Few bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available. We report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1’s N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98–100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen. Our recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.

中文翻译：

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弓形虫体内生物素化重组抗原 SAG1 和 SAG2A 的表达，用于改进的基于血清流行病学珠的多重检测


在大规模血清流行病学调查中，很少有基于微珠的多重检测能够检测针对原生动物寄生虫弓形虫的抗体。此外，每种多重测定都有特定的变化或限制，例如使用截短或融合蛋白作为抗原，可能掩盖重要的表位。因此，这种测定方法必须由感兴趣的团体开发，因为没有一种方法可以商业化。我们报道了 N 端无融合、可溶、体内生物素化重组表面抗原 SAG1 和 SAG2A 的细菌表达和用途，用于检测抗 T 细胞。弓形虫 IgG 抗体。该表达系统依赖于三个兼容的质粒。表达构建体产生麦芽糖结合蛋白与 SAG1（或 SAG2A）的融合体，由 TEV 蛋白酶切割位点分隔，后面是大肠杆菌生物素连接酶 BirA (AviTag) 识别的肽序列，以及末端六个组氨酸标签亲和纯化。 TEV 蛋白酶和 BirA 在第二个质粒上编码，它们的表达导致 BirA 对融合蛋白和 AviTag 内的单个生物素化赖氨酸进行蛋白水解切割。寄生虫蛋白的正确折叠取决于正确的二硫键，这由第三个质粒上编码的巯基氧化酶和蛋白质二硫键异构酶促进。 C 端生物素化允许纯化的表面抗原与磁性 Luminex 珠进行定向、可重复的偶联，每次测定仅需要微量的蛋白质。我们发现，N 端融合伴侣（例如麦芽糖结合蛋白）会对抗体结合产生负面影响，这证实了接触 SAG1 的 N 端表位对于抗体识别非常重要。 我们使用之前通过商业诊断检测进行测试的人血清验证了基于微珠的多重检测，发现即使仅使用生物素化的 SAG1 作为抗原，灵敏度和特异性也达到 98-100% 的一致性。与先前描述的用于弓形虫多重血清学测定的蛋白质相比，我们的重组体内生物素化弓形虫抗原具有明显的优势。它们为 BBMA 和其他格式提供了一种廉价、特异且敏感的替代方案，可替代寄生虫裂解物或真核细胞表达的 SAG1/SAG2A。所描述的通用表达策略也可用于其他抗原，其中定向固定是抗体和配体灵敏识别的关键。