tailieunhanh - Antibody Phage Display Methods and Protocols - part 7

Sau khi rửa sâu rộng, thể thực khuẩn bị ràng buộc eluted và khuếch đại trước khi được sử dụng cho vòng lựa chọn tiếp theo (Hình 1). Sau ba và bốn vòng lựa chọn, chúng tôi đã phân tích scFv Abs từ nhân bản thể thực khuẩn cá nhân cho phản ứng chống lại tuyến ức và các cơ quan lymphoid và nonlymphoid khác nhau | 236 Radosevic and van Ewijk splenocytes absorber cells in order to remove phage of undesired specificities. The thymic tissue was fixed using total body perfusion fixation 6 then minced into small fragments and nonadherent thymocytes were removed by vigorous shaking. The selection of the preabsorbed library on the thymic fragments was performed overnight at 4 C in the presence of a fresh batch of fixed absorber cells. After extensive washing the bound phages were eluted and amplified before being used for the next selection round Fig. 1 . Following three and four rounds of selection we analyzed scFv Abs from individual phage clones for reactivity against thymus and various lymphoid and nonlymphoid organs using immunohistochemistry. Using this subtractive selection protocol we were able to isolate scFv Abs that bind to murine thymic stromal cells selector tissue Abs reactive with lymphoid cells absorber cells were not detected. Furthermore some of the isolated clones crossreacted with human thymic stromal cells indicating that Abs recognizing evolutionary conserved epitopes were recovered Fig. 2 . The subtractive selection of phage Ab libraries on tissue fragments should be adaptable for use against tissues other than the thymus with the aim of generating Abs against tissue-specific antigens. The choice of selector tissue and absorber cells tissue as well as incubation conditions will depend on the individual research question and desired application. In general this approach could be applied in the studies of all disease processes that involve qualitative changes in the histology of the affected tissue. One possible application is in tumor biology in which tumor-cell-specific markers might easily be lost during the preparation of single-cell suspensions because of the isolation procedure. Furthermore abnormalities related to a tumor may not only be located on the tumor cells but also in the extracellular matrix. Therefore using this approach with tumor tissue as .