An acid-treatment method for the enhanced detection of GDNF in biological samples.
Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-beta (TGFbeta) family, is a protein that is essential for the survival of dopaminergic, motor, and peripheral neurons. To facilitate its study, we and others have developed sensitive (low pg/ml) enzyme-linked immunosorbant assays (ELISA) to quantitate endogenous concentrations of GDNF, along with neurotrophin-3 (NT-3) and nerve growth factor (NGF). However, endogenous tissue levels of GDNF in adult animals are not readily detected by ELISA and do not correlate well with message RNA. Based upon previously described methods for the extraction of TGFbeta from tissue samples, we have developed an acid-treatment procedure to allow the quantification of total endogenous GDNF. This procedure also was evaluated for use when measuring total endogenous levels of NT-3 and NGF from biological samples. The acid-treatment procedure increases the detectable amounts of GDNF, NT-3, and NGF in all tissue samples and most of the serum samples tested. Moreover, these values were as much as 35 times greater than those detected using traditional extraction buffers. Such elevated concentrations likely resulted from the acid treatment promoting the dissociation of ligands from receptors or binding proteins, thereby making more of the analyte available to be measured in the ELISA. These findings indicate that appropriate sample treatment is essential for the measurement of total endogenous neurotrophic factors.
Okragly
A J
AJ
Department of Immunology, Research and Development, Promega Corporation, Madison, Wisconsin 53711, USA.
Haak-Frendscho
M
M
D000143:Acids / Q000494:pharmacology*; D000818:Animals; D000918:Antibody Specificity; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006041:Goats; D006736:Horses; D006801:Humans; D051379:Mice; D009414:Nerve Growth Factors / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000737:chemistry; D018696:Neuroprotective Agents / Q000032:analysis*; D020933:Neurotrophin 3; D051381:Rats; D012756:Sheep; D013045:Species Specificity
D000143:Acids; C495039:GDNF protein, human; C495040:Gdnf protein, mouse; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D020933:Neurotrophin 3