Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse.
P-selectin is an important adhesion molecule involved in leukocyte migration. However, to date, no monoclonal antibodies (MAb) generated against rat P-selectin have been identified which block P-selectin mediated leukocyte adhesion. Most studies in the rat have utilized crossreacting antibodies generated against P-selectin in higher species. In a P-selectin deficient mouse we generated an anti-rat/mouse P-selectin MAb, designated RMP-1, by immunization with activated rat platelets. This IgG2a MAb immunoprecipitates a 140 kDa protein under reducing conditions from rat platelet lysate. By ELISA and immunofluorescence flow cytometry, MAb RMP-1 reacts with thrombin-activated but not unactivated rat platelets. In addition, by ELISA MAb RMP-1 binds to activated mouse platelets and recombinant rat and mouse P-selectin. MAb RMP-1 inhibited adhesion of HL-60 myeloid cells to immobilized mouse P-selectin by 97% and to activated rat and mouse platelets by 100% under static conditions, confirming the adhesion function blocking activity of MAb RMP-1. This novel MAb should be useful for studying P-selectin function in vitro and in vivo in both rat and mouse inflammation models.
Walter
U M
UM
Department of Pediatrics, Dalhousie University, Halifax, NS, Canada.
Ayer
L M
LM
Wolitzky
B A
BA
Wagner
D D
DD
Hynes
R O
RO
Manning
A M
AM
Issekutz
A C
AC
D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D001792:Blood Platelets / Q000276:immunology; D015153:Blotting, Western; D002448:Cell Adhesion / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D008024:Ligands; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D019007:P-Selectin / Q000276:immunology*; D015539:Platelet Activation / Q000187:drug effects; D051381:Rats; D011917:Rats, Inbred Lew; D011994:Recombinant Proteins / Q000276:immunology
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011994:Recombinant Proteins