Abstract
Significant levels of cytokine-induced neutrophil chemoattractant (CINC) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC-50 0.5 nM). A fusion protein of glutathione-S-transferase and CINC (GST-CINC) was produced in E. coli. Anti-CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST-CINC affinity chromatography. Both goat and rabbit anti-CINC antibody preparations at 4 micrograms/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity-purified blocking anti-CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells.
MeSH terms
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Animals
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Antibodies / isolation & purification*
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Antibodies / metabolism*
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Base Sequence
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Cell Line, Transformed
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Chemokines, CXC*
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Chemotactic Factors / analysis*
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Chemotactic Factors / genetics
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Chemotactic Factors / immunology
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Chemotaxis, Leukocyte / physiology
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Chromatography, Affinity
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Chromatography, High Pressure Liquid
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Culture Media, Conditioned / analysis
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Culture Media, Conditioned / pharmacology
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Culture Media, Serum-Free / analysis
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Culture Media, Serum-Free / pharmacology
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DNA / genetics
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Electrophoresis, Polyacrylamide Gel
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Enzyme-Linked Immunosorbent Assay
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Growth Substances / analysis*
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Growth Substances / genetics
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Growth Substances / immunology
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Inflammation / pathology
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Intercellular Signaling Peptides and Proteins*
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Kidney / chemistry*
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Kidney / metabolism
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Kidney / pathology*
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Molecular Sequence Data
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Neoplasm Metastasis / pathology
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Neutrophils / physiology
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Rats
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Recombinant Proteins / pharmacology
Substances
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Antibodies
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Chemokines, CXC
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Chemotactic Factors
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Culture Media, Conditioned
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Culture Media, Serum-Free
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Growth Substances
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Intercellular Signaling Peptides and Proteins
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Recombinant Proteins
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DNA