Preparation Of Three Anti-erythropoietin Monoclonal Antibodies And Analysis Of Their Biologial Properties

Erythropoietin(EPO),a 34.4 kD glycoprotein hormone, is an important hematopoiesis factor. The kidney is the mainly production site hi adult, the brain, lung, liver also produce a small amount of EPO. EPO is given by liver in fetus.The mechanisms of the biological function of EPO are not completely known. Some experiments indicated that EPO regulated the proliferation and differentiation of the target cells and avoided them from apoptosis by EPO-induced EPO receptor(EPOR) dimerization and juxtaposition of their cytoplasmic domains and by the signal transduction in sequence via the molecules including JAK2, STATS, STAT1, SHC, SHIP, REK, JNK, P38 and PI-3 Kinase.The biological functions of EPO are including : Accelerating the erythroid progenitor cells proliferation , differentiation and maturation, avoiding them from apoptosis. promoting megakaryocyte maturationpromoting the production of megakaryocyte precursors from CD34+ cells, enhencing the synthesis of reticulated platelets and the adhensive activity of these newly produced platelets.(3)Prolonging life-span of the center nervous system cells.EPO is one of the earliest used cytokine. Since its cDNA was successfully cloned in 1985, rhEPO has been successfully used to treat anemia in various diseases including chronic renal failure, cancer , rheumatoid arthritis , HIV , pregnancy and post parturition , premature infants , myelodysplasia , bone marrow transplantation . EPO is also applied as an adjuvant of autologous blood transfusion . Today , scientists are exploring to treat the damage of brain injure or ischemia , Alzheimer's disease and platelet dysfunction with EPO.The problems of the EPO abusing of athletes in endurance competitions and the side-effects of EPO including hypertension , high viscosity of blood ,stoke , heart and cardio-vascular damage are focused on.Anti-EPO monoclonal antibodies (MoAb) and EPO ELISA kit are necessarily to the mechanism research of EPO bioactivities and EPO quantitative exam. Researchers have been devoting to preparing the anti-EPO MoAbs for nearly 20 years, but a few of them have got success because EPO is the low immunogenic antigen.The spleen cells of Balb/c mice immunized with rhEPO fused with SP2/0 myeloma cells. The hybridoma screened by an indirect enzyme-linkedimmunoabsorbent assay (ELISA). Three anti-EPO MoAbs were obtained . 1D1 is IgG2b MoAb , 4A8 and 7B6 belongs to the IgM class. The ascetic fluids of these MoAbs were induced in vivo in homogenous Balb/c mice. 1D1 ascetic MoAb was purified by chromatograph with protein G, 4A8 and 7B6 MoAb were purified by euglobulin precipitation. Western blot and the antibody competitive inhibiting assay were applied , which verified that these three MoAbs can specifically recognize EPO and bind the same or similar epitopes . The role of EPO in proliferation of TF-1 cells which belong to the human multiple-factor dependent heamatoleukemic cell lines was inhibited by these three MoAbs in vary degree.Our experiments suggested that the low immunogenicity can be altered by antigen presenting process with homogenous APC induced from bone marrow in vitro.We will establish the Sanwich ELISA assay to EPO by using these three MoAbs and the anti-EPO mutiple antibodies or the MoAbs against another epitopes .The three MoAbs prepared in our lab will be useful for futher study of the biological properties and signal transduction of EPO.