Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution.
Flow cytometry-based methods for assessing soluble scFv activities and detecting antigens in solution.
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DOI：
10.1002/bit.22607
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发表时间：
2010-04-01
影响因子：
3.8
通讯作者：
Cangelosi, Gerard A.
中科院分区：
文献类型：
--
作者：
Gray, Sean A.;Weigel, Kris M.;Miller, Keith D.;Ndung'u, Joseph;Buscher, Philippe;Tran, Thao;Baird, Cheryl;Cangelosi, Gerard A.
关键词：
报道了评估和利用来自酵母展示文库的单链片段可变抗体(ScFv)的新方法。用酵母展示法筛选布氏锥虫不变表面糖蛋白(ISG)特异性单链抗体。从非免疫库中分离单链抗体的一个限制步骤是将高活性酵母展示的单链抗体转化为可用于标准免疫分析的可溶性抗体。面临的挑战包括分泌和纯化单链抗体后的溶解度或活性有限。因此，很少有来源于酵母展示平台的scFv作为诊断试剂进入开发和实施阶段。为了解决这个问题，开发了同时使用酵母展示单链抗体和分泌单链抗体作为分析试剂的分析方法。第一种是竞争抑制流式细胞术(CIFC)，它通过竞争性抑制生物素化抗原与酵母展示的ScFv的结合来检测分泌的ScFv。第二种是表位结合试验，它使用分泌的单链抗体来识别额外的酵母展示的单链抗体，这些单链抗体与抗原上的非重叠或非竞争表位结合。表位结合试验不仅用于鉴定酵母展示的单链抗体的夹心分析对，而且还用于鉴定低浓度存在于表达粗提物中的活性可溶性单链抗体。最后，开发了一种CIFC方法，通过使用酵母展示的scFv来检测样品中的未标记抗原，完全绕过了对可溶性scFv表达的需要。这些方法将有助于酵母展示文库来源的单链抗体的继续开发和实际应用。
Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. Alimiting step in the isolation of scFv from non-immune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeast-displayed and -secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of their ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv to identify additional yeast-displayed scFv that bind non-overlapping or non-competing epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast-displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.
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