Purification of neuronal cell surface proteins and generation of epitope-specific monoclonal antibodies against cell adhesion molecules.
To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.
Schlosshauer
B
B
Max-Planck-Institut für Entwicklungsbiologie, Tübingen, F.R.G.
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D015815:Cell Adhesion Molecules; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009474:Neurons / Q000032:analysis*; D013696:Temperature
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D015815:Cell Adhesion Molecules; D000939:Epitopes; D008565:Membrane Proteins