The Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, United States.
Front Immunol. 2019 Aug 13;10:1851. doi: 10.3389/fimmu.2019.01851. eCollection 2019.
Antibodies serve as the primary correlate of protection following most clinically approved vaccines and are thought to confer protection in part through their ability to block (neutralize) infection. Increasingly, studies have shown that beyond their blocking activities, the ability of antibodies to leverage the innate immune response may serve a vital role in protection from infection. Specifically, antibodies can drive phagocytosis, complement activation, and cellular cytotoxicity by interacting with Fc-receptors found on all innate immune cells. Measuring the capacity of antibodies to induce these functions has become critical for the identification of correlates of protection in large-scale vaccine trials. Therefore, there is a growing need to develop robust, high throughput assays able to interrogate the functional capacity of innate immune recruiting antibodies. However, in many instances, only small sample volumes are available. Nevertheless, profiling antibody functions across many pathogen-associated antigens or across global intra-pathogen variants is in high demand, making sample sparing approaches to perform this antibody evaluation critical. Here we describe the development of an approach to interrogate the functional activity of antibodies in serum against up to 5 antigen targets simultaneously. A single bead-based cellular assay was adapted to accommodate 5 different fluorescently colored beads, allowing for the concurrent investigation of antibody responses directed against multiple antigens in a single well. The multiplexed assay was as sensitive, specific, and accurate as the single antigen assay and robustly able to assess functional differences mediated by antibodies across different samples. These findings show multiplexing allows for accurate and more efficient analysis of antibody-mediated effector profiles.
抗体是大多数经临床批准的疫苗接种后产生的主要保护相关物，被认为通过其阻断（中和）感染的能力提供部分保护。越来越多的研究表明，除了阻断作用外，抗体利用先天免疫反应的能力可能在防止感染方面发挥重要作用。具体而言，抗体可以通过与所有先天免疫细胞上发现的 Fc 受体相互作用，驱动吞噬作用、补体激活和细胞毒性。测量抗体诱导这些功能的能力对于在大规模疫苗试验中确定保护相关物变得至关重要。因此，迫切需要开发能够探究先天免疫募集抗体功能能力的强大、高通量检测方法。然而，在许多情况下，只有少量的样本可用。尽管如此，对许多病原体相关抗原或整个病原体内部变异体的抗体功能进行分析的需求很高，因此采用节省样本的方法来进行这种抗体评估至关重要。在这里，我们描述了一种方法的开发，该方法可同时检测血清中针对多达 5 个抗原靶标的抗体的功能活性。我们对单个基于珠子的细胞检测方法进行了改编，以适应 5 种不同的荧光珠，从而可以在单个孔中同时研究针对多种抗原的抗体反应。这种多重检测方法与单个抗原检测方法一样敏感、特异和准确，并且能够稳健地评估来自不同样本的抗体介导的功能差异。这些发现表明，多重检测方法可实现抗体介导的效应因子谱的准确和更高效分析。
