Characterization of Two Distinct Monoclonal Antibodies Specific for Glial Cell Line‐Derived Neurotrophic Factor

Abstract

Abstract:Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-i 531, 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 BlAcore data suggested that G-90 and B-1531 recognize different epitopes: POE confirmed that B-1531 binds to NH 2-terminal peptides between amino acids 18 and 37, whereas 0-90 does not; BlAcore data showed that B-i 531 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-i 531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by 0-90, 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 downregulate GDNF-induced biological activity. Key Words: Glial cell line-derived neurotrophic factor-Monoclonal antibodies-Motoneurons-Dopaminergic neuronsWestern blot-ELISA.

Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

“…To further evaluate the role of GFR-α1 and RET in immobilized GDNF-induced cell spreading, we preincubated the GDNF-coated plate and the SHEP cells with GDNF function-blocking antibodies. These antibodies prevent GDNF binding to GFR-α1–RET but do not affect GDNF interaction with HS of syndecan-3 as they block the central part of GDNF (Xu et al, 1998), which is dispensable for interaction with HS. Neither this treatment nor NCAM function-blocking antibodies had any effect on attachment and spreading of SHEP cells induced by immobilized GDNF (Fig.…”

“…To prepare protein-coated surfaces, the protein of interest (10 µg/ml in PBS) was applied to 96-well ELISA plates (Greiner) or to glass coverslips for 12–18 h at 4°C. After incubation, the surfaces were washed with PBS and blocked with 1% globulin-free BSA for 2 h. Some GDNF-coated surfaces were incubated with 20 µg/ml G-90 GDNF function-blocking antibodies (Xu et al, 1998) for 2 h at room temperature.…”

Role for GDNF in Biochemical and Behavioral Adaptations to Drugs of Abuse

“…Two anti-GDNF antibodies were used in this study: G90 (Amgen), which is known to block GDNF action in vitro, and 1531 (Amgen), which does not block anti-GDNF action in vitro (Xu et al, 1998). However, the two antibodies are equivalent in terms of their ability to detect GDNF in immunoassays.…”

“…To determine whether endogenous GDNF is normally involved in drug-induced plasticity, we infused an anti-GDNF antibody into the rat VTA via osmotic minipump. This antibody (termed "active" antibody) was raised against a moiety of GDNF that is required for binding to its receptor and has been shown to block the physiological activity of GDNF in vitro (Xu et al, 1998). As a control, we infused a second anti-GDNF antibody (termed "inactive" antibody), which was raised against a moiety of GDNF that is not involved in receptor binding and does not block the physiological activity of GDNF.…”

GDNF fusion protein for targeted‐drug delivery across the human blood–brain barrier

“…The fusion of the GDNF monomer to the carboxyl terminus of each HC is depicted in Figure 1. This design sterically restricts the GDNF to a dimeric configuration, which replicates the native dimeric conformation of GDNF that binds to the GFRa1 (Eketjall et al, 1999;Xu et al, 1998). The GDNF reverse (REV) PCR Figure 1.…”