Department of Biological Sciences, University of Limerick, Castletroy, Ireland
LEGTPA Bordeaux Blanquefort (Lycée Agro-Viticole de Blanquefort), Blanquefort, Bordeaux, France.
Appl Environ Microbiol. 2020 Aug 18;86(17). doi: 10.1128/AEM.01435-20.
and other coagulase-positive spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non- spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non- species. This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of and of all antibodies to be incorporated into any immunoassay designed to detect a non- spp.
以及其他凝固酶阳性 spp. 通过表达蛋白 A（SpA）结合 IgG 抗体的 Fc 区域。这些物种在设计用于检测其他靶细菌的基于抗体的检测中一直是假阳性信号的来源。在这里，流式细胞术用于研究许多因素对单个细胞与抗人 IgG 抗体的 SpA 介导结合的影响，包括菌株、热杀伤、过夜储存、生长阶段、细胞生理状态、表面粘附和在模型食品系统中的生长。通过用通透性染料碘化丙啶和 calcein 紫 AM 对抗体染色的细胞进行双重染色，将细胞生理状态与 SpA 介导的抗体结合相关联。通常，缺乏酯酶活性的透化细胞不会强烈结合抗体。还对一些市售的多克隆 IgG 抗体与非 spp.的结合进行了特征描述。并非所有表达 SpA 的物种都强烈结合小鼠 IgG，而一种不被认为表达 SpA 的物种则强烈结合。大多数表达 SpA 的菌株在某种程度上结合兔 IgG 抗体，而只有一个菌株结合山羊 IgG 抗体。为了减少或消除 SpA 介导的 IgG 结合，评估了以下产品作为阻断试剂，并在与初级或次级抗体染色之前应用：正常兔血清、小鼠 IgG 同种型对照、山羊 IgG 和商业 FcR 阻断试剂。只有 FcR 阻断试剂能够一致地减少 spp.与针对其他物种的抗体的 SpA 介导结合，并且可以推荐作为设计用于检测非 spp.的免疫测定中的阻断试剂。本研究描述了与基于抗体的微生物检测相关的一个普遍但研究甚少的问题，即蛋白 A（SpA）介导的 IgG 抗体结合，并提供了一种解决方案：使用商业 FcR 阻断试剂。通过应用本研究的结果，可以消除临床、食品或环境样品中微生物检测中常见的假阳性信号源。使用流式细胞术，作者展示了培养物中 SpA 介导的抗体结合的异质性程度，并且 SpA 介导的抗体结合程度取决于菌株、生长阶段和食品基质，并受到模拟食品加工处理和细胞粘附的影响。此外，我们对 spp.与针对其他细菌物种的抗体的 SpA 介导结合的研究产生了非常细致的图片，导致我们建议针对所有要纳入设计用于检测非 spp.的免疫测定的抗体测试多个 spp.和 菌株。
