PMID- 15033519
OWN - NLM
STAT- MEDLINE
DCOM- 20041101
LR  - 20161124
IS  - 0003-2697 (Print)
IS  - 0003-2697 (Linking)
VI  - 327
IP  - 1
DP  - 2004 Apr 1
TI  - Development of a microplate bioassay for monocyte chemoattractant protein-1 based 
      on activation of p44/42 mitogen-activated protein kinase.
PG  - 119-25
AB  - 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.
FAU - Hirata Terra, Juliana
AU  - Hirata Terra J
AD  - Biotechnology Development, Novartis Pharma AG, CH-4002 Basel, Switzerland.
FAU - Montano, Irene
AU  - Montano I
FAU - Schilb, Alain
AU  - Schilb A
FAU - Millward, Thomas A
AU  - Millward TA
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Amides)
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Carbazoles)
RN  - 0 (Chemokine CCL2)
RN  - 0 (Chromones)
RN  - 0 (DNA, Complementary)
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (Indoles)
RN  - 0 (Morpholines)
RN  - 0 (Pyridines)
RN  - 0 (Receptors, CCR2)
RN  - 0 (Receptors, Chemokine)
RN  - 136194-77-9 (Go 6976)
RN  - 138381-45-0 (Y 27632)
RN  - 31M2U1DVID (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one)
RN  - EC 2.7.11.24 (Mitogen-Activated Protein Kinase 1)
RN  - EC 2.7.11.24 (Mitogen-Activated Protein Kinase 3)
RN  - EC 2.7.11.24 (Mitogen-Activated Protein Kinases)
SB  - IM
MH  - Amides/pharmacology
MH  - Animals
MH  - Antibodies, Monoclonal/analysis
MH  - Biological Assay/*methods
MH  - Carbazoles/pharmacology
MH  - Cell Line
MH  - Chemokine CCL2/*analysis/pharmacology
MH  - Chromones/pharmacology
MH  - Cricetinae
MH  - DNA, Complementary/isolation & purification
MH  - Enzyme Inhibitors/pharmacology
MH  - Indoles/pharmacology
MH  - Microchemistry/methods
MH  - Mitogen-Activated Protein Kinase 1/analysis/*metabolism
MH  - Mitogen-Activated Protein Kinase 3
MH  - Mitogen-Activated Protein Kinases/analysis/*metabolism
MH  - Morpholines/pharmacology
MH  - Phosphorylation
MH  - Promoter Regions, Genetic
MH  - Pyridines/pharmacology
MH  - Receptors, CCR2
MH  - Receptors, Chemokine/genetics/metabolism
MH  - Transfection
EDAT- 2004/03/23 05:00
MHDA- 2004/11/02 09:00
CRDT- 2004/03/23 05:00
PHST- 2003/10/06 00:00 [received]
PHST- 2004/03/23 05:00 [pubmed]
PHST- 2004/11/02 09:00 [medline]
PHST- 2004/03/23 05:00 [entrez]
AID - S0003269704000703 [pii]
AID - 10.1016/j.ab.2003.12.027 [doi]
PST - ppublish
SO  - Anal Biochem. 2004 Apr 1;327(1):119-25. doi: 10.1016/j.ab.2003.12.027.