A new reliable bioassay for determining the biological activity of human interleukin-12 by using human NK cell line NKG cells

A specific and accurate bioassay for determining the biological activity of human interleukin-12 (hIL-12), an important typical Th1 cytokine in both innate and adaptive immunity, is extensively required for biomedical and clinical study. In this paper, we used a new established NK cell line NKG cells as the responder to hIL-12 stimulation by detecting their IFN-gamma production. It was found that NKG cells produced high level of IFN-gamma when simulated by hIL-12, and the dose response curve became the best Sigmoid curve (R-2=0.9977, p<0.0001) when stimulated for 24 h. The intra-assay CVs (<10%) and inter-assay CVs (<5%) demonstrated that the bioassay was precise and reproducible. Furthermore, no obvious cross-effects of other cytokines such as IL-2 and IL-18 was observed on the bioassay. Addition of the neutralizing anti-hIL-12 antibody to the bioassay significantly inhibited the IFN-gamma production in a dose dependent manner, indicating that the bioactivity was actually mediated by hIL-12. The bioassay by using NKG cells was also suitable for determining the biological activity of recombinant hIL-12 in the form of purified product or culture supernatant by CHO-hIL-12 cell line. In conclusion, a reliable hIL-12 bioassay for determining its biological activity was established by using NKG cells as a responder and measuring their production of IFN-gamma. (C) 2012 Elsevier B.V. All rights reserved.