July 1, 2008 (Vol. 28, No. 13)

Specific Bioassays Are Designed for TNFα Biological Activity Studies

Tumor necrosis factor alpha (TNFα) is a potent cytokine involved in immune surveillance and function. Dysregulated TNFα levels have been implicated in chronic diseases such as rheumatoid arthritis, Crohn’s disease, and several neurological diseases.

A number of recombinant anti-TNFα biologicals have been developed and licensed for management of human disease. Simple and predictive bioassays for potency and dose standardization, though, are still sought by the biopharma industry. Automatable, homogeneous bioassays are desirable as they can help streamline this aevaluative process. Implementing predictive automation-friendly bioassays as demonstrated in this tutorial allows effective assessment of anti-TNFα and likely other biologicals for quality control.

Figure 1

Predictability and Productivity

Two predictive bioassays from Promega (www.promega.com) are highlighted in this article. The CellTiter-Glo® Luminescent Cell Viability Assay provides a homogeneous bioluminescent assay for quantitation of ATP, an indicator of metabolically active cells, present inside cells in culture. The assay is designed for use in multiwell plate formats, making it ideal for automated cell viability, cell proliferation, and cytotoxicity studies.

The ONE-Glo™ Luciferase Assay provides a homogeneous bioluminescent assay for quantitation of luciferase expression in mammalian cells containing genes for firefly luciferase. The assay contains a new luciferase substrate, resulting in a reagent that is more stable, more tolerant to sample components, and has less odor than standard luciferase assay reagents, which makes it suitable for high-throughput applications.

Both assays are rapid, simple, and sensitive. The protocols involve adding the reagent directly to cells cultured in growth medium (without washing or preconditioning), mixing, and reading the glowing luminescent signal on a luminometer. For the CellTiter-Glo Assay, the luminescent signal is proportional to the amount of ATP present in the sample, which is directly proportional to the number of cells present. For the ONE-Glo Assay, luminescence is proportional to the upregulation of a specific genetic element and subsequent expression of firefly luciferase.

Figure 2a

TNFα-Mediated Activation of NF-kB

One of the most important downstream signaling targets activated by TNFα is the NF-kB transcription factor, which is involved in diverse biological processes including inflammatory, antiapoptotic, and immune responses. In resting cells, the activity of NF-kB is controlled through its cytoplasmic sequestration by inhibitor of kappa B (IkB). Binding of TNFα to its cell surface receptors initiates a series of kinase cascades, which lead to phosphorylation of IkB, ubiquitination, and proteasome-dependent degradation. The released NF-kB then undergoes nuclear translocation and activation, which induces the transcriptional expression of target genes (Figure 1).

Luciferase reporter assays have been used widely to investigate cellular signaling pathways. In this study, the GloResponse NF-kB-RE-luc2P HEK293 cell line from Promega was used. This line, a clonal derivative of HEK293 cells, contains a luciferase gene (luc2P) under the control of a minimal TATA promoter with multiple Nuclear Factor-kB Response Elements (NF-kB-REs). This NF-kB reporter cell line is designed for rapid analysis of any cellular response that results in modulation of NF-kB activities.

To evaluate TNFα-mediated activation of NF-kB, GloResponse NF-kB-RE-luc2P HEK293 cells were robotically dispensed using Eppendorf’s epMotion 5075 robotic liquid-handling workstation at a density of 10,000 cells per well into white-bottom, white-opaque 96-well Costar #3917 microplates from Corning in 50 µL volumes of DMEM supplemented with 10% fetal bovine serum (FBS).

Threefold serial dilution treatments of TNFα in 50 µL media per well were robotically dispensed across the plate. Treated cells were allowed to incubate for five hours at 37ºC, 5% CO2. Controls were included on each plate: one column of wells containing untreated cells and one cell-free column.

To evaluate anti-TNFα-mediated neutralization of NF-kB, GloResponse NF-kB-RE-luc2P HEK293 cells were dispensed as described previously using Tecan’s Freedom EVO robotic workstation and treated immediately with 10 ng/mL of TNFα and increasing concentrations of antihuman TNFα mAb from R&D Systems. Threefold serial dilutions of the anti-TNFα mAb plus TNFα in 50 µL media per well were robotically dispensed across the plate. Treated cells were allowed to incubate for five hours at 37ºC, 5% CO2. Controls were also included on the plate.

Following treatment, 100 µL of the ONE-Glo reagent was robotically added to each well of all plates and contents were mixed on a rotating platform for 10 minutes. Luminescence associated with luciferase gene expression due to modulation of NF-kB activities was measured using Promega’s GloMax-Multi Plate Reader.

Z´-factor analysis, a statistical measure of reproducibility and robustness, was also performed for each assay. A concentration of 10 ng/mL TNFα or 10 ng/mL TNFα plus 1 µg/mL anti-TNFα in 50 µL media per well was used for induction in part of the plate, whereas the other portion of the plate was left untreated. Cells were robotically prepared and treated similarly to the previously described methods.

Dose-response curve results using the ONE-Glo Assay are shown in Figure 2 for TNFα- and anti-TNFα-treated GloResponse NF-kB-RE-luc2P HEK293 cells dispensed robotically in 96-well plates. The EC50 value, or half maximal effective concentration of TNFα induction, obtained for suspension cell conditions with TNFα-treatment alone was 1.1 ng/mL.

The ND50 value, or half neutralization dose, obtained for suspension-cell conditions for TNFα neutralization was 78 ng/mL. Z´-factor values for both automated treatment conditions were >0.71, indicating excellent assay quality. In addition, both treatment conditions showed a great dynamic response.

Figure 2b

Anti-TNFα Potency Determination

In addition to initiating and augmenting transcriptional activation events, TNFα can promote programmed cell death via the extrinsic apoptosis pathway. Engagement of TNF receptors by the TNFα protein ligand initiates procaspase-8 processing into active caspase-8, whereby active caspase-8 activates procaspase-3 (and -7) to produce catalytically active caspase-3/7. Ultimately, the proteolytic action of caspase-3/7 on various structural elements within a cell leads to apoptosis (Figure 1).

The biological activity of TNFα, or potency of anti-TNFα agents in the presence of a standardized concentration of TNFα, can be measured by determining the number of viable cells remaining after treatment. CellTiter-Glo is well suited for this application.

The mouse cell line L-929 from ATCC was selected due to its historical sensitivity to TNFα. L-929 cells were seeded into clear-bottom, white-opaque 96-well Costar #3903 microplates in 50 µL volumes of DMEM supplemented with 10% FBS. Cells were allowed to adhere for four hours in a sterile incubator at 37ºC, 5% CO2.

TNFα was diluted to 1 ng/mL in culture medium supplemented with actinomycin D from Sigma-Aldrich, an antineoplastic antibiotic. Anti-TNF mAb was diluted to 5 µg/mL in the actinomycin D-containing culture medium, and twofold serial dilutions were created.

The TNFα and antibody dilutions were combined and preincubated for 30 minutes at 37°C, 5% CO2. Fifty microliters of each dilution was delivered to the assay plate containing cells, mixed briefly using an orbital shaker, and incubated overnight at 37°C, 5% CO2. A parallel plate was prepared for Z´-factor analysis using 5 µg/mL anti-TNFα with 1 ng/mL TNFα and medium, with TNFα serving as the cytotoxicity control.

After the overnight treatment, 100 µL CellTiter-Glo reagent per well was added to each plate using the Freedom EVO robotic workstation, and contents were mixed briefly on an orbital shaker. Luminescence associated with cell viability was measured using Tecan’s Safire2 Microplate Reader.

Dose-response curve results are shown in Figure 3 for TNFα- and anti-TNFα-treated L929 cells using the CellTiter-Glo Assay dispensed robotically in 96-well plates.

The ND50 value obtained with adherent cell conditions for TNFα neutralization was 45 ng/mL. The Z´-factor value for this automated assay was >0.76, once again indicating excellent assay quality.

The homogeneity and simplicity of both bioluminescent assays presented in this tutorial demonstrates that they are well-suited for various applications in assessing cell viability and luciferase expression, including when used on automated platforms (all Z´>0.71).

The biological potency of TNFα and anti-TNFα agents can be easily and reproducibly determined using the combination of the GloResponse NF-kB-RE-luc2P HEK293 cell line and the ONE-Glo Luciferase Assay or an established cell model for cytotoxicity combined with the CellTiter-Glo Assay.

Both assays deliver functional measures of TNFα bioactivity, which should prove useful for potency characterization in research and biomanufacturing environments.

Figure 3

Sarah Shultz ([email protected]) is a senior automation scientist in the integrated solutions and engineering department, Andrew Niles and Jey Cheng, Ph.D., are senior R&D scientists in the cellular analysis department, and Simon T. M. Allard, Ph.D., is a product associate in the cellular analysis and HTS/pharma marketing department at Promega. Web: www.promega.com.

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