HIGH-LEVEL EXPRESSION OF CYTOKINE-INDUCED NEUTROPHIL CHEMOATTRACTANT (CINC) BY A METASTATIC RAT-CELL LINE - PURIFICATION AND PRODUCTION OF BLOCKING ANTIBODIES

Significant levels of cytokine-induced neutrophil chemoattractant (CIN C) were found in serum-free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastas is, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoa ttractant for rat neutrophils (EC-50 0.5 nM). A fusion protein of glut athione-S-transferase and CINC (GST-CINC) was produced in E. coli. Ant i-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 mug/mL (an 11-fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat ne utrophil 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 investigatio ns into the role played by CINC in rodent inflammation models and in t he metastasis of RC20 cells. (C) 1993 Wiley-Liss, Inc.