Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen
Serologic Assay To Quantify Human Immunoglobulin G Antibodies to the Staphylococcus aureus Iron Surface Determinant B Antigen
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DOI：
10.1128/cvi.00478-08
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发表时间：
2009-05-01
影响因子：
--
通讯作者：
Esser, Mark T.
中科院分区：
文献类型：
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作者：
Raedler, Michael D.;Heyne, Samantha;Esser, Mark T.
开发并验证了一种直接结合Luminex检测试剂盒，用于检测自然感染或使用试验用酵母菌衍生的IsdB疫苗免疫后血清中金黄色葡萄球菌铁表面决定簇B蛋白（IsdB）的人免疫球蛋白G（IgG）抗体。为了确保在用S.在测定中使用大肠杆菌产生的IsdB抗原----来源于大肠杆菌的IsdB。IsdB抗原通过工程化羧基末端半胱氨酸残基共价缀合至马来酰亚胺微球。以直接结合形式测定抗体滴度，其中藻蓝蛋白标记的IgG 1至IgG 4特异性单克隆抗体（HP 6043）与人血清IgG抗体结合。从结合的HP 6043发射的荧光信号与个体的抗体水平成正比。使用来自对IsdB具有高滴度的疫苗接种者的混合人参考血清来生成12点标准曲线。平均荧光强度（MFI）单位与μ g/ml IsdB特异性IgG的相关性通过四参数曲线拟合算法对MFI数据进行内插而得到。该测定法对1.06 μ g/ml敏感，动态范围为2.1至10，625 μ g/ml。测定的总体特异性>96%，测定的线性（平行性）为每10倍稀释约4%。在三个不同的IsdB抗原批次、三个不同的微球批次、两个二抗批次和三个不同的操作员之间，测定的总精密度为16.6%相对标准偏差。该试验已被证明可用于评价不同剂量和配方的研究性IsdB疫苗接种后的免疫应答。
A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual's antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to mu g/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 mu g/ml with a dynamic range of 2.1 to 10,625 mu g/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is -4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.