Use of proteoliposomes to generate phage antibodies against native AMPA receptor

To isolate antibodies against ionotropic glutamate receptors (GluRs), we pr epared a phage antibody library from mice immunized with proteoliposomes co ntaining purified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective GluRD receptor. Specific binders were selected by repea ted rounds of affinity panning against immobilized GluRD liposomes. Using t his approach, we obtained a panel of high-affinity antibody fragments that immunoprecipitated both recombinant and native GluRD receptors, but not Glu R6, a kainate receptor subunit with a 40% sequence similarity. The antibody fragments showed subunit selectivity, some being strictly specific for Glu RD, whereas others also recognized the GluRB and GluRC but not GluRA subuni ts. Further experiments indicated that the epitopes recognized were conform ational in nature and reside in the N-terminal extracellular 400-residue X domain of GluRD. Our results suggest that proteoliposomes, in combination w ith phage display technology, provide an effective tool for the generation of high-affinity conformation-sensitive monoclonal antibodies against prede termined membrane proteins.