title: Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles.
abstract: Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.
authors.affiliation: IBEC-Institute for Bioengineering of Catalonia, Nanobioengineering Group, C/Baldiri Reixac, 15-21, 08028 Barcelona, Spain; Department of Electronics, University of Barcelona, C/Martí i Franquès 1, 08028 Barcelona, Spain. Electronic address: msanmarti@ibecbarcelona.eu.
authors.forename: Marta
authors.initials: M
authors.lastname: Sanmartí-Espinal
authors.affiliation: Nb4D-Nanobiotechnology for Diagnostics, IQAC-CSIC, C/Jordi Girona, 18-26, 08034 Barcelona, Spain; Centro de Investigación Biomédica en Red en Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), C/María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain.
authors.forename: Roger
authors.initials: R
authors.lastname: Galve
authors.affiliation: IBEC-Institute for Bioengineering of Catalonia, Nanobioengineering Group, C/Baldiri Reixac, 15-21, 08028 Barcelona, Spain.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Iavicoli
authors.affiliation: NBO, INRA, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
authors.forename: Marie-Annick
authors.initials: MA
authors.lastname: Persuy
authors.affiliation: NBO, INRA, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
authors.forename: Edith
authors.initials: E
authors.lastname: Pajot-Augy
authors.affiliation: Nb4D-Nanobiotechnology for Diagnostics, IQAC-CSIC, C/Jordi Girona, 18-26, 08034 Barcelona, Spain; Centro de Investigación Biomédica en Red en Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), C/María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain.
authors.forename: M-Pilar
authors.initials: MP
authors.lastname: Marco
authors.affiliation: IBEC-Institute for Bioengineering of Catalonia, Nanobioengineering Group, C/Baldiri Reixac, 15-21, 08028 Barcelona, Spain; Department of Electronics, University of Barcelona, C/Martí i Franquès 1, 08028 Barcelona, Spain; Centro de Investigación Biomédica en Red en Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), C/María de Luna 11, Edificio CEEI, 50018 Zaragoza, Spain. Electronic address: jsamitier@ibecbarcelona.eu.
authors.forename: Josep
authors.initials: J
authors.lastname: Samitier
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007120:Immunochemistry / Q000379:methods*; D018796:Immunoconjugates / Q000737:chemistry; D057230:Limit of Detection; D008051:Lipid Bilayers / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry* / Q000276:immunology; D018035:Receptors, Odorant / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D017481:Receptors, Somatostatin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D012015:Reference Standards; D013194:Staining and Labeling / Q000379:methods; D013388:Succinimides / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D008051:Lipid Bilayers; C489427:MYC protein, human; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D018035:Receptors, Odorant; D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; C574734:SSTR2 protein, human; D013388:Succinimides; C514389:odorant receptor OR17-40, human
keywords: Bioelectronic nose; Competitive ELISA; G-protein-coupled receptors quantification; Natural vesicles; Olfactory receptors; Transmembrane proteins