A Practical Cytopathic Effect/Dye-Uptake Interferon Assay for Routine Use in the Clinical Laboratory

Yousefi, Shookooh; Escobar, Mario R.; Gouldin, Catherine W.

doi:10.1093/ajcp/83.6.735

Cited by 23 publications

(4 citation statements)

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“…A standard biological assay based on reduction of the cytopathic effect (CPE) of vesicular stomatitis virus (VSV) on Vero cells was carried out using serially diluted rHu-IFN-c. The results were compared with those obtained from commercial rHu-IFN-c (Imukin, Boehringer, Germany) [32].…”

Section: Analytical Proceduresmentioning

confidence: 99%

Process development for production of recombinant human interferon-? expressed in Escherichia coli

Khalilzadeh

Shojaosadati

Maghsoudi

et al. 2004

Journal of Industrial Microbiology and Biotechnology

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A simple fed-batch process was carried out using constant and variable specific growth rates for high-cell-density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma(hIFN-gamma). The feeding rate was adjusted to achieve an appropriate specific growth rate. The dissolved oxygen level was maintained at 20-30% of air saturation by control of airflow and stirrer speed and, where necessary, by enrichment of inlet air with pure oxygen. Glucose was the sole source of carbon and energy and was provided by following a simple exponential feeding rate. The final cell density in the fed-batch fermentation with constant and variable specific growth rate feeding strategies was ~100 g dry cell wt l(-1) after 36 and 20 h, respectively. The final specific yield and overall productivity of recombinant hIFN-gamma in the variable specific growth rate strategy were 0.35 g rHu-IFN-gamma g(-1) dry cell wt and 0.9 g rHu-IFN-gamma l(-1) h(-1), respectively. A new chromatographic purification procedure involving anion exchange and cation exchange chromatographies was developed for purification of rHu-IFN-gamma from inclusion bodies. The established purification process is reproducible and the total recovery of rHu-IFN-gamma was ~30% (100 mg rHu-IFN-gamma g(-1) dry cell wt). The purity of the rHu-IFN-gamma was determined using HPLC. Sterility, pyrogenicity, and DNA content tests were conducted to assure the absence of toxic materials and other components of E. coli in the final product. The final purified rHu-IFN-gamma has a specific antiviral activity of ~2 x 10(7) IU/mg protein, as determined by viral cytopathic effect assay. These results certify the product for clinical purposes.

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Section: Analytical Proceduresmentioning

confidence: 99%

Process development for production of recombinant human interferon-? expressed in Escherichia coli

Khalilzadeh

Shojaosadati

Maghsoudi

et al. 2004

Journal of Industrial Microbiology and Biotechnology

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“…This, in turn, may explain the marked difference in sensitivity seen in the two methods: The dye-uptake method has a sensitivity for interferon determination of 1 unit/ml, in contrast to the MTT-method which can easily detect below 0.1 units/ml. Another reason for the success of the MTT-method is undoubtedly that contrary to the neutral red dyeuptake method (27) or other methods ( 1 1, 12,20,24,26,27) no washing steps are introduced; these often lead to unpredictable loss of cells which, in turn, will influence the final readings of the ODs.…”

Section: Discussionmentioning

confidence: 99%

A new sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT‐method)

Berg

Hansen

Nielsen

1990

APMIS

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A biological method for precise quantification of interferons has been developed. The method is based upon the dehydrogenase system of the intact target cells, which will normally convert an artificial substrate, MTT, into formazan (blue), which, in turn, can be measured spectrophotometrically. This conversion is greatly reduced by cytocidal viruses in a dose‐dependent manner. The protection of target cells by interferon against challenge virus is reflected in a diminished reduction in the production of formazan, thus giving a very precise method for quantification of interferon. The lowest level of detection is around 0.10 international units. The intra‐ and inter‐assay variability appear to be below 10%. The assay, which makes no use of expensive ingredients, is performed in 96‐well micro‐trays and read in an inexpensive ELISA‐scanner.

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“…The standard biological assay of purified samples was based on the reduction of the cytopathic effect of vesicular stomatitis virus on Vero cell lines by serially diluted rhIFN‐γ samples, and the results obtained were then compared with those obtained with commercial rhIFN‐γ (Imukin®; Boehringer Ingelheim) [21]. Purification of samples was performed as described by Khalilzadeh et al [20].…”

Section: Methodsmentioning

confidence: 99%

A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli

Babaeipour

Shojaosadati

Khalilzadeh

et al. 2008

Biotech and App Biochem

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Different feeding strategies for the production of human interferon-gamma using an isopropyl beta-D-thiogalactoside-inducible expression system in recombinant Escherichia coli BL21(DE3) (plasmid pET3a-ifngamma) were studied. Four fed-batch modes were designed to compare the effect of mu (specific growth rate) on recombinant-protein production, substrate consumption, by-product formation and plasmid stability during pre- and post-chemical induction in high-cell-density cultures of E. coli. It was found that Y(p/s), the product/substrate yield of interferon-gamma was significantly affected by mu throughout the process, but product/biomass yield (Y(p/x)) was influenced by mu at the pre-induction stage. By applying an efficient feeding strategy, in which the mu was maintained at the maximum attainable level, recombinant protein was accumulated up to a level of 60% of the total cell protein and its productivity was increased significantly. In this case, the overall productivities of biomass and recombinant protein were 6.36 g l(-1) h(-1) and 2.1 g l(-1) h(-1) respectively, in comparison with 1.91 g l(-1) h(-1) and 0.16 g l(-1) h(-1) during exponential feeding, in which the specific growth rate was kept constant throughout the entire process.

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A Practical Cytopathic Effect/Dye-Uptake Interferon Assay for Routine Use in the Clinical Laboratory

Cited by 23 publications

References 0 publications

Process development for production of recombinant human interferon-? expressed in Escherichia coli

Process development for production of recombinant human interferon-? expressed in Escherichia coli

A new sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT‐method)

A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli

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