Development of a microplate bioassay for monocyte chemoattractant protein-1 based on activation of p44/42 mitogen-activated protein kinase.
Monocyte chemoattractant protein-1 (MCP-1) is a potential therapeutic target for the treatment of several inflammatory conditions, including rheumatoid arthritis and chronic obstructive pulmonary disease. Current cell-based assays for MCP-1 use monocyte chemotaxis or calcium flux as a readout. Here, we describe an alternative bioassay based on MCP-1-induced phosphorylation of the mitogen-activated protein kinases (MAPK) p44 (ERK1) and p42 (ERK2). Adherent cells expressing the MCP-1 receptor CCR2B are treated with MCP-1 in 96-well plates in the presence or absence of inhibitors, fixed and permeabilized with methanol, and then probed with a monoclonal antibody that selectively recognizes the doubly phosphorylated form of p44/42 MAPK. Bound antibody is detected with a secondary antibody-peroxidase conjugate and a chromogenic substrate. The phosphorylation of p44/42 MAPK as detected in this assay peaks after 3-5 min of MCP-1 treatment, and the concentration of MCP-1 required for half-maximal p44/42 MAPK phosphorylation is 1-3 nM. MCP-1-induced phosphorylation of p44/42 MAPK is dependent upon the expression of CCR2B. The assay can be used for screening and characterization of small molecule inhibitors and antibodies blocking the binding of MCP-1 to its receptor. Since the assay is rapid and simple, it may represent a useful alternative to chemotaxis or calcium mobilization assays for the analysis of MCP-1 inhibitors.
Hirata Terra
Juliana
J
Biotechnology Development, Novartis Pharma AG, CH-4002 Basel, Switzerland.
Montaño
Irene
I
Schilb
Alain
A
Millward
Thomas A
TA
D000577:Amides / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D001681:Biological Assay / Q000379:methods*; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D018932:Chemokine CCL2 / Q000032:analysis* / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D006224:Cricetinae; D018076:DNA, Complementary / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008832:Microchemistry / Q000379:methods; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011725:Pyridines / Q000494:pharmacology; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000577:Amides; D000911:Antibodies, Monoclonal; D002227:Carbazoles; D018932:Chemokine CCL2; D002867:Chromones; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007211:Indoles; D009025:Morpholines; D011725:Pyridines; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; C081021:Go 6976; C108830:Y 27632; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases