Zhang H.-J.,Shenzhen Xinpeng Bio Technology Co.
Chinese Journal of Biologicals | Year: 2012
Objective: To develop a MTS/PMS colorimetric assay for activity of recombinant human interferon β1a (IFNβ1a). Methods: A method for determination of biological activity of IFNβ1a was developed using MTS coupled with PMS as staining solution. The concentration and CPE time of cells, working concentration of MTS as well as time for staining were optimized, based on which dose-effect curves were plotted. The developed method was verified for reproducibility and accuracy, and the results were compared with those of crystal violet staining. Results: The optimal concentration and CPE time of cells were 1 × 105 cells/ml and 24-36 h, while the optimal working concentration of MTS and time for staining were 2 mg/ml and 40 min, respectively. The relationship coefficients (R2) of S-shaped effect curves of IFNβ1a for protection of Wish cells were more than 0.99. The coefficient of variation (CV) of activities of samples added to the wells in the same positions of different plates was 6%-20%, while that to the different wells on the same plate was 13%-17%. The recovery rate of cells for determination of IFNβ1a activity was 86%-121%. The effect curve of the developed method for determination of recombinant human IFNβ1a activity was in a reverse S shape, with a mean R2 value of more than 0.99 which was higher than that by crystal violet staining. In addition, the developed method was more stable than crystal violet staining. Conclusion: A MTS/PMS colorimetric assay was successfully developed, which was suitable for routine determination of activity of recombinant human IFNβ1a.
Chen H.-X.,Shenzhen Xinpeng Bio Technology Co. |
Yu Y.-G.,Shenzhen Xinpeng Bio Technology Co. |
Zhang H.-J.,Shenzhen Xinpeng Bio Technology Co. |
Huang L.-Q.,Shenzhen Xinpeng Bio Technology Co.
Chinese Journal of Biologicals | Year: 2011
Objective: To develop a stable and reliable MTS staining method for determination of bioactivity of recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL). Methods: Three batches of test samples of rhTRAlL were determined by induction of human non-small cell lung cancer NCI-H460 cell apoptosis and MTS staining, and calculated for potency by four-parameter regression method, based on which the specificity, precision and accuracy of the developed method were verified. Results: The potencies of the three batches of test samples, with lot numbers of 20071101, 20071102 and 20071103, were 7. 3 × 104, 8. 1 × 104 and 9. 3 × 10 4 U/ml, while the correlation coefficients (R2) were 0. 998, 0. 996 and 0. 998, respectively. The antibody against rhTRAIL completely blocked the reaction of rhTRAIL with NCI-H460 cells. The method showed high specificity, of which the mean variation coefficients of determination results using various test plates, at various sample loading sites, on various dates and by various personnel were 12%, 26%, 24% and 7% respectively, with a mean recovery rate of 114%. Conclusion: MTS staining showed high accuracy and reliability and was suitable for the routine determination of bioactivity of rhTRAIL.