title: Panel of synaptic protein ELISAs for evaluating neurological phenotype.
abstract: The purpose of this study was to develop ELISAs for key neural proteins, three synaptic and one glial, that exist in different intracellular compartments, which would be used as a measure of synaptic phenotype. These assays would be valuable to neurologically phenotype transgenic mouse models of human disease and also human disease itself using minimal amounts of post-mortem tissue. We showed that supernatant from crude brain tissue homogenates extracted in RIPA buffer containing 0.1% SDS bind to synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25), post-synaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) antibody pairs with high affinity and selectivity. Overall, RIPA + 0.1% SDS were more efficient than RIPA + 2% SDS or a buffer containing only 1% Triton-X-100. Diluting the brain extracts resulted in dose-dependent binding to the antibody pairs for each neural protein, with EC50s that varied from 8.6 microg protein for PSD-95 to 0.23 microg for GFAP. The assays were used to measure synaptic marker protein levels at various times during mouse development and GFAP in a model of disease accompanied by neuroinflammation. Comparison of ELISAs with Western blots by measuring marker levels in brain extract from developing mice showed a greater relative difference in values derived from ELISA. These ELISAs should be valuable to phenotype the synapse in neurological disease and their rodent models.
authors.affiliation: Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Slot #611, 4301 West Markham Street, Little Rock, AR, USA. pegottschall@uams.edu
authors.forename: Paul E
authors.initials: PE
authors.lastname: Gottschall
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Ajmo
authors.forename: Autumn K
authors.initials: AK
authors.lastname: Eakin
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Howell
authors.forename: Hina
authors.initials: H
authors.lastname: Mehta
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Bailey
mesh_terms: D000818:Animals; D001921:Brain / Q000254:growth & development / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013569:Synapses / Q000235:genetics / Q000378:metabolism*; D016708:Synaptophysin / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism
chemical_list: D005904:Glial Fibrillary Acidic Protein; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D016708:Synaptophysin; D050825:Synaptosomal-Associated Protein 25