A Novel ELISPOT Assay to Quantify HLA-Specific B Cells in HLA-Immunized Individuals
A Novel ELISPOT Assay to Quantify HLA-Specific B Cells in HLA-Immunized Individuals
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DOI：
10.1111/j.1600-6143.2011.03982.x
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发表时间：
2012-06-01
影响因子：
8.8
通讯作者：
Mulder, A.
中科院分区：
文献类型：
--
作者：
Heidt, S.;Roelen, D. L.;Mulder, A.
体液免疫应答的定量通常是通过测定血清HLA抗体来实现的，它不能提供有关参与体液免疫应答的细胞的信息。因此，我们开发了一种HLA特异性B细胞ELISPOT检测方法，用于定量产生HLA抗体的B细胞。我们使用重组HLA单体作为ELISPOT检测的靶标。用产生HLA抗体的人b细胞杂交瘤进行验证。随后，我们定量了HLA免疫个体、非HLA免疫个体和血清HLA抗体移植患者中产生HLA抗体的B细胞。b细胞杂交瘤对相应特异性的HLA分子形成特异性斑点，敏感性与总IgG ELISPOT检测结果相似。hla免疫的健康个体每100万个B细胞中有182个hla特异性B细胞，而未免疫的个体则没有。接受hla - a2不匹配移植物免疫的患者每100万个B细胞中有143个hla - a2特异性B细胞。总之，我们已经开发并验证了一种高度特异性和敏感性的hla特异性b细胞ELISPOT检测方法，该方法需要在更大的移植患者中进一步验证。该技术是定量体液免疫反应的新工具。
Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B-cell hybridomas producing HLA antibodies. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non-HLA-immunized individuals and transplant patients with serum HLA antibodies. B-cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA-immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while nonimmunized individuals had none. Patients who were immunized by an HLA-A2-mismatched graft had up to 143 HLA-A2-specific B cells per million total B cells. In conclusion, we have developed and validated a highly specific and sensitive HLA-specific B-cell ELISPOT assay, which needs further validation in a larger series of transplant patients. This technique constitutes a new tool for quantifying humoral immune responses.