title: A new reliable bioassay for determining the biological activity of human interleukin-12 by using human NK cell line NKG cells.
abstract: 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-γ production. It was found that NKG cells produced high level of IFN-γ 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 24h. 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-γ 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-γ.
authors.affiliation: Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China.
authors.forename: Min
authors.initials: M
authors.lastname: Cheng
authors.forename: Baozhen
authors.initials: B
authors.lastname: Fei
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zheng
authors.forename: Yongyan
authors.initials: Y
authors.lastname: Chen
authors.forename: Rui
authors.initials: R
authors.lastname: Sun
authors.forename: Haiming
authors.initials: H
authors.lastname: Wei
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Tian
mesh_terms: D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D018664:Interleukin-12 / Q000276:immunology* / Q000494:pharmacology*; D007694:Killer Cells, Natural / Q000187:drug effects* / Q000276:immunology*; D015203:Reproducibility of Results
chemical_list: D018664:Interleukin-12; D007371:Interferon-gamma