Production of a common epitope specific anti-ankyrin monoclonal antibody
Article Sidebar
Main Article Content
Abstract
Background: Ankyrin (Ank) is a protein family with crucial roles in retaining normal cellular physiology. In addition, ankyrin offers the potential as a non-antibody binder against various biomolecules. The designed ankyrin repeat protein (DARPin) selected from phage display libraries is useful for molecular detection and therapy. Monoclonal antibodies (mAbs) specific to the common epitope of DARPin are required to detect protein-protein interaction.
Objectives: This study aimed to establish mAbs against common antigenic determinant of ankyrins for further application in immunological techniques.
Materials and methods: Ank1D4 monomer and dimer were generated in the Escherichia coli expression system for immunogen preparation and validation of established mAbs. The binding activity of anti-Ank mAb obtained from different hybridoma clones was characterized using Ank1D4 by indirect ELISA. Candidate anti-Ank mAbs were validated for their cross-reactivity against irrelevant ankyrin (Ank2D3). The binding kinetic of mAbs from three candidate hybridoma clones (Ank-54, Ank-59, and Ank-94) was evaluated using bio-layer interferometry (BLI). The highest affinity clone (Ank-94 mAb ) was further validated for its specificity against Ank1D4 and dimeric Ank1D4 using indirect ELISA. The interaction of three anti-Ank mAbs and ankyrins was compared by western immunoblotting analysis. The specificity of Ank-94 mAb was determined using a closely related scaffold, i.e., alpha-helicoidal HEAT-like repeat protein scaffold (αRep) by indirect ELISA. Ankyrins were detected by sandwich ELISA using Ank-94 mAb.
Results: The culture supernatant from hybridoma clones were characterized for their anti-ankyrin binding properties. Using indirect ELISA, three clones exhibited positive reactivity against the immunized ankyrin antigen (Ank1D4). The interactive epitope was found to rely on common antigenic determinants found in Ank1D4, dimeric Ank1D4, and an irrelevant ankyrin, Ank2D3. The immunoblotting results suggest that all mAbs interact with the sequential epitope of ankyrins. The cross-reactivity of Ank-94 mAb was not observed with αRep. Ank-94 mAb was selected for further purification and evaluation of binding properties due to its highest degree of binding affinity against Ank1D4.
Conclusion: The establishment of a novel Ank-94 mAb could be a valuable research tool in tracing the target of DARPins or developing immunoassays. Ank-94 mAb is superior over formerly produced Ank mAbs since it recognizes a common epitope on DARPins and relies on sequential epitope. Ank-94 mAb has no cross-reactivity with another scaffold, αRep.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Personal views expressed by the contributors in their articles are not necessarily those of the Journal of Associated Medical Sciences, Faculty of Associated Medical Sciences, Chiang Mai University.
References
Koenig S, Mohler P. The Evolving Role of Ankyrin-B in Cardiovascular Disease. Hear Rhythm [Internet]. 2017;176(10):1884–1889. Available from: file:///C:/Users/Carla Carolina/Desktop/Artigos para acrescentar na qualificação/The impact of birth weight on cardiovascular disease risk in the.pdf
Ranasinghe SL, Fischer K, Gobert GN, McManus DP. Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni. Parasites and Vectors [Internet]. 2015;8(1):1–10. Available from: http://dx.doi.org/10.1186/s13071-015-1022-z
Bon H, Hales P, Lumb S, Holdsworth G, Johnson T, Qureshi O, et al. Spontaneous Extracellular Matrix Accumulation in a Human in vitro Model of Renal Fibrosis Is Mediated by αv Integrins. Nephron. 2019; 142(4): 329-50.
Müller-Pillasch F, Wallrapp C, Bartels K, Varga G, Friess H, Büchler M, et al. Cloning of a new Kunitz-type protease inhibitor with a putative transmembrane domain overexpressed in pancreatic cancer. Biochim Biophys Acta - Gene Struct Expr. 1998; 1395(1): 88-95.
Arai Y, Suzuki A, Mizuguchi M, Takashima S. Developmental and aging changes in the expression of amyloid precursor protein in Down syndrome brains. Brain Dev. 1997; 19(4): 290-4.
Suzuki A, Takashima S, Mizuguc M, Kunishita T, Tabira T. High Expression Cerebral Syndrome Vessels on Kunitz-Type Substances of Patients with Down Syndrome. Tohoku J Exp Med. 1994; 174: 181-7.
Nakamura K, Abarzua F, Hongo A, Kodama J, Nasu Y, Kumon H, et al. Hepatocyte growth factor activator inhibitor-2 (HAI-2) is a favorable prognosis marker and inhibits cell growth through the apoptotic pathway in cervical cancer. Ann Oncol [Internet]. 2009;20(1):63–70. Available from: https://doi.org/10.1093/annonc/mdn556
Satchwell TJ, Bell AJ, Hawley BR, Pellegrin S, Mordue KE, van Deursen CTBM, et al. Severe Ankyrin-R deficiency results in impaired surface retention and lysosomal degradation of RhAG in human erythroblasts. Haematologica. 2016; 101(9): 1018-27.
