Monoclonal antibodies to human interferon‐gamma: production, affinity purification and radioimmunoassay

Human interferon‐gamma (IFN‐gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mo...

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Bibliographic Details
Published in:The EMBO journal 1983-01, Vol.2 (9), p.1527-1530
Main Authors: Novick, D., Eshhar, Z., Fischer, D.G., Friedlander, J., Rubinstein, M.
Format: Article
Language:English
Subjects:
Animals
Antibodies, immunoglobulins
Antibodies, Monoclonal - biosynthesis
Antibodies, Monoclonal - isolation & purification
Biological and medical sciences
Chromatography, Affinity
Fundamental and applied biological sciences. Psychology
Fundamental immunology
gamma -interferon
Humans
hybridoma
Hybridomas - immunology
Interferon-gamma - immunology
Mice
Molecular immunology
monoclonal antibodies
Neutralization Tests
radioimmunoassay
Radioimmunoassay - methods
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Summary:Human interferon‐gamma (IFN‐gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN‐gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN‐gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN‐gamma. Moreover, IFN‐gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN‐gamma subtypes are immunologically cross‐reactive. Immune precipitation of partially purified 125I‐labelled IFN‐gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN‐gamma gave a 50‐fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid‐phase radioimmunoassay, allowing the detection of IFN‐gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h.
ISSN:0261-4189
1460-2075
DOI:10.1002/j.1460-2075.1983.tb01618.x