Use of proteoliposomes to generate phage antibodies against native AMPA receptor

L. Jespersen, Arja Kuusinen, Adelina Orellana, Kari Keinänen (Corresponding Author), J. Engberg

Research output: Contribution to journalArticleScientificpeer-review

24 Citations (Scopus)

Abstract

To isolate antibodies against ionotropic glutamate receptors (GluRs), we prepared a phage antibody library from mice immunized with proteoliposomes containing purified α‐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.

Original languageEnglish
Pages (from-to)1382 - 1389
Number of pages8
JournalEuropean Journal of Biochemistry
Volume267
Issue number5
DOIs
Publication statusPublished - 2000
MoE publication typeA1 Journal article-refereed

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Immunoglobulin Fragments
Bacteriophages
Ionotropic Glutamate Receptors
Kainic Acid Receptors
Acids
Antibodies
Liposomes
Binders
Epitopes
Membrane Proteins
Display devices
Monoclonal Antibodies
Technology
Experiments
proteoliposomes
Gluk2 kainate receptor

Cite this

Jespersen, L. ; Kuusinen, Arja ; Orellana, Adelina ; Keinänen, Kari ; Engberg, J. / Use of proteoliposomes to generate phage antibodies against native AMPA receptor. In: European Journal of Biochemistry. 2000 ; Vol. 267, No. 5. pp. 1382 - 1389.
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abstract = "To isolate antibodies against ionotropic glutamate receptors (GluRs), we prepared a phage antibody library from mice immunized with proteoliposomes containing purified α‐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.",
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Jespersen, L, Kuusinen, A, Orellana, A, Keinänen, K & Engberg, J 2000, 'Use of proteoliposomes to generate phage antibodies against native AMPA receptor', European Journal of Biochemistry, vol. 267, no. 5, pp. 1382 - 1389. https://doi.org/10.1046/j.1432-1327.2000.01137.x

Use of proteoliposomes to generate phage antibodies against native AMPA receptor. / Jespersen, L.; Kuusinen, Arja; Orellana, Adelina; Keinänen, Kari (Corresponding Author); Engberg, J.

In: European Journal of Biochemistry, Vol. 267, No. 5, 2000, p. 1382 - 1389.

Research output: Contribution to journalArticleScientificpeer-review

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AB - To isolate antibodies against ionotropic glutamate receptors (GluRs), we prepared a phage antibody library from mice immunized with proteoliposomes containing purified α‐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.

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