Generation of a novel monoclonal antibody that recognizes the alpha (α)-amidated isoform of a valine residue

BMC Neuroscience

Research Article

Juan Carlos Calva Nieves¹  Ricardo Hernández Miramontes¹  Benito Antón Palma¹  Jorge Alberto Hernández Calderón¹  Mayra Medecigo Ríos¹  Anabel Flores Zamora¹  Maura Epifanía Matus Ortega¹  Rodolfo Acevedo Ortuño¹  Alberto Salazar Juárez¹  Philippe Leff Gelman² 

[1] Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico;Molecular Neurobiology and Addictive Neurochemistry Laboratory, National Institute of Psychiatry, Calzada México-Xochimilco #101, 14370, México D.F., Mexico;Department of Neuroscience, National Institute of Perinatology, Montes Urales # 800, 11000, México D.F., Mexico;

关键词: Valine;    Leucine;    Antibody;    Antisera;    Hybridoma;    α-amidation;    Cross-reactivity;    Immunoassay;    Immunoconjugate;    Neuropeptide;   

DOI  :  10.1186/s12868-015-0206-y

 received in 2015-03-27, accepted in 2015-10-01,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundAlpha (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.MethodsThe 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.ResultsP18C5 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.ConclusionsOur 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.

【 授权许可】

CC BY   
© Palma et al. 2015