Development of an Opsonophagocytic Killing Assay Using HL-60 Cells for Detection of Functional Antibodies against Streptococcus pyogenes
Development of an Opsonophagocytic Killing Assay Using HL-60 Cells for Detection of Functional Antibodies against Streptococcus pyogenes
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DOI：
10.1128/msphere.00617-18
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发表时间：
2018-11-01
期刊：
影响因子：
4.8
通讯作者：
Dale, James B.
中科院分区：
文献类型：
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作者：
Salehi, Sanaz;Hohn, Claudia M.;Dale, James B.
A组链球菌（GAS）疫苗的临床开发将需要实施标准化的高通量测定，以测量疫苗接受者中功能性调理素抗体的活性。在本研究中，我们调整和修改了HL-60为基础的协议，开发用于检测调理素抗体肺炎链球菌使用多种M型GAS。对测定条件的修改允许在测定中评价21种不同M类型的GAS。抗体介导的调理作用的特异性通过用同源而非异源的M蛋白抑制来证明。在全血和HL-60测定中，兔抗血清对30价M蛋白疫苗促进的14种不同M类型的调理吞噬细胞杀伤（OPK）的最大速率相当。还提供了显示IVIG [静脉内免疫球蛋白（人）]中存在的天然获得的人抗体的OPK血清滴度（调理素指数）的数据。使用21种不同M型GAS和IVIG作为抗体来源在不同日期进行的HL-60测定结果显著一致。这份报告表明，OPK测定条件可以优化的调理素抗体对一些流行病学上重要的M型气体的测量，一旦标准化，应促进有效的疫苗，以防止这些infection.IMPORTANCE的临床开发测量功能调理素抗体对A组链球菌是一个重要组成部分的有效疫苗的临床开发路径。先前的研究使用了60多年前开发的一种测定法，该测定法依赖于全血作为吞噬细胞和补体的来源，这两者都是抗体介导的杀伤测定法的关键组分。在这项研究中，我们采用了一种检测方法，使用HL-60人早幼粒细胞白血病细胞系作为吞噬细胞和幼兔血清作为补体的来源，用于检测针对A组链球菌的调理素抗体。在细菌的一些已知生物学特性的基础上，我们修改了测定条件以支持21种流行病学上重要的M类型的评价，并证明了测定功能调理素抗体水平的实用性和重现性。
The clinical development of group A streptococcal (GAS) vaccines will require the implementation of a standardized, high-throughput assay to measure the activity of functional opsonic antibodies in vaccine recipients. In the present study, we adapted and modified the HL-60-based protocol that was developed for the detection of opsonic antibodies against Streptococcus pneumoniae for use with multiple M types of GAS. Modifications of the assay conditions permitted the evaluation of 21 different M types of GAS in the assay. The specificity of the antibody-mediated opsonization was demonstrated by inhibition with homologous, but not heterologous, M proteins. Maximum rates of opsonophagocytic killing (OPK) of 14 different M types promoted by rabbit antiserum against the 30-valent M protein-based vaccine were comparable in whole-blood and HL-60 assays. Data are also presented showing OPK serum titers (opsonic index) of naturally acquired human antibodies present in IVIG [intravenous immune globulin (human)]. Results of the HL-60 assay performed on different days using 21 different M types of GAS and IVIG as the antibody source were significantly concordant. This report indicates that the OPK assay conditions may be optimized for the measurement of opsonic antibodies against a number of epidemiologically important M types of GAS and, once standardized, should facilitate the clinical development of effective vaccines to prevent these infections.IMPORTANCE Measuring functional opsonic antibodies against group A streptococci is an important component of the clinical development path for effective vaccines. Prior studies have used an assay developed over 60 years ago that relied on whole human blood as the source of phagocytes and complement, both of which are critical components of antibody-mediated killing assays. In this study, we adapted an assay that uses the HL-60 human promyelocytic leukemia cell line as phagocytic cells and baby rabbit serum as a source of complement for detection of opsonic antibodies against group A streptococci. On the basis of some of the known biological characteristics of the bacteria, we modified the assay conditions to support the evaluation of 21 epidemiologically important M types and demonstrated the utility and reproducibility of the assay for measurement of functional opsonic antibody levels.