POLYCLONAL LIBRARY--RESOURCES FOR ANTIBODIES & CATALYSIS

多克隆文库--抗体资源

• 批准号: 3438708
• 负责人: Richard A Goldsby
• 金额: $ 10.76万
• 依托单位: AMHERST COLLEGE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438708
• 关键词: antibody; antibody formation; antibody specificity; antigen antibody reaction; bacterial antigens; biomedical resource; calmodulin; catalyst; digoxin; enzyme linked immunosorbent assay; hybridomas; insulin; surface antigens; thyroxine; tissue /cell culture

Antibodies with catalytic activity for a particular reaction can be elicited in the Balb/c mouse after immunization with an appropriate transition state analog. However, the inability to determine precisely the states of many reactions is a barrier to the general exploration of the novel phenomenon of antibody catalysis. A way around the general lack of precise structural information about the transition state might be found by pursuing the alternative approach of searching a large sample of the Balb/c repertoire for antibodies which can catalyze a particular reaction. This proposal seeks to demonstrate the versatility and power of a large hybridoma library which incorporates a significant fraction of the repertoire of specificities (not clonotypes) in the Balb/c mouse. The library, whose composition is substantially underway, is constructed by the PEG-assisted fusion of polyclonally activated B cell blasts from unimmunized Balb/c mice. The library will ultimately contain a large number (ca. 10.000) of discrete hybridoma populations, each of which is composed of at least 1 and usually 3 to 5 or more clones. This library will be screened to demonstrate that some of its members show catalytic activity. Chromogenic or radioactive substrates will be used to identify hybridoma populations that catalyze the hydrolysis of any one of a variety of structural motifs. In addition there is the possibility that among its members one can find a diverse spectrum of antibodies whose affinity or antigen specificity qualifies them as useful. The diversity of antigen reactivities represented in the library will be determined by screening its members against an antigen panel that includes differentiation antigens, different bacterial species, and solutions of soluble antigens (thyroxine, insulin, calmodulin, and digoxin) at submicromolar concentrations. The significance of this approach lies in its potential to provide an alternative route to the derivation of antibodies to immunization programs. Also, there is the intriguing possibility that antibodies whose expression is normally blocked by constraints of tolerance could be accessed by this technology. This might make it possible to derive antibodies to highly conserved determinants on particular types of molecules or specific categories of cells that are of interest in many different species.

可以将对特定反应具有催化活性的抗体 在Balb/c小鼠中，在用适当的 过渡态类似物 然而，无法精确地确定 许多反应的状态是一般探索的障碍。 抗体催化的新现象。 绕过将军的方法 缺乏关于过渡状态的精确结构信息可能 通过寻找大样本的替代方法来发现 Balb/c的抗体库，可以催化特定的 反应 该提案旨在展示一个大型 杂交瘤文库，其掺入了显著部分的 Balb/c小鼠中的特异性（非克隆型）库。 的 图书馆，其组成是基本上正在进行中，是由 PEG辅助的多克隆活化的B细胞母细胞的融合， 未免疫Balb/c小鼠。 图书馆最终将包含一个大型的 数（ca. 10.000）的离散杂交瘤群体，每个杂交瘤群体是 由至少1个和通常3至5个或更多个克隆组成。 此库 将被筛选，以证明它的一些成员表现出催化作用， 活动 将使用显色或放射性底物来鉴定 杂交瘤群体，所述杂交瘤群体催化以下任一种的水解： 各种结构图案。 此外，还有一种可能性， 在其成员中，可以发现多种抗体谱， 亲和性或抗原特异性使它们成为有用的。 之多样 文库中代表的抗原反应性将通过 针对抗原组筛选其成员，所述抗原组包括 分化抗原，不同的细菌物种，以及 可溶性抗原（甲状腺素、胰岛素、钙调蛋白和地高辛）. 亚微摩尔浓度。 这种方法的意义在于 它的潜力，以提供一个替代路线的推导， 免疫计划的抗体。 此外，还有一个耐人寻味的 表达通常被抗体阻断的抗体 可以通过该技术访问公差的约束。 这 这可能会使我们有可能获得针对高度保守的 决定因素对特定类型的分子或特定类别的 在许多不同的物种中都有意义的细胞