Background: Infeliximab is a form of chimeric antibody which neutralizes the most important inflammatory cytokine, TNF-a, in inflammatory disorders. The aim of current study was to pilot expression of chimeric infliximab in Chinese Hamster ovary (CHO) cells.
Methods: In this research study, pVITRO2-neo-mcs vector that consist of infliximab light chain and heavy chain was used to transform into the E.coli by CaCl2 method. The plasmid was then purified and transfected to cultured CHO cells by Lipofectamine 2000® (Invitrogen GmbH, Germany). Transfected cells were selected upon G-418 treatment after 2 weeks and the level of expression, based on standard curve, was measured using IgG ELISA kit after 48 hours for each clone. High level expressed clone was then cultured in roller bottles and recombinant chimeric product was purified by protein A affinity chromatography. The purity of the product was analyzed by 10% gel SDS-PAGE from eluted samples. The efficacy of the purification was analyzed by ELISA before and after purification step. This article is a master's student thesis from February 2015 to August 2016 in pharmaceutical technology development center, Tehran University of Medical Sciences, Tehran, Iran.
Results: The purified plasmid was analyzed on 2% agarose gel. After selective pressure of G-418, 10 stable transfect clones were assessed for infliximab secretion by IgG ELISA kit at 450 nm. The maximum and minimum expression which detected by ELISA were 23 ng/ml and 6 ng/ml, respectively. The band width of infliximab fraction during purification procedure was observed at 0.7-0.8 min. The efficiency of the purification by ELISA was 70%. On SDS-PAGE analysis, two bands, 25 and 50 kDa, respect to light and heavy chains of Infliximab, was confirmed the expression of recombinant protein.
Conclusion: In the current study, the construct for infliximab monoclonal antibody production was designed using genetic engineering techniques and the expression was confirmed by conventional molecular biology methods. The high yield production was carried out in semi-industrial scale using roller bottles with a 70 percentage of purification efficiency.
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Background: Nowadays, stroke can be considered as the one of the major causes of disability and mortality worldwide. However, relationship between serum C-reactive protein (CRP) level with stroke early prognosis has not been well studied, especially in Iran. Therefore, the present study aimed to study the relationship between CRP level of serum in patients with acute stroke at first 24h of admission and stroke early prognosis. Methods: This prospective cross-sectional study was performed on 50 patients with acute stroke who were admitted at Emergency Ward of Shahid Rajaie Hospital, in Tonekabon City, Iran, between May 2013 to July 2014. In first step, valid clinical diagnosis was made based on CT scan and magnetic resonance imaging (MRI) of the patients. The ethical observations were considered for all patients. The serum level of CRP was measured by standard method, at first 24h of the admission. Clinical information and risk factors (age, gender, type of stroke, high blood pressure, diabetes mellitus) was detected for each patient. On discharge, early prognosis by modified Rankin Scale (mRS) (mRS< 3= good and mRS≥ 3= bad) was also recognized. In this study, statistical analysis was performed using SPSS software, and logistic regression method was used (P< 0.05). Results: The results of this study showed that 38% of the patients were 70-80 years old. Also, 52% of the patients were male and 48% were female. The serum CRP level of patients at the first 24h of admission increased in all studied patients. The mean of the serum CRP level was 12.82 that were higher than the normal range. The statistical analysis showed that there was no significant relationship between the serum CRP level and the stroke early prognosis. Conclusion: Although the serum CRP level was not recognized as an anticipator factor for stroke prognosis at this study, it is recommended to performance of more studies by case-study method on this setting. |
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