AlphaScreen TNFα Binding Assay Kit: A Homogeneous, Sensitive and High-Throughput Assay for Screening TNFα Receptors Bouchard N., Legault M. and Wenham D. PerkinElmer BioSignal, 1744 William, suite 3, Montréal, Québec, Canada, H3J 1R4 e-mail: dean.wenham@perkinelmer.com
1 Abstract Tumor necrosis factor alpha (TNFα) is a cytokine involved in a wide variety of inflammatory and immunological processes. Downstream biological effects of TNFα are produced following its binding to specific cell surface receptors. Two distinct forms of TNF receptors have been identified: TNF receptor 1 (p55 or TNFR1) and TNF receptor 2 (p75 or TNFR2). The extracellular domain of TNFR1 is proteolytically cleaved to produce a soluble fragment (stnfr1) detectable in serum and urine samples. The stnfr1 acts as a physiological inhibitor of TNF analogs when it scavenges these molecules and prevents their interactions with membrane-bound receptors. Here we describe how a bead based non-radioactive, homogeneous Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen ) has been developed for the screening of TNFα receptors. The AlphaScreen TNFα /stnfr1 assay kit is intended for the screening of molecules that can displace the TNFα -stnfr1 interaction and the assay employs a system where stnfr1 is captured by anti-stnfr1 Acceptor beads. The stnfr1 in turn binds to a biotinylated derivative of TNFα captured by the Streptavidin Donor beads to generate the AlphaScreen signal. Molecules binding to either TNFα or the stnfr1, at either site of interaction, will compete for binding and therefore result in a signal decrease. The AlphaScreen TNFα /stnfr1 assay kit has been optimized in order to allow assays to be performed homogeneously in 384 well plates with a total of four additions and a total of 1 hr 45 min incubation time. Pharmacological characterization of the system demonstrated a profile consistent with that described in other assays where TNFα and TNFα exhibited Ki=.1 and 4.1 nm respectively. The assay yielded a signal to background (S/B) of 2 and this as well as the affinity of TNFα were relatively unaffected by DMSO up to a concentration of 1%. Precision analysis performed in the presence of 1% DMSO revealed a highly robust assay were the difference in S/B generated between nm (bound) and 1 nm TNFα (displaced) was 15.3 with a Z of.71. In conclusion, the attributes of the AlphaScreen TNFα Binding Assay kit suggest that it is the leading technology commercially available for the HTS application of TNFα binding and further, the data from this study open up the opportunity of developing additional cytokine based AlphaScreen assays. 2
2 TNFα Binding Assay Principle AlphaScreen is a bead based nonradioactive Amplified Luminescent Proximity Homogenous Assay. When biological interactions bring the Donor and Acceptor beads into close proximity, reactive oxygen generated by irradiation of the Donor beads, initiates a luminescence / fluorescence cascade in the Acceptor bead. This process leads to a greatly amplified signal with light output in the 52-62 nm range. When the Acceptor and Donor beads are not in close proximity, the reactive oxygen decays and only a very low background signal is generated. The AlphaScreen TNFα /stnfr1 assay kit is intended for the screening of molecules that can displace the ligand-receptor interaction. stnfr1, captured by anti-stnfr1 Acceptor bead, binds to biotinylated TNFα derivative which is captured by the Streptavidin donor beads to generate the AlphaScreen signal. Molecules binding to either TNFα or the stnfr1 will compete for binding and therefore result in a signal decrease. 3 Methods AlphaScreen TNFα assay kit (Cat# 676622) is available in three sizes (5, 1 and 5 assay points) and is composed of biotinylated- TNFα, anti-stnfr1 Acceptor beads, Streptavidin Donor beads and control buffer. The soluble TNFR1 is not provided in the kit. Competition assay Components were added into a 384-well white opaque plate (Corning cat# 375) as follows: 5 µl of competitor (.1 µm 1 pm final) (TNFα, PeproTech cat# 3-1A) 5 µl of stnfr1 (.3 nm final) (R&D Systems cat# 636-R1) 1 µl of anti-stnfr1 Acceptor beads (2 µg/ml final) / biotin- TNFα mix (.12 nm final) (incubate in the dark 45 minutes at 23ºC) 5 µl of Streptavidin Donor beads (2 µg/ml final) (incubate in the dark 6 minutes at 23ºC and read on AlphaQuest TM or Fusionα TM readers) Assay Buffer: 25 mm HEPES ph7.4, 25 mm NaCl and.3% Tween-2 DMSO tolerance Screening conditions were mimicked by diluting competitor (.1 µm 1 pm final) in assay buffer supplemented with DMSO (,.5, 1, 2 and 5% final) Assay performance Evaluation of the Z values were performed using the following modifications to the competition assay described above: Maximum signal is obtained with 5 µl of buffer supplemented with DMSO (1% final) Maximal displacement is obtained with 5 µl of TNFα (3 and 1 nm final) diluted in buffer supplemented with DMSO (1% final) Assay specificity Competition assays were performed with TNFα, TNFα (PeproTech cat# 3-1B) and EGF (PeproTech cat# 1-15) as competitors 3
4 Assay Specificity 25 AlphaScreen Signal (cps) 2 15 1 5 TNFα TNFβ EGF - -12-11 -1-9 -8-7 -6 Log [ligand (M)] The pharmacology of the binding event was characterized using TNFα, TNFβ and EGF. IC 5 values obtained for TNFα and TNFβ were.27 nm and 4.52 nm respectively. These values are in agreement with data obtained by other groups using different detection technologies. Concentrations of EGF up to.1 µm had no effect on the system. The assay was performed on 3 separate occasions, representative data are shown. 5 DMSO Tolerance 2 [DMSO] AlphaScreen Signal (cps) 15 1 5 %.5% 1% 2% 5% - -13-12 -11-1 -9-8 -7-6 [DMSO] (%) IC 5 (nm) S/B Log [TNFα (M)].5 1 2 5.35.49.52.59 3.23 19.2 18.1 15.6 12.7 6.4 The tolerance of the assay to DMSO was tested in competition assays. DMSO concentration dependently decreased the signal. At 1% DMSO, the signal and pharmacology where both acceptable and thus suitable for screening. 4
6 Assay Performance 2 Total TNFα at 3 nm TNFα at 1 nm AlphaScreen Signal (cps) 15 1 5 12 24 36 48 6 72 84 96 # well TNFα (nm) (Total) Mean Signal (cps) 149 Standard Deviation (cps) 1166 CV (%) Z 8.3 3 nm (IC7) 465 25 5.4.55 1 nm (NS) 92 96 1.4.71 The Z (in the presence of 1% DMSO) was estimated to determine the screening suitability of an assay. A total of 96 wells for each conditions (total binding, IC 7 displacement and maximum displacement) were performed on 2 separate plates. 7 Conclusions A homogeneous and non-radioactive assay to screen compounds acting specifically to inhibit the TNFα /stnfr1 interaction has been developed. This simple assay can be performed with only 4 successive reagent additions and completed within 2 hours. Data from pharmacological characterization is in agreement with data obtained by other groups using radioactive technologies. Different sources of receptor can be used. Using the recommended assay protocol, Z >.5 in the presence of 1% DMSO can be achieved indicative of a performance that is suitable for HTS applications. Data from this study open up the opportunity of developing additional cytokinebased AlphaScreen assays. 5
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