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Abstract

The production of antibodies for stem cell research is one of the prospective directions in immunobiotechnology. The aim of this study was to optimize biotechnological process for production of high-affinity rabbit polyclonal antibodies to human recombinant transcription factors Nanog and Oct4 which are specific markers of pluripotent stem cells. For production of polyclonal antibodies we used recombinant Nanog and Oct4 which were obtained from bacterial strains Rosetta(DE3)/pNanog and Rosetta(DE3)/pOct4. In this work we optimize immunization scheme of laboratory rabbits using minimal amount of recombinant antigens for production of high levels of polyclonal antibodies. After antibody purification with HiTrap� Protein G chromatographic columns the concentrations of purified IgG antibodies were 0.79 mg/ml for Nanog and 1.18 mg/ml for Oct4. ELISA and western blot revealed that obtained polyclonal antibodies are highly specific to recombinant Nanog and Oct4. The maximum antibody titer in western blot and ELISA was 1:5000 and 1:12800 respectively. Results from western blot analysis also showed that sensitivity of developed polyclonal antibodies to Nanog and Oct4 was higher in comparison to commercial analogs. Moreover, immunocytochemistry showed that rabbit polyclonal IgG antibodies were able to detect native forms of Oct4 and Nanog in nuclei of human pluripotent stem cells at dilution ratio 1:2000. Thus, our results showed that developed polyclonal antibodies are highly specific and more sensitive to recombinant and native Oct4 and Nanog than commercial analogs.

Biography

Li Yelena Alexandrovna has completed her Master’s degree from Karagandy State University. Curently she works at National Center for Biotechnology as a Researcher. Her scientific field of interests are stem cell research and cell biotechnology.

Email: hellenala@gmail.com