Serologic analysis of the mouse beta chemokine JE/monocyte chemoattractant protein-1.
Mouse monocyte chemoattractant protein-1 (MCP-1), previously termed JE, is a member of the beta chemokine gene family and a homologue of the human monocyte chemoattractant protein, MCP-1. Mouse rMCP-1 was used to immunize hamsters for the production of mAb. Seven mouse MCP-1-specific mAbs were characterized: two of these mAbs cross-reacted with the human MCP-1, as determined by ELISA. A sensitive and specific capture ELISA for MCP-1 quantitation, which allowed measurement of mouse MCP-1 levels in supernatants from cells stimulated with inflammatory agents, was developed. LPS-stimulated astrocytes produce the highest levels of MCP-1 (80 ng/ml); macrophages and mesangial cells produce lower levels of MCP-1 (2 to 14 ng/ml) after LPS stimulation. IL-1 and TNF-alpha stimulation also can induce low levels of MCP-1 production. Western blot analysis demonstrated that the predominant native form of mouse MCP-1 is a 30-kDa glycoprotein. Two mAbs (2H5 and 6C7) demonstrated dose-dependent neutralization of mouse MCP-1 chemotactic activity. To localize the epitope recognized by one of these neutralizing Abs, the mAb was used to bind a series of genetically engineered truncated variants of human MCP-1. The C-terminal residues 62 to 67 on human MCP-1 molecules seem to be critical to express the epitope recognized by the neutralizing 2H5 anti-MCP-1 mAb. However, multiple sites on the MCP-1 molecule seem to be critical for bioactivity. Thus, these Ab reagents provide a useful tool to explore the biology of the mouse MCP-1 beta chemokine.
Luo
Y
Y
Department of Pathology, Harvard Medical School, Boston, MA 02115.
Laning
J
J
Hayashi
M
M
Hancock
P R
PR
Rollins
B
B
Dorf
M E
ME
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000276:immunology* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D000939:Epitopes; D005260:Female; D007249:Inflammation / Q000276:immunology; D008262:Macrophage Activation; D051379:Mice
D000911:Antibodies, Monoclonal; D018932:Chemokine CCL2; D002630:Chemotactic Factors; D000939:Epitopes