C-MALISA (cellular magnetic-linked immunosorbent assay), a new application of cellular ELISA for MRI

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 5 N HCl, the samples were analyzed by MRI. Paramagnetic relaxation rate enhancements (ΔR2) were measured on images. ΔR2 was converted in values of iron concentration based on a calibration curve. The apparent dissociation constants (Kd∗) of the three contrast agents were estimated based on the MRI measurement of ΔR2. Kd∗ of 1.22 × 10−7 M, of 7.00 × 10−8 M, and of 1.13 × 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).