Gibson NJ, Tolbert LP, Oland LA. Roles of specific membrane lipid domains in EGF receptor activation and cell adhesion molecule stabilization in a developing olfactory system. PLoS One. 2009; 4(9).
Kretschmer T, Nguyen DH, Beuerman RW, Tiel RL, Kline DG. Elevated ankyrin G in a plexiform neurofibroma and neuromas associated with pain. J Clin Neurosci. 2004; 11(8): 886-9.
Urakami K, Okada A, Takahashi K, Ohno K, Kitaguchi N, Tanaka S, et al. Amyloid Beta Protein Precursor with Kunitz-Type Protease Inhibitor Domains (APPI) in Cerebrospinal Fluid and APPI mRNAs in Cultured Skin Fibroblasts of Patients with Alzheimer’s Disease. Tohoku J Exp Med. 1994; 174(3): 199-207.
Epa VC, Dolezal O, Doughty L, Xiao X, Jost C, Plückthun A, et al. Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2. PLoS One. 2013; 8(3): 1-10.
Nangola S, Urvoas A, Valerio-Lepiniec M, Khamaikawin W, Sakkhachornphop S, Hong SS, et al. Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein. Retrovirology [Internet]. 2012;9(1):17. Available from: http://www.retrovirology.com/content/9/1/17
Plückthun A. Designed ankyrin repeat proteins (DARPins): Binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol. 2015; 55: 489-511.
Binz HK, Stumpp MT, Forrer P, Amstutz P, Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J Mol Biol. 2003; 332(2): 489-503.
Forrer P, Stumpp MT, Binz HK, Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins. FEBS Lett. 2003; 539(1-3): 2-6.
Michaely P, Tomchick DR, Machius M, Anderson RGW. Crystal structure of a 12 ANK repeat stack from human ankyrinR. EMBO J. 2002; 21(23): 6387-96.
Boersma YL. Advances in the Application of Designed Ankyrin Repeat Proteins (DARPins) as Research Tools and Protein Therapeutics. Methods Mol Biol. 2020.
Milovnik P, Ferrari D, Sarkar CA, Plückthun A. Selection and characterization of DARPins specific for the neurotensin receptor 1. Protein Eng Des Sel. 2009; 22(6): 357-66.
Praditwongwan W, Chuankhayan P, Saoin S, Wisitponchai T, Lee VS, Nangola S, et al. Crystal structure of an antiviral ankyrin targeting the HIV-1 capsid and molecular modeling of the ankyrin-capsid complex. J Chem PhysJ Chem Phys. 2014/07/06. 2014; 28(8): 869-84.
Hadpech S, Nangola S, Chupradit K, Fanhchaksai K, Furnon W, Urvoas A, et al. Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation. Sci Rep. 2017; 7(1): 1-19.
Sievers F, Higgins DG. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 2018; 27(1): 135-45.
Mugabo Y, Lim GE. Scaffold proteins: From coordinating signaling pathways to metabolic regulation. Endocrinology. 2018; 159(11): 3615-30.
Marlor CW, Delaria KA, Davis G, Muller DK, Greve JM, Tamburini PP. Identification and cloning of human placental bikunin, a novel serine protease inhibitor containing two Kunitz domains. J Biol Chem [Internet]. 1997;272(18):12202–8. Available from: http://dx.doi.org/10.1074/jbc.272.18.12202
Tomasini-Johansson BR, Zbyszynski PW, Toraason I, Peters DM, Kwon GS. PEGylated pUR4/FUD peptide inhibitor of fibronectin fibrillogenesis decreases fibrosis in murine Unilateral Ureteral Obstruction model of kidney disease. PLoS One. 2018; 13(10).
Jiang X, Seo YD, Chang JH, Coveler A, Nigjeh EN, Pan S, et al. Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment. Oncoimmunology [Internet]. 2017; 6(7): 1–12. Available from: https://doi.org/10.1080/2162402X.2017.1333210
Kretschmer T, England JD, Happel LT, Liu ZP, Thouron CL, Nguyen DH, et al. Ankyrin G and voltage gated sodium channels colocalize in human neuroma - Key proteins of membrane remodeling after axonal injury. Neurosci Lett. 2002; 323(2): 151-5.
Siegel PM, Bojti I, Bassler N, Holien J, Flierl U, Wang X, et al. A DARPin targeting activated Mac-1 is a novel diagnostic tool and potential anti-inflammatory agent in myocarditis, sepsis and myocardial infarction. Basic Res Cardiol [Internet]. 2021;116(1): 1-25. Available from: https://doi.org/10.1007/s00395-021-00849-9
Nangola S, Thongkum W, Saoin S, Ansari AA, Tayapiwatana C. An application of capsid-specific artificial ankyrin repeat proteinproduced in E. coli for immunochromatographic assay as a surrogate for antibody. Appl Microbiol Biotechnol. 2014; 98(13): 6095-103.