title: Development of ELISA to measure TRPV1 protein in rat tissues.
abstract: The transient receptor potential vanilloid receptor type 1 (TRPV1) is a non-selective cation channel expressed in both the peripheral and the central nervous systems. To quantitatively determine TRPV1 protein levels in native rat tissues, novel monoclonal antibodies were raised against full-length recombinant human TRPV1 protein and utilized to develop a sandwich ELISA assay. Monoclonal antibody 10E3-1A2 specifically recognized TRPV1 protein and the recognition epitope was determined to reside in amino acids 45-58 of human and rat TRPV1. Using the TRPV1 polyclonal antibody ABRK4 as the capturing antibody and the monoclonal antibody 10E3-1A2 as the detection antibody, a sandwich ELISA that detected both human and rat TRPV1 protein was established. Recombinant human TRPV1 heterologously expressed in mammalian HEK293-F cells, which showed high ligand-binding affinity, was purified by TRPV1 monoclonal antibody affinity chromatography and used as protein standard to quantify TRPV1 protein levels. This ELISA detected TRPV1 protein as low as 1.5ng/ml (15pM), and was able to determine TRPV1 protein levels in native rat tissues such as DRG and spinal cord. This is the first TRPV1 sandwich ELISA that determines the abundance of TRPV1 protein in different tissues. It provides a powerful tool to quantify changes of TRPV1 protein levels in pathological states.
authors.affiliation: Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA. hanping08@gmail.com
authors.forename: Ping
authors.initials: P
authors.lastname: Han
authors.forename: Alla V
authors.initials: AV
authors.lastname: Korepanova
authors.forename: Melissa H
authors.initials: MH
authors.lastname: Vos
authors.forename: Ana
authors.initials: A
authors.lastname: Pereda-Lopez
authors.forename: Marc R
authors.initials: MR
authors.lastname: Lake
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Bianchi
authors.forename: Robert B
authors.initials: RB
authors.lastname: Moreland
authors.forename: Connie R
authors.initials: CR
authors.lastname: Faltynek
authors.forename: Mark L
authors.initials: ML
authors.lastname: Chiu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005727:Ganglia, Spinal / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas; D016056:Immunodominant Epitopes; D047468:Immunoprecipitation; D007546:Isoquinolines / Q000493:pharmacokinetics; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D013116:Spinal Cord / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics; D014508:Urea / Q000031:analogs & derivatives / Q000493:pharmacokinetics
chemical_list: C523980:1-(5-tertbutylindan-1-yl)-3-isoquinolin-5-ylurea; D000911:Antibodies, Monoclonal; D016056:Immunodominant Epitopes; D007546:Isoquinolines; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D014316:Tritium; D014508:Urea