Generation of Functional Blocking Monoclonal Antibodies Against Mouse Interleukin-12 p40 Homodimer and Monomer

Abstract

The role of interleukin (IL)-12 (p40:p35) and IL-23 (p40:p19) is becoming clear in immune response and inflammation. However, biological functions of IL-12 p40 homodimer (p40(2)) and monomer (p40) remain poorly understood due to the lack of specific monoclonal antibodies (MAb). Earlier we have demonstrated that both p40(2) and p40 activate microglia and macrophages to induce the expression of iNOS and TNF-alpha. To facilitate the studies on p40(2) and p40 further, we here describe the production of neutralizing MAb against mouse p40(2) and p40 for the first time after immunization of Armenian hamsters with recombinant p40(2). Antibodies produced from clones a3-1d and d7-12c specifically recognized p40(2) but not p40, IL-12, and IL-23. These MAbs also inhibited p40(2)- but not p40-, IL-12-, and IL-23-induced production of inflammatory molecules and activation of NF-kappaB. On the other hand, antibodies produced from clones a3-3a and a3-7g specifically recognized p40 and inhibited p40- but not p40(2)-, IL-12-, and IL-23-induced production of inflammatory molecules and activation of NF-kappaB. While MAbs a3-1d and d7-12c were used to establish p40(2)-specific ELISA, we utilized MAbs a3-3a and a3-7g to develop p40-specific ELISA. Interestingly, the production of p40(2) and p40 but not IL-12 in mouse peritoneal macrophages and primary microglia was an immediate early response to bacterial lipopolysaccharides. Furthermore, double-stranded RNA, the active component of a viral infection, induced the production of p40(2) and p40 but not IL-12 in macrophages and microglia. These results indicate the presence of different regulatory mechanisms for the production of IL-12p40(2)/p40 and IL-12p70.