Generation of a new monoclonal antibody and its application for determination and purification of biologically active human gamma-interferon.
Three monoclonal antibodies (MAbs) were raised against recombinant human gamma-interferon (rHuIFN-gamma). They were specific for rHuIFN-gamma 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-gamma. 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-gamma, because after treatment with 1 N HCl or 0.2% SDS, or at 95 degrees 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-gamma with almost the same sensitivity. Therefore, the ELISA is useful for detecting the biologically active conformation of rHuIFN-gamma, and possibly may be useful in both industrial production and clinical studies. rHuIFN-gamma molecules bind specifically to the immunoaffinity column using the MAb KM48 as ligands. After elution with 7 M Urea/1 M NaCl, the rHuIFN-gamma can be renatured in phosphate-buffered saline with high recovery. Thus, the column provides simple one-step purification of biologically active rHuIFN-gamma.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011994:Recombinant Proteins; D007371:Interferon-gamma