Quality control of widely used therapeutic recombinant proteins by a novel real-time PCR approach

Background: Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase [rSK], and alpha interferon [IFN-alpha] preparations

Methods: A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilu ons of DNA extracted fromE. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-alpha samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry

Results: The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-alpha preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA

Conclusion: Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers' minds on refining drugs, and provides consumers with safer biopharmaceuticals

Effects of rutin on oxidative stress in mice with kainic acid-induced seizure / 中西医结合学报

<p><b>OBJECTIVE</b>Flavonoids are present in foods such as fruits and vegetables. Several studies have demonstrated a relationship between the consumption of flavonoid-rich foods and prevention of human disease, including neurodegenerative disorders. We assessed the effect of rutin (quercetin-3-O-rutinoside) on oxidative stress in kainic acid (KA)-induced seizure.</p><p><b>METHODS</b>Thirty-six BALB/c mice were randomly divided into three groups. In the control group, saline (intra-peritoneal, i.p.) was administered for 7 d, and on the last day, KA (10 mg/kg, i.p.) was injected 30 min after administration of saline. In rutin groups, mice were pretreated with rutin (100 and 200 mg/kg, i.p.) for 7 d, and on the last day, KA (10 mg/kg, i.p.) was injected 30 min after administration of rutin. Subsequently, behavioural changes were observed in mice. Lipid peroxidation and oxidative stress were measured respectively in the early and late phases after KA-induced seizures.</p><p><b>RESULTS</b>Seizure scores in the rutin groups were significantly lower than those in the control group (P < 0.01). Furthermore, rutin dose-dependently inhibited the number of wet-dog shakes (WDS) (P < 0.05). Malondialdehyde level in the hippocampus of the rutin groups was significantly lower than that in the hippocampus of the control group on days 1 and 21 after KA administration. In the rutin groups, the thiol levels observed on day 1 after KA administration were higher than that in the control group (P < 0.01).</p><p><b>CONCLUSION</b>These results indicate that rutin has potential anticonvulsant and antioxidative activities against oxidative stress in KA-induced seizure in mice.</p>

Evaluation of T-SPOT[registered] serology test for the diagnosis of pulmonary tuberculosis

Tuberculosis is a chronic contagious infectious disease which is fatal within 5 years in more than half of cases if not diagnosed. Since the fight against tuberculosis is based on early diagnosis and complete treatment of all TB patients, useful biochemical methods are emphasized to find a more rapid diagnostic method. This study aims to evaluate the impact of diagnostic value of T-SPOT[registered] serology in patients suspected or diagnosed with tuberculosis admitted in Bou-Ali Hospital of Zahedan, Iran. The descriptive-analytic study conducted on 60 patients, 30 of whom had AFB sputum smear positive pulmonary tuberculosis, and 30 patients had AFB sputum smear negative pulmonary tuberculosis. The results were stated as sensitivity, specificity, positive and negative predictive value and likelihood ratio using conventional epidemiological table. In 23 out of 30 patients with AFB sputum smear positive pulmonary tuberculosis, T-SPOT[registered] serology became positive and in 12 out of 30 patients with AFB sputum smear negative pulmonary, T-SPOT[registered] serology became positive. According to the epidemiological table in this study, sensitivity, specificity, positive and negative predictive value and likelihood ratio of this test were determined respectively 76%, 40%, 56%, 63% and 1.25%. According to the results, this test is not able to distinguish active pulmonary tuberculosis from latent infection. Moreover, considering high contact of regional people with TB patients and pulmonary involvement of people due to factors other than TB, the test value with this likelihood ratio is low.