Peti ThuwajitChanitra ThuwajitPornpimon Yuti2024-01-102024-01-10201920192024Thesis (M.Sc. (Immunology))--Mahidol University, 2019https://repository.li.mahidol.ac.th/handle/20.500.14594/92203Immunology (Mahidol University 2019)Trophoblast cell surface antigen 2 (Trop2) overexpression has been reported in various human cancers. A single chain variable fragment (ScFv) is a common type of antibody fragments that can be developed and modified to use for many purposes. Circulating tumor cells (CTCs) play a role in tumor progression and become the interesting biomarker for detection among cancer patients because it can be simplified for monitoring progression of patients. This study aimed to develop an easy usage of ScFv against Trop2 to detect Trop2-expressing cancer cells. The ScFv against Trop2 with mCherry labelling (Trop2mCherry ScFv) was constructed by phage display technique and expressed by bacterial system (Escherichia coli). After production process, it was tested for the binding efficiency on many types of cancer cell lines to screen for Trop2 expression by immunofluorescence analysis (IFA) and confocal microscopy analysis, in comparison to the staining by commercial anti-Trop2 antibody. For application, situation of CTCs was performed by mixing the Trop2-expressing cancer cell with normal blood and stained by this ScFv and then detected under fluorescence microscope. The results showed KKU-213, as the highest, and KKU-055, as the lowest Trop2-expressing cells. The staining of Trop2mCherry ScFv was significantly reduced in Trop2-knockdown KKU-213 cells when compared with un-transfected cells. Overexpression of Trop2 were performed in KKU-055 cells when observed by confocal microscope analysis. The results showed the co-localization between Trop2-GFP tag and binding of antibody had no difference between commercial antibody and Trop2mCherry ScFv. When the situation of CTCs was performed by KKU-213 cells mixing together with blood from heathy donor, only KKU-213 cells were stained by Trop2mCherry ScFv. Therefore, Trop2mCherry ScFv could stain Trop2 expressing cells compatible to commercial antibody. Moreover Trop2mCherry ScFv may detect Trop2-expressing cancer cells in blood circulation. In summary, Trop2mCherry can be produced for detection of Trop2 expression on surface of cancer cells and should be developed for simplified utilization.xiii, 89 leaves : ill.application/pdfengผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้าCancer cells -- Laboratory manualsTumors -- Biopsy -- Laboratory manualsTrophoblast -- ImmunologyMaster ThesisMahidol University