A critical factor in achieving success with any assay is to ensure that the assay detection instrumentation is compatible with the reagents being used. In addition to overall compatibility with the reader detection mode, assay reagents must also demonstrate scaleability from larger-volume to smaller-volume wells to accommodate higher-throughput processing.
Figure 1 - Principle of the Transcreener ADP2 FP assay, a universal, second-generation far-red fluorescence polarization assay that detects the ADP nucleotide byproduct from any ATP-utilizing enzyme reaction.
This article presents data showing the validation of the Transcreener® ADP2 FP assay (Figure 1) (BellBrook Labs, Madison, WI) on the Synergy 2 Multi-Mode Microplate Reader and Synergy 4 Hybrid Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT). The Transcreener ADP2 assay is a universal, second-generation far-red fluorescence polarization assay that detects the adenosine diphosphate (ADP) nucleotide byproduct from any adenosine triphosphate (ATP)-utilizing enzyme reaction. The assay has been validated in both 96- and 384-well microplate formats on the Synergy 2 Multi-Mode Microplate Reader and Synergy 4 Hybrid Multi-Mode Reader. Data generated with a standard curve in both microplate formats meet BellBrook Labs’ Instrument Validation Program criteria, which include Z′ ≥ 0.7 with Δ mP shift ≥ 95 at 10% conversion of 10 μM ATP to ADP. The 96-well assay met the validation criteria using the standard photomultiplier tube (PMT). Although the 384-well assay performed well and achieved a Z′ ≥ 0.6 using the standard PMT, the validation criteria were met using an optional red-shifted PMT.
Protein kinase A (PKA) was used as an enzyme model to further demonstrate scaleability of the Transcreener Assay on BioTek detection instrumentation. Enzyme titrations, Z′-factor determinations, and inhibitor potency studies with PKA in 96- and 384-well formats are shown. Results indicate that researchers can have confidence in both reagent and reader performance regardless of which well format or detection instrumentation they are using.
Transcreener ADP2 FP assay
The Transcreener ADP detection mixture comprises an ADP-Alexa® 633 tracer bound to an ADP2 antibody. The tracer is displaced by ADP formed from the phosphorylation of substrate. The displaced tracer freely rotates in solution, leading to a large decrease in fluorescence polarization. Therefore, ADP production is proportional to a decrease in polarization. The far-red tracer in this assay minimizes interference from fluorescent compounds and light scattering.
Instrument Validation Program
Figure 2 - Synergy 2 and Synergy 4 meet BellBrook Labs’ Instrument Validation Criteria. The validation criteria include: Z′ ≥ 0.7 at 10% conversion (1 μM ADP) of 10 μM ATP, Δ mP shift ≥ 95 mP at 10% conversion, and read time ≤ 5 min.
Bellbrook Labs has developed an Instrument Validation Program with specific validation criteria for running the Transcreener ADP2 FP assay. These criteria include: Z′ ≥ 0.7 at 10% conversion (1 μM ADP) of 10 μM ATP, Δ mP shift ≥ 95 mP at 10% conversion, and read time ≤ 5 min. Both the Synergy 2 and Synergy 4 were tested against these criteria (Figure 2). The 96-well format assay passed validation under the following conditions: 15 flashes per well, Z′ = 0.74, and Δ mP of 174 in 1:39 min. The 384-well format assay passed validation with the red-shifted PMT under the following conditions: 3 flashes per well, Z′ = 0.81, and Δ mP of 168 in 1:52 min. The 384-well format assay using the standard PMT did not meet the validation criteria of the program, but results yielded acceptable data by many standards: 12 flashes per well, Z′ ≥ 0.6, and Δ mP of 166 in 4:11 min.
Assay validation with protein kinase A
Figure 3 - PKA titrations in 96- and 384-well microplate formats show comparable EC50. PKA was titrated 1:2 and reactions were performed with 10 μM ATP and 50 μM kemptide substrate for 1 hr. The final 100-μL (96-well) and 20-μL (384-well) assay volume with the detection module consisted of 2 nM Alexa 633 tracer, 0.5x Stop and Detect Buffer B, 8.5 μg/mL ADP2 antibody, and 0.5x enzyme reaction mixture (25 mM HEPES pH 7.5, 2 mM MgCl2, 1 mM EGTA, 0.5% DMSO, 0.01% Brij-35, 0.5x ATP, and substrate). EC50 for the 96-well assay was 2.8 mU/rxn. EC50 for the 384-well assay was 1.3 mU/rxn (red-shifted PMT) and 1.2 mU/rxn (standard PMT).
Figure 4 - Z′ values achieved at low % ATP conversion. Z′-factor experiments were run to confirm % conversion achieved in the PKA titration. Enzyme concentrations were chosen as indicated on the graphs, and reactions were performed under the same conditions used for the enzyme titration. Z′-factor was determined by comparing to a no enzyme control on each plate. Enzyme concentrations chosen for subsequent inhibitor titration experiments achieved a Z′ ≥ 0.7, highlighted by the dashed lines. 96-well format (a): 7 mU/rxn achieved a Z′ = 0.8 at 7% conversion; 384-well format, standard PMT (b): 12 mU/rxn achieved Z′ = 0.7 at 16% conversion; 384-well format, red-shifted PMT (c): 6 mU/rxn achieved Z′ = 0.8 at 10% conversion.
Figure 5 - Potency of PKA inhibitors is comparable between well formats. GO-6983, H-89, and staurosporine (known PKA inhibitors) and Tyrphostin AG1478 (a no inhibition control for PKA) were serially titrated into the PKA reaction following the assay conditions noted previously for the Z′-factor confirmation study (all reagents procured from Sigma-Aldrich [St. Louis, MO]).
Validation of the Transcreener assay was performed with PKA as a model system. Assay optimization in the form of enzyme titrations (Figure 3) and determination of Z′ assay performance relating to % conversion of ATP (Figure 4) allowed for the establishment of appropriate assay conditions for pharmacology verification and high-throughput screening in general. Results show that the potency of compound inhibition (nM) of the known inhibitors is all comparable between the different well formats tested (Figure 5). There is also good agreement between the inhibitor results obtained in 384-well format using the standard and red-shifted PMT.
Ms. Worzella, Mr. