Mouse Monoclonal Antibody with Specificity for Human Interferon Gamma

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Abstract
A monoclonal antibody (MAb) specific for human interferon-.gamma. (HuIFN-.gamma.) was developed 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 Ig and remaining IFN activity in the supernatants was determined by virus yield reduction assay. (NH4)2 SO4 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.. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the MAb immunoprecipitates a molecule of MW = 47 kD) [kilodaltons] under nonreducing conditions. Under reducing conditions, 2 additional bands of MW = 26 kD (major band) and MW = 21 kD (minor band) were observed. A sepharose affinity column was constructed using this MAb and was able to retain .apprx. 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.