PROCESSED ANTIGEN CHARACTERIZATION BY MASS SPECTROMETRY

研究开发用于序列分析的仪器和方法 呈递给免疫系统的多肽抗原与 主要组织相容性复合体的I类和II类分子是 建议。这是发展的必要的第一步。 合成/重组疫苗或其他免疫系统调节剂 对细菌和病毒感染，癌症， 自身免疫性疾病和组织移植排斥反应。最高优先级 是描述特定疾病状态的方法的发展 在数千个自身的复杂混合物中发现的相关抗原 多肽。多阶段层析与两者联用 微毛细管高效液相色谱/高 高效毛细管电泳电喷雾串联质谱仪 光谱分析将用于这一目的。上述两种系统 将直接与细胞毒性检测相结合。具体目标包括 以下内容：(A)进一步提高串联质量的灵敏度 一种便于I类序列分析的光谱方法 低分子水平的多肽，(B)发展蛋白水解性消化 有助于对第II类抗原进行序列分析的方法 低Femtomol水平，(C)对I类HLA-呈递的多肽进行序列测定 黑色素瘤细胞表面可被黑色素瘤识别的A2.1分子 特异性细胞毒性T淋巴细胞，(D)对由以下呈现的多肽进行序列测定 人类I类分子，人类白细胞抗原-B7，Aw68，和Aw69，(E)以确定 多肽与I类、MHC分子相互作用的结构基础 并确定异种反应性的人类白细胞抗原A2.1识别的多肽表位 限制性CTL，(F)对HIV病毒蛋白加工的多肽进行序列测定 并且关联地呈现1类、人类白细胞抗原A2.1分子、(G)至 确定内源性流感血凝素激动剂的范围 与野生动物产生的小鼠MHC 1kD分子有关 血凝素基因和合成微型基因及其内源性鉴定 结合KD但不具有免疫原性的血凝素多肽。

Research to develop instrumentatIon and methods for the sequence analysis of peptide antigens presented to the immune system in association with class I and class II molecules of the major histocompatibility complex is proposed. This is a necessary first step in the development of synthetic/recombinant vaccines or other modulators of the immune system that are effective against bacterial and viral infections, cancer, autoimmune disorders and tissue transplant rejection. Of highest priority is the development of methods for characterizing specific disease state associated antigens found in complex mixtures of several thousand self peptides. Multistage chromatography in conjunction with both microcapillary high performance liquid chromatography/ and high performance capillary electrophoresis/ electrospray ionization tandem mass spectrometry will be employed for this purpose. Both of the above systems will be coupled directly to cytotoxicity assays. Specific goals include the following: (a) to further improve the sensitivity of the tandem mass spectrometry method so as to facilitate sequence analysis of class I peptides at the low femtomole level, (b) to develop proteolytic digestion methods that will facilitate sequence analysis of class II antigens at the low femtomole level, (c) to sequence peptides presented by class I HLA- A2.l molecules on human melanoma cells that are recognized by melanoma specific cytotoxic T lymphocytes, (d) to sequence peptides presented by the human class I molecules, HLA-B7, Aw68, and Aw69, (e) to determine the structural basis of peptide interaction with class I, MHC molecules and to define peptide epitopes recognized by xenoreactive, HLA-A2.1 restricted CTL, (f) to sequence peptides processed from HIV virus proteins and presented in association the class 1, HLA-A2.l molecules, (g) to identify the range of endogenous influenza hemagglutinin pep tides associated with the murine MHC class 1 Kd molecule produced by the wild type hemagglutinin gene and synthetic minigenes and to identify endogenous hemagglutinin peptides that bind Kd but are not immunogenic.