Use of proteoliposomes to generate phage antibodies against native AMPA receptor.
To isolate antibodies against ionotropic glutamate receptors (GluRs), we prepared a phage antibody library from mice immunized with proteoliposomes containing purified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective GluRD receptor. Specific binders were selected by repeated rounds of affinity panning against immobilized GluRD liposomes. Using this approach, we obtained a panel of high-affinity antibody fragments that immunoprecipitated both recombinant and native GluRD receptors, but not GluR6, a kainate receptor subunit with a 40% sequence similarity. The antibody fragments showed subunit selectivity, some being strictly specific for GluRD, whereas others also recognized the GluRB and GluRC but not GluRA subunits. Further experiments indicated that the epitopes recognized were conformational in nature and reside in the N-terminal extracellular 400-residue X domain of GluRD. Our results suggest that proteoliposomes, in combination with phage display technology, provide an effective tool for the generation of high-affinity conformation-sensitive monoclonal antibodies against predetermined membrane proteins.
Jespersen
L K
LK
The Royal Danish School of Pharmacy, Department of Pharmacology, Copenhagen, Denmark.
Kuusinen
A
A
Orellana
A
A
Keinänen
K
K
Engberg
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000276:immunology*; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region; D051379:Mice; D008969:Molecular Sequence Data; D011510:Proteolipids; D011869:Radioligand Assay; D018091:Receptors, AMPA / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011510:Proteolipids; D018091:Receptors, AMPA; C020485:proteoliposomes