Comparison of potency assays using different read-out systems and their suitability for quality control.
ABSTRACT In this study, three of the most commonly used non-radioactive read-out systems for bioassays, the tetrazolium salt MTS/PMS, the fluorescent dye Alamar Blue and the ATP bioluminescence assay were compared regarding their suitability for quality control purposes. In this regard, three different potency assays using murine CTLL-2, as well as human DiFi and Kit 225 cells were performed. No major differences regarding accuracy and precision were detected between the different read-out systems. Both workload and hands-on time were similar for all three assays used. All read-out systems were suitable for use in quality control. However, luminescence and fluorescence techniques were much more sensitive than the colorimetric system. The first two could determine approximately ten times lower drug concentrations, and the assay could be performed by using considerably lower cell numbers. Moreover, in two of the three potency assays, the luminescence and fluorescence read-out systems provided higher signal to noise ratios leading to a higher precision of the assays. Regarding the comparison of the luminescence and fluorescence system, the ATP assay has the advantage to be much faster than the Alamar Blue assay. Consequently, in this study, the luminescence technique turned out to be the most advantageous of the three read-out systems.
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ABSTRACT: A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose-response curves in the concentration range of 0.02-3.3 micrograms/ml. With intra-assay variability of 5-12%, interassay variability of 6-10% and spike recoveries of 101-109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability. The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb.Journal of Immunological Methods 04/1997; 202(2):163-71. · 2.01 Impact Factor
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ABSTRACT: Accurate assessment of the anti-growth effects of chemotherapeutics is immensely importance in cancer research with regard to drug discovery and toxicological safety. A number of in vitro cytotoxicity assays are used for these purposes. However, there is the possibility for different results in the assessments because the way they measure the viability of cancer cells is specific to each assay. In the present study, the performance of two common assays (MTT and ATP) in the assessment of anti-growth effects of chemotherapeutics on a lung cancer cell line (A549) was compared. The cells were treated with paclitaxel, docetaxel, gemcitabine, 5-fluorouracil (5-FU), etoposide, doxorubicin, epirubicin, cisplatin, 4-hydroperoxycyclophosphamide (4-HC) and carboplatin in six different concentrations. When taking all the drugs and inhibitions into account, a moderate correlation (r=0.670; p=0.01) between the assays was found. However, IC 50 values by the MTT assay were higher in 90% of the drugs than those found by the ATP assay. In addition to this, there was a statistically significant difference between the dose response curves of the assays, which was dependent on the drugs of choice. We recommend caution in comparing these assays to evaluate the anti-growth effects of chemotherapeutics because the MTT assay seem to give rise to relatively lower inhibition (higher viability) levels than the ATP assay, depending on the drugs of choice.Toxicology in Vitro 03/2008; 22(1):232-9. · 3.21 Impact Factor
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ABSTRACT: A one-step non-radioactive assay to determine the proliferation of murine lymphocytes, lymphoid tumor cells and hybridoma cells is described. This assay requires the addition of Alamar Blue dye to cell cultures and the degree of change in its color, which is reflective of the extent of cellular proliferation, can be determined by an ELISA plate reader. Alamar Blue must be added during the initial phase of cell culture. The pattern of concanavalin A (ConA) or anti-CD3 antibody-induced proliferative response of murine lymphocytes as assessed by Alamar Blue was similar to that of a [3H]thymidine assay. Similarly, the spontaneous proliferation curve of anti-CD3 antibody secreting cell line (YCD3-1), monocytic macrophage cell lines (PU5-1.8, P388D1, J774.1) and myeloma cells (Sp2/0) as determined by Alamar Blue closely resembled that of the [3H]thymidine assay. The minimum detectable number of proliferating cells was comparable in Alamar Blue and [3H]thymidine assays. Since cell lysis/extraction and washing procedures are not involved in the Alamar Blue assay, this approach has several distinct advantages over currently available assays (eg. [3H]thymidine). First, it allows daily monitoring of proliferation without compromising the sterility of cultures. An indication of proliferation can be evaluated (spectrophotometrically or visually) as early as 24 h after ConA stimulation. Second, unlike previously reported assays, Alamar Blue permits further analysis of proliferating cells by other methods. Analysis of cells in culture with Alamar Blue for various surface antigens (CD44, CD45RB, CD4, heat stable antigen) by flow cytometry revealed that the fluorescent profile and relative percentage of cells in cultures with the Alamar Blue were comparable to those without this reagent. The salient advantages of Alamar Blue assay over the [3H]thymidine assay include: (i) non-radioactivity; (ii) simplicity; (iii) less costly; (iv) non-labor intensive; (v) rapidity of assessment of proliferation of large number of samples; (vi) non-toxicity; (vii) usefulness in determining the kinetics of cell growth of hybridomas; and (viii) non-interference of secretion of antibodies by a hybridoma cell line.Journal of Immunological Methods 05/1994; 170(2):211-24. · 2.01 Impact Factor