فهرست مطالب seyedeh marzieh hosseini

Avian Influenza disease annually entails a significant economic loss to the poultry industry around the world. Influenza virus is a polymorphic virus of the orthomyxoviridae family (single-stranded RNA genome), and nucleoprotein (NP) is the structural and internal protein of the virus. The aim of the work was to purify nucleoprotein for further investigations with a simple, low-cost, fast and practical method. In this study, H9N2 influenza virus was isolated in specific pathogen-free embryonated chicken eggs by allantoically inoculating 103 to 105 egg-infective doses (EID50) for 9 to 11 days, purified by 10% (W/V) polyethylene glycol (PEG) 6000 with a sucrose gradient of 60% to 30%. The influenza virus proteins were collected and prepared as fractions by preparative electrophoresis. Finally, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot procedures. The protein analysis with SDS-PAGE and silver nitrate staining indicated that the desired samples contained purified nucleoprotein and lacked other viral proteins. The results of the investigation of lyophilized fractions containing nucleoprotein on the SDS-PAGE revealed the absence of viral RNA in nucleoprotein and its high purity. According to this study, purified nucleoprotein can be used to produce nucleoprotein vaccines, as well as to study structural, molecular and diagnostic and therapeutic materials.

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.
To produce in-house polyclonal antibody against active pharmaceutical ingredient (API) of recombinant human erythropoietin (rh-EPO) was the aim of this study.
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.
The purity of antibodies after ion exchange chromatography, obtained from caprylic acid and ammonium sulfate precipitation were 97 and 80%, respectively.
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.

One of the most important aspects in recombinant biologic production, based on GMP rules, is the accuracy of final product quality control, especially assessment of host cell macromolecules contamination rate in final product. The purification requirement can be eliminated when the yeast cell containing the recombinant protein is used as a host cell. It is possibile that the final product contaminated to the host cell protein during purification stages of HBsAg (HBV vaccine). The protein purification costs depend on the purification procedures required. Nowadays several companies produce commercial kits for identification and assessment of host cell protein contamination based on ELISA and Western blotting methods. But high prices, difference in sensitivity and lack of easy access to these kits sometimes create problems. So, in this study, two methods of Ammonium sulphate and caprilic acid precipitation technique were used separately for IgG purification. The results showed that IgG purification increased by 97% in caprylic acid method, compared with only a 77% increase in ammonium sulphate method. There were also significant differences in specificity and sensitivity between our standardized ELISA technique and using commercial kit (Cygnus CHO HCP).