Sekiya Shunsuke, Murata Makoto, Arai Satoshi, Murayama Hiroshi, Kawasaki Atushi, Ashida Noriyuki, Okada Kohki, Ikemoto Masaki
Research and Development Section, Diagnostics Department, Yamasa Corporation, 10-1 Araoi-cho 2-Chome, Choshi, Chiba 288-0056, Japan.
Research and Development Section, Diagnostics Department, Yamasa Corporation, 10-1 Araoi-cho 2-Chome, Choshi, Chiba 288-0056, Japan.
J Immunol Methods. 2016 Dec;439:44-49. doi: 10.1016/j.jim.2016.09.009. Epub 2016 Sep 28.
Calprotectin, a heterodimer of S100A8 and S100A9, has been reported to be a useful biomarker in inflammatory bowel disease (IBD); however, the relationship between the fecal level of S100A9 and the extent of inflammation in IBD remains unclear. Our aim was to develop a new enzyme-linked immunosorbent assay (ELISA) for rat S100A9, and to investigate whether changes in fecal S100A9 levels reflect the inflammatory conditions in the intestinal tracts of rats with dextran sulfate sodium (DSS)-induced colitis. Anti-rat S100A9 monoclonal antibodies were raised in mice and used for the development of a novel ELISA for rat S100A9. The performance of our ELISA was assessed by dilution and recovery tests, and the detection range was 3.75-240ng/mL. The dilution test showed good linearity. The recovery of fecal S100A9 was 95.1% (mean), with a range of 86.1%-108.8%. Colitis was induced in rats by oral administration of 3% DSS/drinking water (DW) for 11days (D group), while DW alone was provided to rats of the control group (C group) during the same period. The extent of inflammation was evaluated with the disease activity index (DAI), and the concentration of fecal S100A9 was determined by ELISA. Both the DAI scores and the fecal S100A9 levels were significantly higher in the D group than in the C group. Microscopic observation revealed that S100A9 was dominantly produced in many immune cells of myeloid origin in rat rectal tissues. These results indicate that the novel ELISA may be applied to clinically evaluate IBD in rats with high sensitivity. In conclusion, our ELISA is useful in toxicological and pharmacological evaluations.
钙卫蛋白是S100A8和S100A9的异二聚体，据报道它是炎症性肠病（IBD）中一种有用的生物标志物；然而，IBD中粪便S100A9水平与炎症程度之间的关系仍不清楚。我们的目的是开发一种针对大鼠S100A9的新型酶联免疫吸附测定（ELISA），并研究粪便S100A9水平的变化是否反映硫酸葡聚糖钠（DSS）诱导的大鼠结肠炎肠道中的炎症状况。抗大鼠S100A9单克隆抗体在小鼠中产生，并用于开发针对大鼠S100A9的新型ELISA。通过稀释和回收率测试评估我们的ELISA性能，检测范围为3.75-240ng/mL。稀释测试显示出良好的线性。粪便S100A9的回收率为95.1%（平均值），范围为86.1%-108.8%。通过给大鼠口服3% DSS/饮用水（DW）11天诱导结肠炎（D组），而在同一时期给对照组（C组）大鼠仅提供DW。用疾病活动指数（DAI）评估炎症程度，并用ELISA测定粪便S100A9的浓度。D组的DAI评分和粪便S100A9水平均显著高于C组。显微镜观察显示，S100A9主要在大鼠直肠组织中许多髓系来源的免疫细胞中产生。这些结果表明，这种新型ELISA可能以高灵敏度应用于临床评估大鼠的IBD。总之，我们的ELISA在毒理学和药理学评估中是有用的。
