Burtea Carmen, Laurent Sophie, Roch Alain, Vander Elst Luce, Muller Robert N
Department of Organic and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons-Hainaut, 24, Avenue du Champ de Mars, B-7000 Mons, Belgium.
J Inorg Biochem. 2005 May;99(5):1135-44. doi: 10.1016/j.jinorgbio.2005.02.009.
A modified cellular ELISA (enzyme-linked immunosorbent assay), named cellular magnetic-linked immunosorbent assay (C-MALISA), has been developed as an application of magnetic resonance imaging (MRI) for in vitro clinical diagnosis. To validate the method, three contrast agents targeted to integrins were synthesized by grafting to USPIO (ultrasmall particles of iron oxide): (a) the CS1 (connecting segment-1) fragment of fibronectin (FN) (USPIO-g-FN); (b) the peptide GRGD (USPIO-g-GRGD); (c) a non-peptidic RGD mimetic (USPIO-g-mimRGD). Jurkat cells and rat mononuclear cells were stimulated to activate their integrins. After cell fixation on ELISA plates, incubation with the contrast agents, rinsing, and digestion in 5N HCl, the samples were analyzed by MRI. Paramagnetic relaxation rate enhancements (delta R2) were measured on images. Delta R2 was converted in values of iron concentration based on a calibration curve. The apparent dissociation constants (K(d)) of the three contrast agents were estimated based on the MRI measurement of delta R2. K(d) of 1.22 x 10(-7) M, of 7.00 x 10(-8) M, and of 1.13 x 10(-8) M were found respectively for USPIO-g-FN, USPIO-g-GRGD, and USPIO-g-mimGRG. The MRI confirmed a statistically significant difference (p < 0.01, p < 0.05) between the stimulated cells incubated with integrin-targeted compounds with respect to the controls (i.e., non-stimulated cells and stimulated cells incubated with non-specific USPIO). The integrin specificity of the three compounds was confirmed by the pre-incubation with GRGD (for USPIO-g-mimRGD and USPIO-g-GRGD) or FN (for USPIO-g-FN).
一种改良的细胞酶联免疫吸附测定法（ELISA），即细胞磁联免疫吸附测定法（C-MALISA），已被开发出来作为磁共振成像（MRI）在体外临床诊断中的应用。为验证该方法，通过将其接枝到超小氧化铁颗粒（USPIO）上，合成了三种靶向整合素的造影剂：（a）纤连蛋白（FN）的CS1（连接段-1）片段（USPIO-g-FN）；（b）肽GRGD（USPIO-g-GRGD）；（c）一种非肽类RGD模拟物（USPIO-g-mimRGD）。刺激Jurkat细胞和大鼠单核细胞以激活其整合素。将细胞固定在ELISA板上，与造影剂孵育，冲洗，并在5N盐酸中消化后，通过MRI对样品进行分析。在图像上测量顺磁弛豫率增强（δR2）。根据校准曲线将δR2转换为铁浓度值。基于δR2的MRI测量估计了三种造影剂的表观解离常数（K(d)*）。USPIO-g-FN、USPIO-g-GRGD和USPIO-g-mimGRG的K(d)*分别为1.22×10(-7) M、7.00×10(-8) M和1.13×10(-8) M。MRI证实，与整合素靶向化合物孵育的刺激细胞与对照（即未刺激细胞和与非特异性USPIO孵育的刺激细胞）之间存在统计学显著差异（p < 0.01，p < 0.05）。通过与GRGD（对于USPIO-g-mimRGD和USPIO-g-GRGD）或FN（对于USPIO-g-FN）预孵育，证实了这三种化合物的整合素特异性。
