MONONUCLEAR CELL FACTORS AFFECTING BASOPHIL/MAST CELLS

影响嗜碱性细胞/肥大细胞的单核细胞因子

• 批准号: 3134666
• 负责人: J. ANDREW GRANT
• 金额: $ 15.47万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3134666
• 关键词: affinity chromatography; antibody specificity; basophils; chemical structure; enzyme linked immunosorbent assay; gel electrophoresis; high performance liquid chromatography; immunologic techniques; interleukin 3; laboratory mouse; mast cell; monoclonal antibody; monocyte; protein purification; protein structure; radioimmunoassay

We have described a group of cytokines that modulate mediator release from basophils and mast cells. Histamine releasing factors (HRF)-mediated activation of mast cells and basophils may represent a amplification mechanism of allergic process. The clinical importance of these cytokines can not be established until their chemical structure is better defined. We have purified one species of HRF (44 kD) by sequential HPLC. The objective of this grant is to purify this and other species of HRF to homogeneity and sequence them. In addition, we plan to raise monoclonal antibodies against HRF, develop immunoassays and affinity columns to aid purification. Crude supernatants containing HRF will be generated by culturing two cell lines, U937 and RPMI 8866, both of which produce HRF similar to that synthesized by mononuclear cells (MNC). Furthermore, MNC-derived HRF will be generated by culturing cells isolated from leukocytes concentrates obtained from Blood Bank donors. HRF will be purified by using sequential HPLC. We have recently demonstrated that a major fraction of HRF binds to heparin-agarose. We have also optimized the running conditions of a C4 reverse phase column, and recovered HRF from the column with satisfactory yield. The strategy for the purification of HRF is to apply sequential HPLC involving affinity, ion exchange and reverse phase columns. The homogeneity will be verified by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Purified HRF will be electroblotted onto Immobilon PVDF filters and used directly for sequencing. The latter will be accomplished using an updated Applied Bio-system Sequenator. Monoclonal antibodies will be raised in mice using purified HRF. Hybridoma cells will be grown in the peritoneum of pristane-treated mice. Purified antibody will be used to develop ELISA and/or RIA as well as affinity columns. Our final goal is to conduct a biochemical and immunological comparison of various species of HRF produced by different subpopulations of MNC. This will be accomplished by comparing the elution profile from HPLC columns and by testing the antigenic cross-reactivity among various species of HRF obtained from different sources.

我们已经描述了一组调节介质释放的细胞因子， 嗜碱性粒细胞和肥大细胞。 组胺释放因子介导 肥大细胞和嗜碱性粒细胞的激活可能代表了 过敏过程的机制。 这些细胞因子的临床意义 在其化学结构得到更好的定义之前，无法建立。 我们用连续高效液相色谱法纯化了一种44 kD的HRF。 的 这项赠款的目的是净化这种和其他物种的HRF， 同质性并将其排序。 此外，我们计划提高单克隆 针对HRF的抗体，开发免疫测定和亲和柱，以帮助 洁净. 通过培养两个细胞产生含有HRF的粗上清液 株系U937和RPMI 8866，这两个株系产生的HRF与 由单核细胞（MNC）合成。 此外，MNC衍生的HRF将 通过培养从白细胞浓缩物中分离的细胞产生 从血库捐献者那里获得。 HRF将通过使用连续 高效液相色谱法 我们最近已经证明，HRF的主要部分结合到 肝素琼脂糖。 优化了C4的运行条件， 反相柱，并从柱中回收HRF， 产率 纯化HRF的策略是应用连续的 HPLC包括亲和柱、离子交换柱和反相柱。 的 将通过一维和二维SDS-聚丙烯酰胺验证均匀性 凝胶电泳 将纯化的HRF电印迹到Immobilon上 PVDF过滤并直接用于测序。 后者将 使用更新的应用生物系统测序仪完成。 将使用纯化的HRF在小鼠中培养单克隆抗体。 杂交瘤 细胞将在降植烷处理的小鼠的腹膜中生长。 纯化 抗体将用于开发ELISA和/或RIA以及亲和力 列. 我们的最终目标是进行生化和免疫 不同亚群产生的各种HRF的比较 在MNC。 这将通过比较以下洗脱曲线来实现： HPLC柱，并通过测试各种抗原之间的抗原交叉反应性， 从不同来源获得的HRF种类。