Production and characterization of mouse monoclonal antibodies against human monocyte chemoattractant protein-1.
We developed five different hybridoma cell lines that produced mAb against human monocyte chemoattractant protein-1 (MCP-1). The subclass of all five antibodies was IgG1. All five mAb formed complexes with metabolically labeled MCP-1 that could be demonstrated by immunoprecipitation. The antibodies were specific for MCP-1. They did not cross-react by immunoprecipitation with structurally related host defense cytokines present in metabolically labeled PHA- or LPS-stimulated mononuclear cell culture fluids, nor did they cross-react in a direct ELISA with neutrophil attractant/activation protein-1, with crude platelet lysate proteins, or with pure platelet proteins that have amino acids sequences similar to that of MCP-1. The mAb also reacted with rMCP-1 expressed in Escherichia coli, suggesting that they recognize protein structure rather than the glycosylated portion of human MCP-1. When the mAb were mixed with MCP-1, the monocyte chemotactic response to MCP-1 was inhibited. A sandwich ELISA was developed to detect MCP-1 in biologic fluids containing relatively high concentrations of other proteins. The sensitivity was 300 pg/ml, or 30 pg/ELISA well. An anti-MCP-1 mAb column was used in an improved method of MCP-1 purification. Approximately 240 micrograms of MCP-1 were purified from 5 liters of FCS-containing U-105MG cell culture supernatant. The yield was at least 60%. In addition to two forms of MCP-1 reported previously by us, two more forms of MCP-1 were found in a mixture of culture supernatants of PHA- and LPS-stimulated human PBMC.
Yoshimura
T
T
Immunopathology Section, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702.
Takeya
M
M
Takahashi
K
K
Kuratsu
J
J
Leonard
E J
EJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D018932:Chemokine CCL2; D002630:Chemotactic Factors / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D002634:Chemotaxis, Leukocyte; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000276:immunology; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D018932:Chemokine CCL2; D002630:Chemotactic Factors; D007074:Immunoglobulin G