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Novel antigen design for the generation of antibodies to G-protein-coupled receptors
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
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2011 (English)In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 370, no 1-2, 14-23 p.Article in journal (Refereed) Published
Abstract [en]

Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

Place, publisher, year, edition, pages
2011. Vol. 370, no 1-2, 14-23 p.
Keyword [en]
GPCR, Antibody, Antigen design
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-38142DOI: 10.1016/j.jim.2011.05.001ISI: 000293481600002Scopus ID: 2-s2.0-79960073239OAI: oai:DiVA.org:kth-38142DiVA: diva2:436335
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20110823

Available from: 2011-08-23 Created: 2011-08-22 Last updated: 2017-12-08Bibliographically approved

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Uhlén, MathiasGräslund, TorbjörnHober, Sophia

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Larsson, KarinHofström, CamillaUhlén, MathiasGräslund, TorbjörnHober, Sophia
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Proteomics (closed 20130101)Molecular Biotechnology (closed 20130101)School of Biotechnology (BIO)
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JIM - Journal of Immunological Methods
Biochemistry and Molecular Biology

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