Please use this identifier to cite or link to this item: http://hdl.handle.net/1942/29814
Title: Characterization and site-specific bio-functionalization of nanobodies for the early detection of ovarian cancer biomarkers
Authors: TRAN, Huong 
GRAULUS, Geert-Jan 
Smiejkowska, Natalia
Vincke, Cecile
Devoogdt, Nick
Muyldermans, Serge
ADRIAENSENS, Peter 
GUEDENS, Wanda 
Issue Date: 2019
Source: CHEMICAL RESEARCH IN FLANDERS CRF-2, Blankenberge-Belgium, 14-16/10/2019
Abstract: Epithelial ovarian cancer (EOC), of which the incidence increases with age, ranks fifth in cancer deaths among women1. The common symptoms of EOC are indistinct and similar to other benign observations2. Most women are diagnosed at advanced stage III or IV of the disease, at which the 5-year relative survival rate is low (around 39% for stage III and only 17% for stage IV)2,3. EOC can be successfully treated (with rate 92%) if it is detected early but the diagnosis of EOC at early stages is difficult since there are no symptoms and no screening test has proven to be effective (only 15% of all ovarian cancers are found at the early stage). In this study, different nanobodies4 targeting EOC markers such as Human epididymis protein 4 (HE4)2, Secretory leukocyte protease inhibitor (SLPI)3 and Progranulin (PGRN)3 were selected based on their expression level as well as their target binding affinity using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methods. The binding properties of the selected nanobodies were also determined by epitope mapping. The nanobodies with high expression level (almost 8 mg/ mL, 21 mg/ mL and 11 mg/ mL for HE4, SLPI and PGRN, respectively) and high binding affinity (KD value around 10-10– 10-8 M) were selected as best candidates for the development of multi-array biosensors that are capable of detecting multiple targets rapidly and combine high selectivity, high sensitivity and specificity for the detection of EOC in early stage. The selected nanobodies were successfully site-specifically alkynated at their C-terminus using the Expressed protein ligation (EPL) technique and were coupled to an azidified PEG counterpart using “click”-chemistry5. The attachment of a click-chemistry functionality at the C-terminus of the nanobodies would pave the way to sensor platforms at which all nanobodies are covalently coupled with a unique and uniform orientation, allowing optimal target binding and resulting in improved sensitivity and selectivity since all nanobodies will have their active region accessible for the target binding. 1. American Cancer Society. Cancer Facts & Figures 2019. Cancer Facts Fig. 2019 (2019). 2. Molina, R. et al. HE4 a novel tumour marker for ovarian cancer: comparison with CA 125 and ROMA algorithm in patients with gynaecological diseases. Tumour Biol. (2011). doi:10.1007/s13277-011-0204-3 3. Han, J. J., Yu, M., Houston, N., Steinberg, S. M. & Kohn, E. C. Progranulin is a potential prognostic biomarker in advanced epithelial ovarian cancers. Gynecol. Oncol. 120, 5–10 (2011). 4. Muyldermans, S. et al. Camelid immunoglobulins and nanobody technology. Vet. Immunol. Immunopathol. 128, 178–183 (2009). 5. Ta, D. T. et al. An efficient protocol towards site-specifically clickable nanobodies in high yield: Cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation. Protein Eng. Des. Sel. 28, 351–363 (2015).
Document URI: http://hdl.handle.net/1942/29814
Category: C2
Type: Conference Material
Appears in Collections:Research publications

Files in This Item:
File Description SizeFormat 
Final - Abstract for Chemical Research in Flanders-CRF-2 (1).pdfConference material290.98 kBAdobe PDFView/Open
Show full item record

Page view(s)

48
checked on Sep 6, 2022

Download(s)

4
checked on Sep 6, 2022

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.