Generation of recombinant, carbohydrate-free intercellular adhesion molecule-1 (ICAM-1) and ICAM-1 fragments in Escherichia coli and mapping of epitopes recognized by anti-ICAM-1 monoclonal antibodies.
Intercellular adhesion molecule-1 (ICAM-1) has been shown to interact with the integrin leukocyte function associated antigen-1 (LFA-1) in a variety of cell-cell adhesion phenomena. Furthermore, it serves as a receptor for the majority of the Rhinoviruses and for Plasmodium falciparum-infected human erythrocytes. We generated recombinant, carbohydrate-free ICAM-1 and several ICAM-1 fragments by expression in Escherichia coli using the fusion protein expression system pUEX1-3. In Western blot and dot blot analyses we tested mAbs (7F7, 8B9, P3.58-BA3, -BA11, -BA14, -BA19, -BA21, -BA23, -BA24, -BA26, CL203.4 and 84H10) and a polyclonal antiserum directed against native ICAM-1 for their reactivity with these constructs. We were able to localize the binding site for the mAbs P3.58-BA3, -BA11, -BA14, -BA19, -BA21, -BA23, -BA24 and -BA26 at domain 5, whereas the mAbs 7F7, 8B9, CL203.4 and 84H10 did not recognize the recombinant, carbohydrate-free ICAM-1. Our findings suggest the presence of an immunodominant epitope on domain 5 of ICAM-1.
Mölgg
M
M
Institut für Hygiene, University of Innsbruck, Austria.
Schwaeble
W
W
Johnson
J P
JP
Dierich
M P
MP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D002241:Carbohydrates / Q000172:deficiency*; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology*; D002874:Chromosome Mapping; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D014162:Transfection / Q000235:genetics
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins