The Application Of The Rat Anti-human RANTES MAb In Reducing The Rejection In Rat Small Bowel Transplant And The Humanization Of The MAb

AIM1. To prepare and identify rat monoclonal antibodies against humanRANTES molecule. Provided a new experiment method for research thedistribution and function of RANTES.2．To master the way of setting up the model of segmental small boweltransplantation heterotopically in rats by tri-cuff anastomosis method, andestablishment of rat model of nutrition support.3. To apply the rat mAb against human RANTES in rat small boweltransplantation model, validate the effect of mAb in alleviating the transplant rejection.4. To primary humanize the rat against human RANTES monoclonalantibody.METHODS1. Rat mAbs were prepared by hybridoma technique. Ascites titer wasidentified by ELISA. The activities of mAbs were identified by Western bolt.The characters of mAbs were identified by FCM, immunohistochemistry andimmunocytochemistry.2. Heterotopic small bowel transplant was carried out between inbredmature male SD and Wistar rats, which was used tri-cuff anastomosis method.All recipients were divided into four groups. Group1, SD; Group2, SDSD;Group 3, SDWistar; Group 4, SDWistar+RANTES mAb (3.0mg·kg^-1·d^-1, ip). The grafts were sampled on the postoperative day 3, 5, 7and hematoxylin eosin staining was conducted on the specimens to observepathological change.3. Checked the concentration of IL-2 , IL-6, TNF- and RANTES in ratserum after operation in four groups at different time by ELISA kit. Checked theconcentration of RANTES in tissue of small bowel of rat receptor byimmunohistochemistry .4. We got VH and VL gene of mAb from hybridoma cell lines ByRT-PCR,and connected to the humanization expression vectors. Then wepurified the humanized mAb from transfected 293 cell line.RESULTS1. Two hybridoma cell lines, RANTES No1, RANTES No2, secreting mAbsagainst human RANTES molecule were obtained. The titers of ascitic mAbsreached to 10-6 and the Ig subclasses of RANTES No1 and RANTES No2 were IgG1 and IgM, respectively. Moreover, RANTES No1 could be used inimmunohistochemistry staining.2. The pathological examination of grafts in Group 3 demonstrated the mild,moderate, and severe rejection on postoperative day 3, 5, and 7. While rats inthe Group 2 and 4 showed no obvious indication of rejection.3. The results of ELISA kit detection showed that concentration of IL-2 ,IL-6, TNF- and RANTES in rat serum of mAb therapy group is obviouslydiminution than group 3. Meanwhile, the concentration of RANTES in tissue ofsmall bowel in group 4 is reduced obviously.4. We got two cell lines which can express humanized monoclonalantibody, and got purified protein by affinity chromatography.CONCLUSION1. Prepared two rat mAbs against human RANTES, Both can be used inWestern blot, FCM and immunocytochemistry. Only No.1 can be used inimmunohistochemistry .2. Applied the rat mAb against human RANTES in rat small boweltransplantation model, validated it can alleviate the transplant rejection.3. Improved the techniques in rat small bowel transplantation model andestablished the rat model of vein nutrition support.4. primary humanized the rat against human RANTES monoclonalantibody by chimeric antibody technique, which can provide a useful tool forfurther application.