TY - JOUR
T1 - Detection of cells producing murine interferon‐α using antipeptide antibodies
AU - Hwang, Seung Y.
AU - Greenway, Alison L.
AU - McMullen, Gabrielle L.L.
AU - Hertzog, Paul J.
PY - 1994/1/1
Y1 - 1994/1/1
N2 - The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN‐α. Rabbits were immunized with a peptide, corresponding to the 15 COOH‐terminal amino acids of MuIFN‐α‐1, conjugated to keyhole limpet haemocyanin (KLH), and the resulting antipeptide antibodies were identified by indirect ELISA. Antipeptide antibodies were purified from rabbit immune sera by immunoadsorption to peptide immobilized on nitrocellulose and any remaining antibodies to KLH removed by immunoadsorption to KLH‐Sepharose. The characterization of the antipeptide antibodies by ELISA, immunoprecipitation, affinity chromatography and immuno‐fluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN‐α molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN‐α This finding is in agreement with the recent demonstration of constitutive IFN‐α production by unstimulated human leucocytes and has important implications for the functions of interferons. The production, characterization and use of antipeptide antibodies as described herein may also have broader application for studies of the expression of other cytokines.
AB - The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN‐α. Rabbits were immunized with a peptide, corresponding to the 15 COOH‐terminal amino acids of MuIFN‐α‐1, conjugated to keyhole limpet haemocyanin (KLH), and the resulting antipeptide antibodies were identified by indirect ELISA. Antipeptide antibodies were purified from rabbit immune sera by immunoadsorption to peptide immobilized on nitrocellulose and any remaining antibodies to KLH removed by immunoadsorption to KLH‐Sepharose. The characterization of the antipeptide antibodies by ELISA, immunoprecipitation, affinity chromatography and immuno‐fluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN‐α molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN‐α This finding is in agreement with the recent demonstration of constitutive IFN‐α production by unstimulated human leucocytes and has important implications for the functions of interferons. The production, characterization and use of antipeptide antibodies as described herein may also have broader application for studies of the expression of other cytokines.
KW - antipeptide antibodies
KW - immunofluorescence
KW - interferon‐alpha
UR - http://www.scopus.com/inward/record.url?scp=0028244535&partnerID=8YFLogxK
U2 - 10.1111/j.1440-1711.1994.tb03783.x
DO - 10.1111/j.1440-1711.1994.tb03783.x
M3 - Article
C2 - 8088862
AN - SCOPUS:0028244535
SN - 0818-9641
VL - 72
SP - 234
EP - 240
JO - Immunology and Cell Biology
JF - Immunology and Cell Biology
IS - 3
ER -