CORE--Monoclonal Antibody Facility

杂交瘤核心设施提供了一个细胞培养设施，以协助调查人员完成项目。我们可以生产用于它们的单克隆抗体，在某些情况下还可以生产多克隆抗体。这包括对小鼠进行免疫，进行出血测试以确定免疫程度，切除脾和淋巴结进行融合和实际融合。杂交瘤在HAT培养基中选择，并在许多检测中进行特异性测试。我们用放射免疫分析、酶联免疫吸附试验和免疫印迹法进行筛选。产生适当特异性抗体的杂交瘤通过限制稀释法克隆两次。为了产生大量的抗体，我们在组织培养中培养杂交瘤，或者将它们注射到Pristane免疫的小鼠体内。通过蛋白A或蛋白G层析、DEAE离子交换或在带有共价连接的抗免疫球蛋白的分离柱上从这些溶液中纯化抗体。所有这些程序都可供计划项目的成员使用。除了生产单抗外，我们还制备了广泛使用的试剂，如针对HA标签的抗体、C-Myc标签、针对小鼠的同型抗体、针对蛋白酶体的抗体以及任何其他与同型和同种异型相匹配的对照抗体。我们还试图开发已知参与区分Th1和Th2免疫的受体抗体。目前普遍的科学思维认为，自身免疫性疾病源于Th1免疫反应的过度激活。我们正试图准备针对IL-12B2受体的小鼠单克隆化，据信IL-12B2受体在Th1细胞的扩张中发挥重要作用，而干扰素γβ受体被认为参与Th2细胞的生成。我们已经生产并测试了针对从已知序列衍生的多肽而产生的抗IL-12B2的单抗。许多检测结果表明，该抗体不能与Th1细胞结合。因此，我们制备了含有IL-12B2链胞外区的GST融合蛋白。这是通过RT-PCR从小鼠体内收集的RNA中完成的，这些RNA是在脂多糖、IL-12和伽马干扰素免疫后收集的。我们成功地制备了PCRed这一片段，并已在细菌中表达。我们将使用仓鼠作为杂交瘤，以避免小鼠可能存在的耐受性。杂交瘤将以另一种GST融合蛋白为阴性对照，通过ELISA筛选融合蛋白进行检测。阳性杂交瘤上清液将在准备主要表达Th1细胞的脾细胞群上进行测试。

The hybridoma core facility provides a cell culture facility to assist the investigators in the completions of the projects. We are available to produce monoclonal and in some cases polyclonal antibodies for their use. This includes the immunization of mice, performing test bleeding to determine the degree of immunization, removal of spleens and lymph nodes for fusion and the actually fusion. Hybridomas are selected in HAT medium and tested in a number of assays for specificity. We screen using radioimmunoassay, ELISA, and western blotting. Hybridomas producing antibodies of the appropriate specificity are cloned two times by limiting dilution. To produce large quantities of antibodies we grow the hybridomas in tissue culture, or injected them into pristane primed mice. The antibodies are purified from these solution by Protein A or Protein G chromatography, DEAE ion exchange or on separose columns with covalently attached anti- immunoglobulins. All of these procedures are available to the members of the program project. In addition to production of monoclonal we prepare widely used reagents such as antibodies to HA tag, C-Myc tag, antibodies to mouse isotypes, antibodies to the proteosomes, and any other control antibodies matched for isotype and allotype. We are also trying to develop antibodies to receptor known to be involved in the discriminating Th1 and Th2 immunity. Common scientific thinking at this time suggests that autoimmune disease stems from overactivation of Th1 immune response. We are trying to prepare mouse monoclonals against the IL-12B2 receptor, believed to play an important role in expansion of Th1 cells and the interferon Gamma Beta receptor believed to be involved in generation of Th2 cells. We have produced and tested monoclonal anti-IL-12B2 produced against a peptide derived from the known sequence. This failed to bind to Th1 cells as determined by a number of assays. Therefore, we prepared a GST fusion protein containing the extracellular domain of the IL-12B2 chain. This was done by RT-PCR from RNA collected from mice following immunization with LPS, IL-12 and gamma interferon. We successful prepared PCRed this fragment and have now expressed it in bacteria. We will use Hamster for the hybridoma in order to avoid the tolerance which might be present in the mouse. The hybridomas will be tested by ELISA screening on the fusion protein with another GST fusion as a negative control. Positive hybridoma supernatnats will be tested on spleen cell populations prepared to express primarily Th1 cells.