Preparation Of A Granzyme B Targeted MR Contrast Agent Gd-DTPA-Granzyme B Monoclonal Antibody

Targeted MR technology is a type of new MRI technologies which developed in recent years. It combines gadolinium diethylenetriamine pentaacetic acid(Gd-DTPA) with some specific carrier molecules such as monoclonal antibody. Lead by those carrier molecules, the MRI contrast agent will be specifically uptaken in the organ, certain part of body or disease organization which is targeted by the carrier molecules. Then the T1 release time of the water molecules around those organizations will be changed to make up a specific MRI image. Because the contrast agent combined with carrier molecules could keep longer in the targeting organ or tissue, the targeted magnetic resonance is more specific compared with the traditional MRI technology and it can avoid the limitation of un-specific contrast agent, which has some value in early diagnosis of disease. So far, targeted magnetic resonance technology is actively studied in Oncology, while in other area, it got less attention.As we all know allograft rejection has obvious immune response activation and inflammatory cell infiltration processes. Granzyme B (GB) is a major cytotoxic T lymphocyte effecter molecule in acute rejection. Activated T-lymphocytes release of granzyme B into target cells, and activate endonuclease system to start the target cell DNA break, which causes target cell apoptosis. At present, many studies have suggested that granzyme B had some clinical value for the early diagnosis of renal allograft rejectionThis study is to use anti-granzyme B monoclonal antibody and Gd-DTPA to prepare a granzyme B targeted MR contrast and to investigate the optimal conditions for synthesis, efficacy and safety in order to find a more specific early non-invasive diagnostic method of acute rejection in renal transplantation .Methods1 Gd-DTPA-GB mAb preparation: The DTPABA dissolved in dimethylformamide, mixing the DTPABA and granzyme B antibody, incubated at room temperature. PD-10 column purification, determine the antibody concentration. GdCl₃ added to NaAc, adding the elution solution at room temperature. PD-10 column purification, eluate collection, sub-mounted, 4℃storage.2 Identification of Gd-DTPA-GB mAb contrast agent: Using matrix assisted laser desorption ionization / time of flight mass Spectrometry (MALDI-TOF-MS) (Voyager-DE STR 4349) to determine of whether the contrast agent was connected and the number of Gd³⁺.3 Cytotoxicity test: collection of the logarithmic phase of human renal tubular epithelial cells, inoculated in 96-well plates,37℃incubate. To fill the hole at the end of cell monolayer and to add a certain concentration gradient of Gd, Gd-DTPA, GdCl₃ and Gd-DTPA-GB mAb respectively, incubated for 12 hours.Using MTT to determine the cytotoxicity.4 Gd-DTPA-GB mAb immune activity test: To take a normal male adult SD rat thyroid tissue, paraffin-embedded and sliced. Divided into experimental group (Gd-DTPA-GB mAb), positive control group (GB mAb) and negative control group (PBS), Using ABC method (Avidin-biotin-peroxidase Complex method). to compare the three groups and determine the immune activity of the Gd -DTPA-GB mAb.5 Statistical Methods: Results were expressed in the text as mean±SD, t test was used to compare between the two groups and Statistical analysis was performed by using SPSS13.0 software package. Statistical significance level isα=0.05, P <0.05 when the difference was significant.ResultsThe molecular weight shift from granzyme B monoclonal antibody (133986.41) to Gd-DTPA-GB mAb (139736.06) in MALDI-TOF-MS represented the successful combination of the antibody with Gd-DTPA. The molar ratio of Gd per IgG molecule was about 20. MTT assay showed that Gd, DTPA, Gd-DTPA or Gd-DTPA-GB mAb had no obvious impact on cell viability, and there were no significant differences on cell proliferation rate between these 4 groups (P >0.05). Immunohistochemistry results showed that combination with Gd-DTPA had no influence on the immune activity of anti granzyme B antibody.Conclusions1 Gd-DTPA could combine with granzyme B monoclonal antibody through a simple response.2 MALDI-TOF-MS identified the conditions that we set can synthesize targeted magnetic resonance contrast agent Gd-DTPA-GB mAb.3 Cytotoxic and immunological experiments showed that the targeted molecular imaging agent had a good immune activity and no significant cytotoxicity.