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Article

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

by
Farah Perveen Mughal
1,
Ann Christina Bergmann
2,
Ha Uyen Buu Huynh
2,
Sarah Hyllekvist Jørgensen
3,4,
Inaam Mansha
2,
Meliha Kesmez
3,
Patrick Mark Schürch
5,
Alexandre Pierre André Theocharides
5,
Paul Robert Hansen
1,
Tina Friis
2,
Morten Orebo Holmström
6,
Evaldas Ciplys
7,
Rimantas Slibinskas
7,
Peter Højrup
3,
Gunnar Houen
2,3,8,* and
Nicole Hartwig Trier
8,*
1
Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark
2
Department of Autoimmunology and Biomarkers, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark
3
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
4
Global Research Technologies, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark
5
Department of Medical Oncology and Hematology, University Hospital Zurich, Rämistrasse 100, 8091 Zurich, Switzerland
6
National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark
7
Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio al. 7, LT-10257 Vilnius, Lithuania
8
Department of Neurology, Rigshospitalet Glostrup, Valdemar Hansens vej 13, 2600 Glostrup, Denmark
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(12), 6803; https://doi.org/10.3390/ijms23126803
Submission received: 7 April 2022 / Revised: 4 June 2022 / Accepted: 15 June 2022 / Published: 18 June 2022
(This article belongs to the Special Issue Advances in Antibody Design and Antigenic Peptide Targeting 2.0)
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Abstract

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.
Keywords: calreticulin; epitope mapping; myeloproliferative neoplasms; peptide antibodies; frameshift mutations calreticulin; epitope mapping; myeloproliferative neoplasms; peptide antibodies; frameshift mutations

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MDPI and ACS Style

Mughal, F.P.; Bergmann, A.C.; Huynh, H.U.B.; Jørgensen, S.H.; Mansha, I.; Kesmez, M.; Schürch, P.M.; Theocharides, A.P.A.; Hansen, P.R.; Friis, T.; et al. Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases. Int. J. Mol. Sci. 2022, 23, 6803. https://doi.org/10.3390/ijms23126803

AMA Style

Mughal FP, Bergmann AC, Huynh HUB, Jørgensen SH, Mansha I, Kesmez M, Schürch PM, Theocharides APA, Hansen PR, Friis T, et al. Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases. International Journal of Molecular Sciences. 2022; 23(12):6803. https://doi.org/10.3390/ijms23126803

Chicago/Turabian Style

Mughal, Farah Perveen, Ann Christina Bergmann, Ha Uyen Buu Huynh, Sarah Hyllekvist Jørgensen, Inaam Mansha, Meliha Kesmez, Patrick Mark Schürch, Alexandre Pierre André Theocharides, Paul Robert Hansen, Tina Friis, and et al. 2022. "Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases" International Journal of Molecular Sciences 23, no. 12: 6803. https://doi.org/10.3390/ijms23126803

APA Style

Mughal, F. P., Bergmann, A. C., Huynh, H. U. B., Jørgensen, S. H., Mansha, I., Kesmez, M., Schürch, P. M., Theocharides, A. P. A., Hansen, P. R., Friis, T., Holmström, M. O., Ciplys, E., Slibinskas, R., Højrup, P., Houen, G., & Trier, N. H. (2022). Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases. International Journal of Molecular Sciences, 23(12), 6803. https://doi.org/10.3390/ijms23126803

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