School of Medical Sciences, The University of Auckland, New Zealand; Maurice Wilkins Centre for Biodiscovery, The University of Auckland, New Zealand.
School of Medical Sciences, The University of Auckland, New Zealand.
J Immunol Methods. 2022 Jan;500:113194. doi: 10.1016/j.jim.2021.113194. Epub 2021 Nov 19.
Group A Streptococcus (GAS) is a major human pathogen responsible for superficial infections through to life-threatening invasive disease and the autoimmune sequelae acute rheumatic fever (ARF). Despite a significant global economic and health burden, there is no licensed vaccine available to prevent GAS disease. Several pre-clinical vaccines that target conserved GAS antigens are in development. Assays that measure antigen-specific antibodies are essential for vaccine research. The aim of this study was to develop a multiplex beadbased immunoassay that can detect and quantify antibody responses to multiple GAS antigen targets in small volume blood samples. This builds on our existing triplex assay comprised of antigens used in clinical serology for the diagnosis of ARF (SLO, DNase B and SpnA). Five additional conserved putative GAS vaccine antigens (Spy0843, SCPA, SpyCEP, SpyAD and the Group A carbohydrate), were coupled to spectrally unique beads to form an 8-plex antigen panel. After optimisation of the assay protocol, standard curves were generated, and assessments of assay specificity, precision and reproducibility were conducted. A broad range of antibody (IgG) titres were able to be quickly and accurately quantified from a single serum dilution. Assay utility was assessed using a panel of 62 clinical samples including serum from adults with GAS bacteraemia and children with ARF. Circulating IgG to all eight antigens was elevated in patients with GAS disease (n = 23) compared to age-matched controls (n = 39) (P < 0.05). The feasibility of using dried blood samples to quantify antigen-specific IgG was also demonstrated. In summary, a robust and reproducible 8-plex assay has been developed that simultaneously quantifies IgG antibodies to GAS vaccine and diagnostic antigens.
A 组链球菌（GAS）是一种主要的人类病原体，可导致从浅表感染到危及生命的侵袭性疾病和急性风湿热（ARF）等自身免疫后遗症。尽管 GAS 疾病给全球带来了巨大的经济和健康负担，但目前仍没有可用的疫苗来预防 GAS 疾病。目前正在开发几种针对保守 GAS 抗原的临床前疫苗。用于疫苗研究的测量抗原特异性抗体的检测方法至关重要。本研究旨在开发一种多重微珠免疫分析方法，该方法可检测和定量小体积血液样本中针对多种 GAS 抗原靶标的抗体反应。这是在我们现有的三联检测方法的基础上进行的，该方法由用于 ARF 临床血清学诊断的抗原（SLO、DNase B 和 SpnA）组成。另外 5 种保守的假定 GAS 疫苗抗原（Spy0843、SCPA、SpyCEP、SpyAD 和 A 组碳水化合物）被偶联到光谱独特的微珠上，形成 8 重抗原谱。优化检测方法方案后，生成标准曲线，并进行了检测方法特异性、精密度和重现性的评估。通过对单个血清稀释液进行快速、准确的定量分析，能够检测到广泛的抗体（IgG）滴度。使用包含成人 GAS 菌血症和儿童 ARF 血清的 62 个临床样本的样本组评估了检测方法的实用性。与年龄匹配的对照组（n=39）相比，患有 GAS 疾病的患者（n=23）的所有 8 种抗原的循环 IgG 均升高（P<0.05）。还证明了使用干血样本来定量抗原特异性 IgG 的可行性。总之，已经开发出一种稳健且可重复的 8 重检测方法，可同时定量检测 GAS 疫苗和诊断抗原的 IgG 抗体。
