AN ACID-TREATMENT METHOD FOR THE ENHANCED DETECTION OF GDNF IN BIOLOGICAL SAMPLES

Glial cell line-derived neurotrophic factor (GDNF), a distant member o f the transforming growth factor-beta (TGF beta) family, is a protein that is essential for the survival of dopaminergic, motor, and periphe ral neurons. To facilitate its study, we and others have developed sen sitive (low pg/ml) enzyme-linked immunosorbant assays (ELISA) to quant itate endogenous concentrations of GDNF, along with neurotrophin-3 (NT -3) and nerve growth factor (NGF). However, endogenous tissue levels o f GDNF in adult animals are not readily detected by ELISA and do not c orrelate well with message RNA. Based upon previously described method s for the extraction of TGF beta from tissue samples, we have develope d an acid-treatment procedure to allow the quantification of total end ogenous 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 de tected using traditional extraction buffers. Such elevated concentrati ons 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 ind icate that appropriate sample treatment is essential for the measureme nt of total endogenous neurotrophic factors. (C) 1997 Academic Press.