title: A novel anti-aldolase C antibody specifically interacts with residues 85-102 of the protein.
abstract: Aldolase C is a brain-specific glycolytic isozyme whose complete repertoire of functions are obscure. This lack of knowledge can be addressed using molecular tools that discriminate the protein from the homologous, ubiquitous paralog aldolase A. The anti-aldolase C antibodies currently available are polyclonal and not highly specific. We obtained the novel monoclonal antibody 9F against human aldolase C, characterized its isoform specificity and tested its performance. First, we investigated the specificity of 9F for aldolase C. Then, using bioinformatic tools coupled to molecular cloning and chemical synthesis approaches, we produced truncated human aldolase C fragments, and assessed 9F binding to these fragments by western blot and ELISA assays. This strategy revealed that residues 85-102 harbor the epitope-containing region recognized by 9F. The efficiency of 9F was demonstrated also for immunoprecipitation assays. Finally, surface plasmon resonance revealed that the protein has a high affinity toward the epitope-containing peptide. Taken together, our findings show that epitope recognition is sequence-driven and is independent of the three-dimensional structure. In conclusion, given its specific molecular interaction, 9F is a novel and powerful tool to investigate aldolase C's functions in the brain.
authors: CEINGE-Biotecnologie Avanzate; Naples, Italy; International Centre for Genetic Engineering and Biotechnology; Trieste, Italy.
authors: Simona
authors: S
authors: Langellotti
authors: Department of Life Sciences; University of Trieste; Trieste, Italy.
authors: Maurizio
authors: M
authors: Romano
authors: International Centre for Genetic Engineering and Biotechnology; Trieste, Italy.
authors: Corrado
authors: C
authors: Guarnaccia
authors: CEINGE-Biotecnologie Avanzate; Naples, Italy.
authors: Vincenzo
authors: V
authors: Granata
authors: Department of Sports Science and Wellness; University of Naples "Parthenope"; Naples, Italy; IRCCS SDN-Foundation; Naples, Italy.
authors: Stefania
authors: S
authors: Orrù
authors: CEINGE-Biotecnologie Avanzate; Naples, Italy.
authors: Adriana
authors: A
authors: Zagari
authors: International Centre for Genetic Engineering and Biotechnology; Trieste, Italy.
authors: Francisco E
authors: FE
authors: Baralle
authors: CEINGE-Biotecnologie Avanzate; Naples, Italy; IRCCS SDN-Foundation; Naples, Italy.
authors: Francesco
authors: F
authors: Salvatore
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D002460:Cell Line; D019295:Computational Biology; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics / Q000378:metabolism
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000939:Epitopes; D007527:Isoenzymes; D016900:Neurofilament Proteins; D010446:Peptide Fragments; C044882:neurofilament protein L; D012313:RNA; D005634:Fructose-Bisphosphate Aldolase
keywords: ELISA; SPR; aldolase C; epitope mapping; monoclonal antibodies; western blot