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Iranian Journal of Biotechnology، جلد ۱۵، شماره ۱، صفحات ۵۰-۵۷
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عنوان انگلیسی Production of Erythropoietin-specific polyclonal Antibodies
چکیده انگلیسی مقاله Background: Erythropoietin, as a principal hormone promotes red blood cell production in bone marrow. Varieties of erythropoietin biosimilar are being produced by recombinant DNA technology in cell cultures. The detection or quantification of these molecules are being performed by diff erent methods which some of theme such as Western blot and enzymelinked immunosorbent assay (ELISA) require specifi c antibodies. High cost, inappropriate shipping (cold chain failures), reduced sensitivity and thus poor detection performance are common pitfalls of using commercial kits for performing immunological tests.Objectives: To produce in-house polyclonal antibody against active pharmaceutical ingredient (API) of recombinant human erythropoietin (rh-EPO) was the aim of this study.Materials and Methods: Two healthy female albino rabbits were injected four times in 14 days interval using rh-EPO API as antigen. The produced antibody was separated from plasma via either caprylic acid or saturated ammonium sulfate precipitation and the results were compared from each purifi cation methodologies. The antibody was further purified by ion exchange chromatography. Acceptable purity and good immunogenicity were detected respectively by SDS-PAGE and western blot analysis. The purifi ed antibody was compared with a commercial kit to determine rh-EPO concentration in different steps of production batches via ELISA.Results: The purity of antibodies after ion exchange chromatography, obtained from caprylic acid and ammonium sulfate precipitation were 97 and 80%, respectively.Conclusions: As producing in house kits is one of the important challenges of bio-pharmaceutical manufacturers, a simple, cost- and time-eff ective, and easy to scale up strategy for making in-house polyclonal antibody was set up. Caprylic acid precipitation resulted higher purity than ammonium sulfate and finally purified antibody (97% purity) used as a capture antibody in sandwich ELISA test was able to detect erythropoietin antigen as sensitive (100%) and specific (100%) as commercial kits.
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نویسندگان مقاله کورش معبودی |
department of biochemistry, ardabil branch, islamic azad university, ardabil, iran
سازمان اصلی تایید شده: دانشگاه آزاد اسلامی علوم و تحقیقات (Islamic azad university science and research branch)

سیده مرضیه حسینی | seyedeh marzieh
immunochemistry laboratory of quality control department, pasteur institute of iran, karaj, iran
سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

مینا سپاهی |
recombinant biopharmaceutical production department, pasteur institute of iran, karaj, iran
سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

هاشم یعقوبی |
biochemistry department, ardabil branch, islamic azad university, ardabil, iran
سازمان اصلی تایید شده: دانشگاه آزاد اسلامی علوم و تحقیقات (Islamic azad university science and research branch)

شاهین حدادیان |
nano-biotechnology department, pasteur institute of iran, no. 69, 12th farwardin ave, tehran. p.o.box 1316943551, iran.
سازمان اصلی تایید شده: انستیتو پاستور ایران (Pasteur institute of iran)

نشانی اینترنتی http://www.ijbiotech.com/article_44224_5446c269fb541700f01b886010ca26c1.pdf
فایل مقاله اشکال در دسترسی به فایل - ./files/site1/rds_journals/441/article-441-485017.pdf
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