High-throughput screening and identification of scFv antibodies against glycosyla

描述（由申请人提供）：本提案的具体目标是开发一种高通量方法来鉴定识别翻译后修饰蛋白的scFv抗体。我们建议完成一种基于乳液、分泌和捕获的新型亲和筛选过程的概念验证，我们称之为“逃逸”。在提出的噬菌体ESCape方法中，用编码scFv抗体- gpiii融合文库的噬菌体转化大肠杆菌，用M13K07-H9 （His6::gpIX）辅助噬菌体感染。然后将受感染的细菌与钴螯合物“捕获头”一起隔离在油包水（W/O）乳剂中。因此，在过夜孵育期间，在每个分离的室中，产生数千个重组噬菌体拷贝，其中一端显示scFv蛋白（与gpIII蛋白融合），另一端显示his6肽（与gpIX蛋白融合）（图1）。显示噬菌体的h6末端将与钴螯合捕获头结合。乳剂会被打破，这些小珠子和附着在上面的噬菌体就会被分离出来。与噬菌体结合的小珠将与Alexa 488标记的抗原一起孵育，然后清洗以去除任何未结合的配体。然后用流式细胞术将标记的珠粒（连同附着在其上的噬菌体）从珠粒群中分选出来。富集的小珠可以被镀在大肠杆菌的草坪上，噬菌体编码的scFv进一步测试特异性和亲和力。我们将把这种噬菌体ESCape方法与传统的噬菌体生物筛选方法进行比较，以鉴定识别c-ErbB2/Her2蛋白糖基化胞外结构域的scFv抗体。将评估鉴定的特异性scFvs的数量、它们的结合亲和力、与筛选方法相关的时间和成本，以及自动化检测的能力。

DESCRIPTION (provided by applicant): The specific goal of this proposal is to develop a high-throughput method to identify scFv antibodies that recognize post-translationally modified proteins. We propose to complete a proof-of-concept for a new type of affinity screening process based on emulsion, secretion, and capture, which we term "ESCape". In the proposed phage ESCape method, E. coli transformed with a phagemid encoding an scFv antibody-gpIII fusion library is infected with M13K07-H9 (His6::gpIX) helper phage. The infected bacterium is then compartmentalized along with a cobalt-chelate "capture-bead" in a water-in-oil (W/O) emulsion. Consequently, in each isolated compartment during overnight incubation, thousands of copies of the recombinant phage displaying an scFv protein at one end (fused to the gpIII protein) and a his6 peptide at the opposite end (fused to the gpIX protein) are produced (Fig. 1). The his6-end of the display phage will bind to the cobalt-chelate capture-bead. The emulsion will be broken, and these beads, along with any phage bound to them, will be isolated. The phage-bound beads will be incubated with an Alexa 488-labeled antigen and then washed to remove any unbound ligand. The labeled beads will then be sorted (together with the phage attached to them) from the population of beads by flow cytometry. Enriched beads can be plated onto a lawn of E. coli and the phagemid-encoded scFv further tested for specificity and affinity. We will compare this phage ESCape method with traditional methods for phage biopanning to identify scFv antibodies that recognize the glycosylated extracellular domain of the c-ErbB2/Her2 protein. The number of specific scFvs identified, their binding affinities, the time and cost associated with the screening method, and the ability to automate the assay will be evaluated. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to develop a high-throughput method to identify scFv antibodies that recognize post-translationally modified proteins. A novel method is described for amplifying single bacteriophage within an emulsion droplet and subsequently capturing and displaying the phage on the surface of a 3 micron bead. We will apply this phage-based surface display method toward identification of single chain variable fragment (scFv) antibodies that specifically recognize a glycosylated protein. The number of specific scFvs identified, their binding affinities, the time and cost associated with the screening method, and the ability to automate the assay will be evaluated and compared with traditional phage biopanning.