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We were able to stably conjugate the prostate cancer-detecting muJ591 antibody and MIRB to generate antibody:SPIO complexes with an iron to antibody concentration that is sufficient to serve as a functional MR contrast agent detectable by clinical MRI in vitro, flow cytometry, and immunofluorescence based methods. Several studies have shown how antibodies conjugated to superparamagnetic iron oxide particles were used for specific MR imaging of human lymphocytes, the Her-2/neu receptor in breast cancer cells, and apoptotic cells [20,25,27]. These studies used ICP-AES to determine levels of iron to determine loading concentration of the conjugates. However, none of these studies explicitly elaborated on the relative amount of iron per antibody or ligands of interest. In the present study, we used a combination of Bradford protein and ICP-AES based assays to determine the total protein content and iron loading per antibody as an indirect measure of conjugation efficacy. This allowed us to confirm the presence of iron in the complex and to characterize the response as a function of iron concentration, which is valuable information for MR imaging since SPIO dosage has been previously established based on iron concentration and this can be then used to ensure enough contrast sensitivity. When 1 mg/mL of muIgG antibody or muJ591 antibody were conjugated to MIRB using our two-step carboiimide reaction, we recovered in average 0.4560.03 mg/mL of muIgG:MIRB and 0.5660.05 mg/mL of muJ591 antibody:MIRB complex. This protein recovery after the conjugation was estimated by Bradford protein assays performed before and after the reaction. The MIRB cluster-conjugated muJ591 antibody had similar values of elemental iron per antibody to the muIgG antibody. It is therefore likely that we have similar nanoparticle per antibody ratio and the muIgG is therefore an acceptable control. However, when the T2 relaxation time was obtained, the muIgG:MIRB had a slower T2 which was very closer to the value obtained for the MIRB nanoparticle alone. The conjugation with the muJ591 antibody seems to have an effect on the relaxation time in a favorable direction to its detection since the T2 relaxation time becomes faster. |
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