We in this study measured the site density of E-selectin in order to explore the practical pliability using radionuclide labeling method and γ-imaging of single photon emission computer tomography (SPECT). This method required labeling of antibody with 125I using Indogen method and binding of the labeled antibody to E-selectin. Labeled E-selectin was separated and purified in a Sephadex G25 column. The different fractions of the eluants were imaged, analyzed and quantified with SPECT method. For measuring the saturation curve of E-selectin, 130 μL of E-selectin solution with different concentrations were added in a 48-well plate and incubated overnight at 4℃. After incubation, 130 μL of labeled antibody solution were added and kept incubated for 30 min. The resulted mixture was washed, and the radioactivity in each sample was detected by SPECT. The levels of radioactivity were translated to site densities, and were used to plot a standard curve. The labeled product was quantitatively analyzed with SPECT. The labeling rate of E-selectin was 78%. The saturation curve of different concentration samples showed that when the concentration was in the concentration range of 0-1 mg/mL, the standard curve was y=6 045.7x—51.166, R2=0.997 9. Based on this finding, it could be concluded that γ-imaging is an important tool for analysis of radiolabeled product and determination of site density.
Citation：
ZHANG Jinhe, LI Quhuan, LING Yingchen, ZHONG Jianqiu, YIN Jilin, XU Hao. Application of γ imaging in chromatographic separation and protein molecular site density determination. Journal of Biomedical Engineering, 2017, 34(3): 445-448. doi: 10.7507/1001-5515.201609035
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Copyright © the editorial department of Journal of Biomedical Engineering of West China Medical Publisher. All rights reserved
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Snook J H, Guilford W H. A High-throughput technique reveals the load-and site density-dependent kinetics of E-selectin. Cell Mol Bioeng, 2012, 5(4): 493-503.
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- 1. Chen S, Alon R, Fuhlbrigge R C, et al. Rolling and transient tethering of leukocytes on antibodies reveal specializations of selectins. Proc Natl Acad Sci U S A. 1997. 94(7): 3172-3177.
- 2. Li Quhuan, Fang Ying, Ding Xiaoru, et al. Force-dependent bond dissociation govern rolling of HL-60 cells through E-selectin. Exp Cell Res, 2012, 318(14): 1649-1658.
- 3. Larsen S W, Rinvar E, Svendsen O, et al. Determination of the disappearance rate of iodine-125 labelled oils from the injection site after intramuscular and subcutaneous administration to pigs. Int J Pharm, 2001, 230(1/2): 67-75.
- 4. Yamamoto S, Hatazawa J. Development of an alpha/beta/gamma detector for radiation monitoring. Rev Sci Instrum, 2011, 82(11): 113503.
- 5. Afanasiev O K, Nagase K, Simonson W, et al. Vascular E-selectin expression correlates with CD8 lymphocyte infiltration and improved outcome in Merkel cell carcinoma. J Invest Dermatol, 2013, 133(8): 2065-2073.
- 6. Snook J H, Guilford W H. A High-throughput technique reveals the load-and site density-dependent kinetics of E-selectin. Cell Mol Bioeng, 2012, 5(4): 493-503.
- 7. Wiblin D J, Roe S D, Myhill R G. Computer aided desk-top scale-up and optimization of chromatography processes. J Chromatogr A, 1995, 702: 8187.
- 8. 鲍时翔, 姚汝华. 蛋白质分离纯化与层析技术进展. 华南理工大学学报: 自然科学版, 1996, 24(12): 98-103.
- 9. Ling Y C, Zhang J H, Li Q H, et al. Measuring site density of P-selectin on planar surface based on 125I labeling. Bio Technology, 2013, 8(3): 428-433.
