title: Immunoassay for glial fibrillary acidic protein: antigen recognition is affected by its phosphorylation state.
abstract: Glial fibrillary acid protein (GFAP) is used commonly as a marker of astrogliosis and astrocyte activation in several situations involving brain injury. Its content may be measured by immunocytochemistry, immunoblotting or enzyme-linked immunosorbent assay (ELISA), usually employing commercial antibodies. Two major post-translational modifications in GFAP (phosphorylation and proteolysis) may alter the interpretation of results or for immunoassay standardization. This study using a non-sandwich ELISA aimed to investigate the putative changes in the immunorecognition due to the phosphorylated state of the antigen by a routinely used polyclonal anti-GFAP antibody from DAKO. Results involving in vitro phosphorylation of purified GFAP or biological samples (brain tissue, cell culture and cerebrospinal fluid) mediated by protein kinase dependent on cAMP indicate that GFAP phosphorylation improves the recognition by the used antibody. These results provide support to the understanding of fast changes in the GFAP-immunoreactivity and suggest that caution is necessary in the interpretation of results using this antibody, as well as indicate that the effect of post-translational modifications must be considered during the standardization of immunoassays with other antibodies.
authors.affiliation: Department of Biochemistry, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2600-Anexo, 90035-003 Porto Alegre, RS, Brazil.
authors.forename: Francine
authors.initials: F
authors.lastname: Tramontina
authors.forename: Marina C
authors.initials: MC
authors.lastname: Leite
authors.forename: Keila
authors.initials: K
authors.lastname: Cereser
authors.forename: Daniela Fraga
authors.initials: DF
authors.lastname: de Souza
authors.forename: Ana Carolina
authors.initials: AC
authors.lastname: Tramontina
authors.forename: Patrícia
authors.initials: P
authors.lastname: Nardin
authors.forename: Ana Cristina
authors.initials: AC
authors.lastname: Andreazza
authors.forename: Carmem
authors.initials: C
authors.lastname: Gottfried
authors.forename: Flavio
authors.initials: F
authors.lastname: Kapczinski
authors.forename: Carlos-Alberto
authors.initials: CA
authors.lastname: Gonçalves
mesh_terms: D000818:Animals; D000906:Antibodies; D001253:Astrocytes / Q000166:cytology / Q000502:physiology*; D001921:Brain / Q000166:cytology / Q000502:physiology*; D001923:Brain Chemistry; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis* / Q000134:cerebrospinal fluid / Q000378:metabolism; D007700:Kinetics; D009457:Neuroglia / Q000166:cytology* / Q000502:physiology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000906:Antibodies; D005904:Glial Fibrillary Acidic Protein