Font Size: a A A

The Original Research Of MR Immuno-imaging On Nude Mouse Bearing Human Hepatocellular Carcinoma By Using Two Step Pretargeting Strategy

Posted on:2005-11-15Degree:DoctorType:Dissertation
Country:ChinaCandidate:X LiuFull Text:PDF
GTID:1104360125951548Subject:Medical Imaging
Abstract/Summary:PDF Full Text Request
The aim of this research is to explore optimal reaction condition of DTPA-coupLed streptavidin labeled with gadolinium ion by means of preparing Gd-DTPA-SA and to improve sensitivity of MR molecular immunoimaging by using targeting and magnifying effects of biotin-avidin system.Materials and Methods The preparation of biotinylated monoclonal antibodyHAblS was conjugated with biotin by using NHS-LC-biotin. Freshly prepared NHS-LC-biotin was added to monoclonal antibody in 0.1M bicarbonate buffered saline, pH 7.4, at molar ratio of 30:1 and incubated for 2 h at 4癈. Then unconjugated biotin was removed by filtering centrifuge. The number of biotin molecules coupled to each antibody was determined by the Green et al. using HABA solution. And the binding capacity of biotinylated antibody was also determined. The preparation of Gd-DTPA-streptavidinStreptavdin was dissolved in 0.1M bicarbonate buffer at different pH value (5, 6, 7, 8). Cyclic anhydride DTPA was dissolved in DMSO and added. The molar ratio of DTPA to streptavidin was respectively 1000, 500,200,100. The mixture incubated at room temperature for 1 h. After added GdCl3 solution and reacted for several minutes, filtering centrifuge was used to eliminate redundant GdCl3, Gd-DTPA. DTPA.The number of gadolinium ion that streptavidin coupled and maximum biotin-binding capacity of Gd-DTPA-SA complex were measured using ICP-AES and biotin binding colorimetry. The preparation of Gd-DTPA-HAbl8Cyclic anhydride DTPA as dissolved in DMSO. HAblS antibody solution (2 mg, 1ml) was added and the mixture incubated at room temperature for 45 minutes. The mole ratio of DTPA to monoclonal antibody was 20:1. The resulting solution was loaded onto filtering centrifuge to remove dissociative DTPA. GdCl3 was dissolved in distilled water. To this solution, DTPA-Habl8 was added. The pH of reaction system was adjusted to 8.0 using 0.1M phosphate buffered saline. After stirring for 1 h at room temperature, centrifuge the solution to eliminate Gd. In Vivo MR imagingTwo step pretargeted tumor imaging was achieved by first injecting intravenously biotinylated monoclonal antibody HablS into 8 BALB/c nude mice bearing QGY-7723 tumor cells line. 24 h later, 80,g avidin was administered as chaser and after 30 min, Gd-DTAP-streptavidin was injected into the tumor bearing mouse. Gd-DTPA-Habl8 and Gd-DTPA were respectively injected intravenously into the other 12 tumor-bearing mice as contrast groups.MR imaging was performed at 10 min, 30 min, 60 min, 90 min, 6 h, 12 h, 24 h and 48 h before and after injection of MR contrast agents. All images were obtained using SE Tl-weighted imaging consequence, with 500ms/15ms/90? 2mm slice thickness, including axial and coronary SE images.After MR imaging, enhancement time course of three different groups were determined by using enhancement data measured in the region of interest in the tumor, liver and muscle. Enhancement ratio, tumor-to-muscle SI ratio and contrast-to-noise of tumor also were calculated. Gadolinium concentration measurementsImmediately after MR imaging, the animals were sacrificed. All the tissues including tumor, liver, spleen and kidney were quickly frozen until used for ICP-AES measurements. The gadolinium content was measured according to the method of Tamat et al. The 342.249 nm atomic emission line of Gd was chosen for ICP-AES analysis. The tissue uptake of Gd was-6-calculated as a percentage of the injected contrast agent percentage ofinjected dose ( I.D.).Results1. The average number of biotin conjugated with each monoclonal antibody molecular was 15. And the immunoactivity of biotinylated antibody was 91%.2. The number of gadolinium ion that streptavidin coupled increased with pH value of the reaction system increasing. When DTPA-to-SA molar ratio was below 500, the number of gadolinium ion that SA-DTPA coupled increased with DTPA to S A molar ratio increasing; but decreased when DTPA to SA molar ratio is 1000.The average number of gadolinium ion that streptavidin coupled was 15.6 when DTPA-to-...
Keywords/Search Tags:Biotin, Avidin, Streptavidin, Pretargeting, Magnetic, Resonance Imaging, Monoclonal Antibody
PDF Full Text Request
Related items