An in-house indirect immunofluorescence test for ensuring that the cell bank is contamination free

 Background: Cross-contamination between cells is a usual mistake at cell culture laboratories and cell banks worldwide. MRC5 diploid cell and Rk-13, Vero and Hela continuous cell lines are used in different stages of human viral vaccines propagation at Razi Vaccine and Serum Research Institute of Iran. However diploid and continuous cells are propagated at separated cell culture laboratory and continuous cells can contaminate MRC5 diploid cells. Therefore, a sensitive test is needed. World Health Organization recommends few molecular and cellular techniques to cell characterization. Materials and Methods: The present study was therefore designed to set up an indirect immunofluorescence (IIF) test as follows: Polyclonal anti-MRC5 cell and anti-rabbit antibodies were prepared in rabbit and goat, respectability. Anti-rabbit IgG was purified using protein G affinity chromatography, conjugated to fluorescein isothiocyanate (FITC), and then further purified to remove unbound FITC using Sephadex G 25 chromatography. Using double immunodiffusion assay, purification of homemade anti-rabbit IgG was asssayed by observation of a single arch. Results: The titer of homemade FITC conjugated goat anti rabbit IgG was measured 1/16 vs 1/8 of commercial type. Fluorescein/protein molar ratio of local made fluorescein goat anti-rabbit IgG was measured 3.44 and its protein concentration and FITC concentration were determined 1.198 mg/ml and 0.01 mg/ml, respectively. Conclusion: Moreover, homemade IIF test showed 100% intra-laboratory reproducibility. Purity of three batches of MRC5 working seed cell was verified using in- house IIF test and no contamination to continuous cell lines was found

Synthesis and Antibacterial Activity of Novel Levofloxacin Derivatives Containing a Substituted Thienylethyl Moiety

Background and the purpose of the study Piperazinyl quinolones such as ciprofloxacin, ofloxacin and levofloxacin are an important group of quinolone antimicrobials which are widely used in the treatment of various infectious diseases. In the present study, we synthesized a new series of levofloxacin derivatives and evaluated their antibacterial activities. Methods:The N-substituted analogs of levofloxacin 6a-j were prepared by nucleophilic reaction of Ndesmethyl levofloxacin 11 with thienylethyl bromide derivatives 8 or 9. All target compounds were tested using conventional agar dilution method in comparison to levofloxacin and N-desmethyl levofloxacin and their MIC values were determined against a panel of Gram-positive and Gram-negative bacteria. Results:All compounds showed significant antibacterial activities against Gram-positive bacteria (MIC = 0.04-6.25 mug/mL); however, the activity against Gram-negative bacteria was lower (MIC = 1.56-100 mug/mL). As is evident from the data, oxime derivatives 6e, 6h and 6i are superior in inhibiting the growth of Gram-positive bacteria (MIC = 0.04-0.19 mug/mL), and their activities were found to be 5-25 times better than N-desmethyl levofloxacin 11 and equal or better than levofloxacin 4. Conclusion:We have designed and synthesized novel quinolone derivatives bearing functionalized thienylethyl moiety on the piperazine ring of levofloxacin. The results of antibacterial screening against Gram-positive and Gram-negative bacteria revealed that the introduction of functionalized thienylethyl moiety on the piperazine ring of levofloxacin can improve the activity against Gram-positive bacteria. Gram-positive bacteria are responsible for a wide range of infectious diseases, and rising resistance in this group is causing increasing concern. Thus, this study introduces structural features of levofloxacin scaffold for development of new candidates in the field of anti-Gram positive chemotherapy