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Rapid characterization of hybridomas producing monoclonal antibodies against platelet beta 3 integrin using ELIspot

机译：使用ELISPOT产生杂交瘤杂交癌的杂交癌的快速表征

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摘要

Generally, B-cell responses against human platelet antigens are assessed by the serological detection of specific platelet antibodies, mostly against beta 3 integrin. However, this approach seems to be of low sensitivity, since platelet autoantibodies against alpha IIb beta 3 are detected in only 50% of all patients with immune thrombocytopenia (ITP). In this study, a novel B-cell ELIspot method was established to characterize the specificity of mouse monoclonal antibodies (moabs) against human beta 3 integrin. Moabs produced by hybridomas were immobilized on membrane and bound antibodies were visualized as spots using biotinylated recombinant proteins alpha IIb beta 3 or alpha v beta 3 and the enzyme labeled streptavidin-substrate system. Three hybridomas, Gi5, Gi16 and AP3, designated previously as anti-alpha IIb beta 3, anti-alpha IIbb and anti-beta 3, respectively, were investigated. Hybridoma producing moab against CD177 was used as the negative control. Whereas AP3 reacted with alpha IIb beta 3 and alpha v beta 3, Gi5 only formed spots with alpha IIb beta 3. Titration analysis showed that the number of spots correlated significantly with the number of seeded cells. Approximately 15 antibody producing hybridoma cells could be identified among 103 nonproducing B-cells. Furthermore, superior correlation with the total number of IgG producing cells was obtained. Analysis of the third hybridoma, Gi16 (anti-aIIb), showed only few spots with alpha IIb beta 3, indicating that this hybridoma contained different clones (producer and non-producer). Significant increased number of spots could be identified after re-cloning of these clones by limiting dilution method. Our results demonstrate that this B-cell ELIspot assay can be used for the identification of a small number of hybridoma cells producing moabs against beta 3 integrin, verification of their monoclonality, productivity and for determining their specificity in the early state of workup steps. In the future, this approach may be useful to define B-cell clones in patients who developed platelet antibodies against different beta 3-integrins and to differentiate their diversities.
机译：一般来说，B细胞对人类血小板抗原的反应是通过血清学检测特异性血小板抗体来评估的，主要是针对β3整合素的抗体。然而，这种方法似乎灵敏度较低，因为在所有免疫性血小板减少症（ITP）患者中，只有50%的患者检测到针对αIIbβ3的血小板自身抗体。在本研究中，建立了一种新的B细胞ELIspot方法来表征小鼠抗人β3整合素单克隆抗体（moabs）的特异性。将杂交瘤产生的MOAb固定在膜上，并使用生物素化重组蛋白αIIbβ3或αvβ3和酶标记的链霉亲和素底物系统将结合抗体显示为斑点。研究了三种杂交瘤Gi5、Gi16和AP3，它们之前分别被指定为抗αIIbβ3、抗αIIbb和抗β3。以产生抗CD177单克隆抗体的杂交瘤作为阴性对照。AP3与αIIbβ3和αvβ3反应，而Gi5仅与αIIbβ3形成斑点。滴定分析表明，斑点数与种子细胞数显著相关。在103个不产生抗体的B细胞中，可鉴定出约15个产生抗体的杂交瘤细胞。此外，获得了与产生IgG的细胞总数的良好相关性。对第三个杂交瘤Gi16（抗亚投行）的分析显示，只有少量带有αIIbβ3的斑点，表明该杂交瘤包含不同的克隆（产生子和非产生子）。在通过限制稀释法重新克隆这些克隆后，可以发现显著增加的斑点数量。我们的结果表明，这种B细胞ELIspot分析可用于鉴定少量产生抗β3整合素单克隆抗体的杂交瘤细胞，验证其单克隆性和生产效率，并在检查步骤的早期状态下确定其特异性。在未来，这种方法可能有助于在开发针对不同β3整合素的血小板抗体的患者中定义B细胞克隆，并区分其多样性。

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  • 来源
    《Platelets》 |2016年第8期|共6页
  • 作者

    Maenner Denis; Werth Silke; Bein Gregor; Santoso Sentot;

  • 作者单位

    Justus Liebig Univ Inst Clin Immunol &

    Transfus Med Langhansstr 7 D-35385 Giessen Germany;

    Justus Liebig Univ Inst Clin Immunol &

    Transfus Med Langhansstr 7 D-35385 Giessen Germany;

    Justus Liebig Univ Inst Clin Immunol &

    Transfus Med Langhansstr 7 D-35385 Giessen Germany;

    Justus Liebig Univ Inst Clin Immunol &

    Transfus Med Langhansstr 7 D-35385 Giessen Germany;

  • 收录信息
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 基础医学;
  • 关键词

    Monoclonal Antibodies; platelets; integrin beta 3; B cells ELISpot;

    机译：单克隆抗体;血小板;整合蛋白β3;B细胞ELISPOT;

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