A new reliable bioassay for determining the biological activity of human interleukin-12 by using human NK cell line NKG cells.
A new reliable bioassay for determining the biological activity of human interleukin-12 by using human NK cell line NKG cells.
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DOI：
10.1016/j.intimp.2012.03.015
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发表时间：
2012-05
影响因子：
5.6
通讯作者：
M. Cheng;Baozhen Fei;Xiaodong Zheng;Yongyan Chen;R. Sun;Haiming Wei;Z. Tian
中科院分区：
文献类型：
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作者：
M. Cheng;Baozhen Fei;Xiaodong Zheng;Yongyan Chen;R. Sun;Haiming Wei;Z. Tian
人白细胞介素12(hIL-12)是天然免疫和获得性免疫中一种重要的典型Th1细胞因子，生物医学和临床研究广泛需要一种特异、准确的生物活性测定方法。本研究以新建立的NK细胞系NKG细胞作为hIL-12刺激的应答细胞，检测其产生的干扰素-γ。结果发现，在hIL-12作用下，NKG细胞产生高水平的干扰素-γ，刺激24小时，量效曲线出现最佳Sigmoid曲线(R2=0.9977，p<；0.0001)。批内和批间变异系数分别为10%和5%，证明该方法准确、重现性好。此外，其他细胞因子如IL-2和IL-18在生物测定中未观察到明显的交叉影响。在生物测定中加入中和性抗hIL-12抗体显著抑制干扰素-γ的产生，并呈剂量依赖关系，表明其生物活性实际上是由hIL-12介导的。NKG细胞生物活性测定也适用于CHO-hIL-12细胞株以纯化产物或培养上清液形式测定重组hIL-12的生物活性。综上所述，以NKG细胞为反应细胞，通过检测其产生干扰素-γ，建立了一种可靠的检测其生物学活性的hIL-12生物活性测定方法。
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 (R2=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.