Characterization of two distinct monoclonal antibodies specific for glial cell line-derived neurotrophic factor.
Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-1531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G-90 and B-1531 recognize different epitopes: PCE confirmed that B-1531 binds to NH2-terminal peptides between amino acids 18 and 37, whereas G-90 does not; BIAcore data showed that B-1531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-1531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by G-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to down-regulate GDNF-induced biological activity.
Xu
R Y
RY
Department of Neuroscience, Amgen, Thousand Oaks, California 91320, USA.
Pong
K
K
Yu
Y
Y
Chang
D
D
Liu
S
S
Lile
J D
JD
Treanor
J
J
Beck
K D
KD
Louis
J C
JC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015374:Biosensing Techniques; D015153:Blotting, Western; D002795:Choline O-Acetyltransferase / Q000493:pharmacokinetics; D004298:Dopamine / Q000493:pharmacokinetics; D029721:Drosophila Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000276:immunology* / Q000494:pharmacology; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D029721:Drosophila Proteins; C495039:GDNF protein, human; C495040:Gdnf protein, mouse; C495041:Gdnf protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D002795:Choline O-Acetyltransferase; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse; C495001:Ret protein, rat; D004298:Dopamine