Premium
High‐level expression of cytokine‐induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: Purification and production of blocking antibodies
Author(s) -
Wittwer Arthur J.,
Carr Linda S.,
Zagorski John,
Dolecki Gregory J.,
Crippes Barbara A.,
De Larco Joseph E.
Publication year - 1993
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041560226
Subject(s) - polyclonal antibodies , antibody , chemotaxis , cytokine , blocking antibody , affinity chromatography , microbiology and biotechnology , inflammation , chemokine , cell culture , immunoassay , in vitro , immune system , receptor , chemistry , biology , immunology , enzyme , biochemistry , genetics
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 μg/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. © 1993 Wiley‐Liss, Inc.