A test for screening monoclonal antibodies to membrane proteins based on their ability to inhibit protein reconstitution into vesicles.
The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.
Gaymard
F
F
Laboratoire de Biochimie et Physiologie Végétales, ENSA-M, INRA, CNRS URA 573, Montpellier, France.
Thibaud
J B
JB
Touraine
B
B
Simon-Plas
F
F
Grouzis
J P
JP
Gibrat
R
R
Sentenac
H
H
Grignon
C
C
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D010940:Plant Proteins