Preparation And Characterization Of Monoclonal Antibody Against Rabbit NADP(H)-dependent Retinol Dehydrogenase/Reductase

All-trans retinoic acid (atRA), the metabolite of vitamin A, is a kind of bioactive compound in many vertebrates. It participates in a wide spectrum of physiological processes by modulating the gene's transcription, including cell growth, cell differentiation, morphogeneisia, immunologic regulatory function etc. In addition, it also shows extensive biological effects in anti-infection, suppressing tumor cell growth and proliferation, inducing (tumor) cell differentiation, apoptosis etc. Retinoids are widely applied to prevent and treat some malignant tumour, for instance, breast carcinoma, cutaneous carcinoma, cervix cancer and acute promyelocytic leukemia etc. The homeostasis of retinoids in vivo was adjusted by many factors such as diets and the relative enzymes, however, the mechanisms of retinoid metabolism with relevant enzymes are not completely clarity.NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) , a enzyme purified and identified from rabbit liver cytosol, is involved in oxidoreduction between retinol and retinal with high activity. atRA can be derived in vivo from retinol in two oxidative steps with retinal as an intermediate. The change from retinol to retinal is the rate-limited step in these reactions.Balb/c mice were immunized subcutaneously with purified recombinant rabbit NRDR expressed by gene engineering. The immunized mouse spleen was removed under the sterile condition, and the spleen cells were dispersed through the strainer to get a single cell suspension. Hybridoma cell lines were obtained by fusing these spleen cells with myeloma NS-1 cells. Positive hybridomas with the capability of secreting mAb were screened by indirect enzyme-linked immunosorbent assay (ELISA) and subcloned by limiting dilution method traditionally. Ascites was produced in order to gain a great quantity of mAb by intraperitoneal injection. The titers and the relative affinities of mAbs were tested by indirect ELISA. Ig subclasses of mAbs were detected by mouse monoclonal antibody isotyping kit. The specificity of mAbs had been assessed by indirect ELISA and Western blot.At the end, three clones of hybridoma were obtained, which were designated as NR1,NR2 and NR5. The Ig subtypes of them are all IgG1. The cell culture supernatant titers of NR1,NR2 and NR5 were1:20,1:40 and 1:20. Ascites titers of NR1,NR2 and NR5 mAb were 1:106, 1:107 and 1:106 respectively. The relative affinity is NR2>NR1>NR5 in order. Western blot analysis showed that mAbs had specific binding abilities with antigen.