方法来实现。该检测法使用二甲基甲酰胺（DMF）分化的人早幼粒细胞白血病细胞（HL-60 细胞）作为中性粒细胞和幼兔补体的来源，从而消除了兰斯菲尔德检测法中的主要变异源。本方案概述了进行 GAS OPK 检测的方法，包括滴定测试血清以生成杀菌指数。这种体外检测方法可以通过证明候选诱导抗体是否导致补体沉积和吞噬细胞杀伤，从而有助于选择疫苗候选物。
