Detection of cells producing murine interferon-alpha using antipeptide antibodies.
The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN-alpha. Rabbits were immunized with a peptide, corresponding to the 15 COOH-terminal amino acids of MuIFN-alpha-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 immunofluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN-alpha molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN-alpha. This finding is in agreement with the recent demonstration of constitutive IFN-alpha 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.
Hwang
S Y
SY
Centre for Molecular Biology and Medicine, Monash University, Clayton, Victoria, Australia.
Greenway
A L
AL
McMullen
G L
GL
Hertzog
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007120:Immunochemistry; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D013154:Spleen / Q000166:cytology* / Q000276:immunology*
D000906:Antibodies; D016898:Interferon-alpha; D010446:Peptide Fragments