Monoclonal Antibody Resources for C. elegans

描述(由申请人提供)： 抗体被广泛用于原位和生化实验中检测特定的细胞成分，目的是确定蛋白质之间的相互作用、蛋白质加工和可逆的蛋白质修饰，如磷酸化。多克隆抗体制备相对简单，在分子和细胞研究中得到广泛应用。然而，这类试剂的数量有限，因此很少广泛使用。单抗的制作比较复杂，但分泌单抗的杂交瘤可以无休止地传代，允许许多不同的研究小组长期使用相同的试剂。市场上有针对大量脊椎动物细胞成分的多克隆和单克隆抗体。相比之下，很少有这样的商业试剂可用于研究秀丽线虫等模式生物，可能是因为这些试剂的市场太小，无法使它们具有商业可行性。我建议开发一种“工具包”，由针对一系列蛋白质的单抗组成，这些蛋白质标记线虫的离散细胞成分和亚细胞隔间。这些隔室包括所有细胞共有的细胞器(如内质网、高尔基体、内小体)、特化细胞的细胞器(如突触小泡)、所有细胞共有的亚细胞结构(如核膜、中心体和小凹)以及标记发育中的胚胎、生殖系和神经系统中的结构域的成分。细胞成分包括大分子复合体(如剪接体、蛋白体、复制起始复合体)和常见的细胞蛋白(如笼蛋白、动蛋白、动力蛋白和中间丝)。抗体将被彻底描述，并通过发展研究杂交瘤银行向公众提供。该工具包将对线虫研究人员非常有用。它将提供一套标准化的可再生参考抗体工具，供整个社区使用。其次，它将极大地促进其他细胞成分的定位，因为它提供了一大套细胞标记，可以与兔多克隆抗体配合使用，进行共同定位研究。最后，它将通过提供检测提取物中特定细胞隔间和免疫沉淀物中特定成分存在的手段，促进生化方法的使用，例如亚细胞分离和免疫沉淀。

DESCRIPTION (provided by applicant): Antibodies (Abs) are widely used to detect specific cellular components both in situ and in biochemical experiments aimed at defining protein-protein interactions, protein processing, and reversible protein modifications such as phosphorylation. Polyclonal Abs are relatively simple to make and are widely used in molecular and cellular studies. However, such reagents are limited in quantity and thus rarely made widely available. Monoclonal Abs are more complicated to make, but hybridomas secreting the monoclonal antibody can be passaged ad infinitum permitting the identical reagent to be used by many different research group over long periods of time. Polyclonal and monoclonal Abs are available commercially that are directed against a huge array of vertebrate cellular components. By contrast, few such commercial reagents are available for work with model organisms such as C. elegans probably because the market for these reagents is too small to make them commercially viable. I propose to develop a 'tool kit' consisting of monoclonal Abs directed against a series of proteins that label discrete cellular components and subcellular compartments of C. elegans. The compartments include organelles common to all cells (e.g. endoplasmic reticulum, the Golgi, endosomes), organelles of specialized cells (e.g. synaptic vesicles), subcellular structures common to all cells (e.g. nuclear membrane, centrosomes and caveoli) and components that label domains in the developing embryo, germline, and nervous system. The cellular components include macromolecular complexes (e.g. splicesomes, proteosomes, replication origin complexes) and common cellular proteins (e.g clathrin, kinesin, dynein and intermediate filaments). The Abs will be thoroughly characterized and made publically available through the Developmental Studies Hybridoma Bank. The tool kit will be extremely useful for C. elegans researchers. It will provide a set of standardized renewable reference antibody tools available to the entire community. Secondly, it will greatly facilitate localizing other cellular components by providing a large set of cellular markers that can be used coordinately with rabbit polyclonal antibodies for co-localization studies. Finally, it will stimulate the use of biochemical approaches, such as subcellular fractionation and immunoprecipitations, by providing means to detect specific cellular compartments in extracts and the presence of specific components in immunoprecipitates.