Research paper Novel antigen design for the generation of antibodies to G-protein-coupled receptors K. Larsson a , C. Hofström b , C. Lindskog c , M. Hansson d , P. Angelidou d , T. Hökfelt e , M. Uhlén a , H. Wernérus d , T. Gräslund b , S. Hober a, ⁎ a Division of Proteomics, School of Biotechnology, KTH/AlbaNova University Center, Stockholm, Sweden b Division of Molecular Biotechnology, School of Biotechnology, KTH/AlbaNova University Center, Stockholm, Sweden c Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden d Atlas Antibodies, AlbaNova University Center, Stockholm, Sweden e Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden article info abstract Article history: Received 27 December 2010 Received in revised form 5 May 2011 Accepted 5 May 2011 Available online 12 May 2011 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. © 2011 Elsevier B.V. All rights reserved. Keywords: GPCR Antibody Antigen design 1. Introduction G-protein-coupled receptors (GPCRs) belong to a protein family characterized by seven transmembrane helices con- nected by loops, an extracellular N-terminal and an intracellular C-terminal tail (Fig. 1A). This family of cellular receptor proteins is the largest protein family with more than 1000 members (Wess, 1997), regulating cellular function through interactions with a diverse selection of ligands, such as neurotransmitters, hormones, light, odors and taste (Lundström, 2005). Due to the regulating activities, GPCRs are very interesting as drug targets and currently 60–70% of the drug development is focused on GPCRs (Lundström, 2005). However, because of difficulties in production and purification of these membrane proteins (Lundström, 2005; Sarkar et al., 2008), only a handful of solved structures exists in the Protein Data Bank, and thus information valuable for the drug development process is often lacking (Berman et al., 2000). In order to further understand and study GPCRs, genetic fusion to tags has commonly been used. To this end, small tags like His 6 -, HA- or Flag have been expressed together with the target receptor (Jongsma et al., 2007). Hence, monoclonal antibodies recognizing the tag can Journal of Immunological Methods 370 (2011) 14–23 Abbreviations: GPCR, G-protein-coupled receptor; B2AR, beta 2-adrenergic receptor; GLP1R, glucagon-like peptide 1 receptor; NPY5R, neuropeptide Y receptor 5; ABP, albumin binding protein; HPA, Human Protein Atlas. ⁎ Corresponding author. Tel.: + 46 8 5537 8330; fax: +46 8 5537 8481. E-mail address: sophia.hober@biotech.kth.se (S. Hober). 0022-1759/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2011.05.001 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim