Mouse monoclonal antibody with specificity for human interferon gamma

Abstract

We developed a monoclonal antibody (MAb) specific for human interferon gamma (HuIFN-gamma) by hybridizing cells from the NS-1 myeloma cell line with spleen lymphocytes from BALB/c mice immunized with partially purified HuIFN-gamma. Hybridoma culture supernatants were screened for neutralization of antiviral activity of HuIFN-gamma by the method determining the inhibition of nucleic acid synthesis assay (INAS), employing human fibroblasts infected with encephalomyocarditis virus (EMC). Clones exhibiting neutralization of antiviral activity of HuIFN-gamma were recloned, retested and an MAb with maximum neutralization activity was selected. This MAb was of IgM subclass and was specific for HuIFN-gamma. Antiviral activities either of human leukocyte-derived (HuIFN-alpha) or human fibroblast-derived interferon (HuIFN-beta) were not affected by this monoclonal antibody as determined by the INAS test. The specificity of the MAb for HuIFN-gamma was further confirmed by an indirect immunoprecipitation method, where monoclonal antibody-HuIFN-gamma complexes were immunoprecipitated with rabbit anti-mouse immunoglobulin and remaining IFN activity in the supernatants was determined by virus yield reduction assay. Ammonium sulfate precipitated preparations of this MAb were able to significantly increase (range of 230- to 1300-fold) the virus yield when compared with that obtained in the presence of IFN-gamma. SDS-PAGE analysis revealed that the MAb immunoprecipitates a molecule of Mr = 47 kD under nonreducing conditions. Under reducing conditions, two additional bands of Mr = 26 kD (major band) and Mr = 21 kD (minor band) were observed. A sepharose affinity column was constructed using this MAb and was able to retain approximately 60% of the partially purified HuIFN-gamma preparation applied. Significant amounts of HuIFN-gamma were eluted by increasing the ionic strength and decreasing the pH. HuIFN-alpha and HuIFN-beta were not retained by this column.