title: Generation of a novel monoclonal antibody that recognizes the alpha (α)-amidated isoform of a valine residue.
abstract: BACKGROUND
  Alpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species.
  
  
  METHODS
  The α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (-CONH2) and free α-carboxylic acid (-COO(-)) isovariant of the valine residue.
  
  
  RESULTS
  P18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/μg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb.
  
  
  CONCLUSIONS
  Our results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others.
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. bapags@gmail.com.
authors.forename: Benito
authors.initials: B
authors.lastname: Antón Palma
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. pleff@ymail.com.|Department of Neuroscience, National Institute of Perinatology, Montes Urales # 800, 11000, México D.F., Mexico. pleff@ymail.com.
authors.forename: Philippe
authors.initials: P
authors.lastname: Leff Gelman
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. maymar@hotmail.com.
authors.forename: Mayra
authors.initials: M
authors.lastname: Medecigo Ríos
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. Instrotwong68@gmail.com.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Calva Nieves
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. rdlfacevedo@gmail.com.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Acevedo Ortuño
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. mematuso@gmail.com.
authors.forename: Maura Epifanía
authors.initials: ME
authors.lastname: Matus Ortega
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. joalhercal@yahoo.com.mx.
authors.forename: Jorge Alberto
authors.initials: JA
authors.lastname: Hernández Calderón
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. richyhm@yahoo.com.mx.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Hernández Miramontes
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. anabelflores@imp.edu.mx.
authors.forename: Anabel
authors.initials: A
authors.lastname: Flores Zamora
authors.affiliation: Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico. azazel_vamp@yahoo.com.mx.
authors.forename: Alberto
authors.initials: A
authors.lastname: Salazar Juárez
mesh_terms: D000577:Amides / Q000737:chemistry; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis*; D005260:Female; D007118:Immunoassay; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D020033:Protein Isoforms; D051381:Rats; D017208:Rats, Wistar; D014633:Valine / Q000276:immunology*
chemical_list: D000577:Amides; D058846:Antibodies, Monoclonal, Murine-Derived; D020033:Protein Isoforms; D014633:Valine