Purification Of Anti-GPⅢa Monoclonal Antibody SZ-21 And Thrombus Imaging In Rabbit Model Using ～(99)Tc～m-SZ-21

Objective: To abstract, purify, and identify monoclonal antibody SZ-21 from abdominal dropsy; to label SZ-21 with 99Tcm and evaluate the value of 99Tcm-SZ-21 in radioimmunoimaging of thrombi in rabbit thrombi module.Methods: High potency SZ-21 abdominal dropsy was screened by indirect ELISA. McAb SZ-21 was obtained by Protein G Affinity Chromatography and concentrated by 30% PEG2000; antibody concentration was determined by ultraviolet light spectro photometer. Antibody was identified and analyzed with SDS-PAGE. Using GPⅢa in platelets of human and rabbit respectively as antigen, FITC-GAM as second antibody and setting mouse IgG negative control as detecting condition, binding capability of SZ-21 and active platelets was detected by FCM. SZ-21 was modified with 2-iminotholane and labeled with 99Tcm-GH. Labeling yield, radiochemical purity and radiochemical purity were determined by ITLC-SG and Sephadex G-25. Thrombi were induced in five rabbit in there right femoral arteries by intubation tube placement of self-made embolization coils, one in right tibial artery. X rays were acquired to confirm the localization of the coils. 99Tcm -SZ-21 was injected through a lateral ear vein in five rabbits. As negative control, 99Tcm-GH was administered in one rabbit. Static images were acquired in a 256×256 matrix and 500Kcounts were accumulated in each image from 30 min up to 3 h after injection. Radioactivity disposition in thrombi locus was observed. Irregularly shaped ROIs were drawn on the images to encompass the area of thrombus. Similarly sized ROIs were applied to the adjacent background muscle area on the thrombus side (T/M) and to the area corresponding to thrombus area on the contralateral side (T/B). Vein blood was drawn at 2min,5min,10min,30min,1h～3h after injection in two of these animals in order to observe the blood clearance of 99Tcm-SZ-21. Animals were sacrificed after 3h of imaging. The vessels including clots were harvested and imaged. Cardiac muscle, liver, spleen, lung, kidney and so on were extirpated. Radioactive counting was acquired to calculate %ID/g.Results: The concentration of monoclonal antibody SZ-21 was 2mg/mL. SDS-PAGE showed purity of the antibody was beyond 95%。The results of FCM demonstrated that the positive cell populations were 98% and 97% (SZ-21 with human platelet and rabbit platelet respectively) .The radiochemical purity was beyond 90% and stable in vitro. Thrombi can be visualized at 30 min after injection, up to 2 h images of thrombi were clear, T/M (2.55±0.72) and T/B (1.94±0.51) ratios were high. Ex vivo imaging showed T/B was 4.43±1.5.Conclusion: As a method of purifying antibody from abdominal dropsy, Protein G Affinity Chromatography is characteristic of high efficiency ,fast and high purified yield. The combination of SZ-21 and platelet, high thrombus uptake and definitive in vitro images show that 99Tcm-SZ-21 has considerable potential as a clinical imaging agent for the detection of thromboemblism.