Generation of a New Monoclonal Antibody and Its Application for Determination and Purification of Biologically Active Human Gamma-Interferon

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Abstract
Three monoclonal antibodies (MAbs) were raised against recombinant human gamma-interferon (rHuIFN-γ). They were specific for rHuIFN-γ and recognized parts of the molecule other than both terminal regions. Among these MAbs, only KM48 has an ability to neutralize the antiviral activity of rHuIFN-γ. Using KM48 in combination with polyclonal antibodies, we have developed a double-sandwich enzyme immunoassay (ELISA). This ELISA preferentially detects the biologically active form of rHuIFN-γ, because after treatment with 1 N HCl or 0.2% SDS, or at 95°C, the reactivity in the ELISA and the biological activity determined by cytopathic effect (CPE) assay decrease in parallel. In addition, the ELISA recognizes both natural and rHuIFN-γ with almost the same sensitivity. Therefore, the ELISA is useful for detecting the biologically active conformation of rHuIFN-γ, and possibly may be useful in both industrial production and clinical studies. rHuIFN-γ molecules bind specifically to the immunoaffinity column using the MAb KM48 as ligands. After elution with 7 M Urea/1 M NaCl, the rHuIFN-γ can be renatured in phosphate-buffered saline with high recovery. Thus, the column provides simple one-step purification of biologically active rHuIFN-γ.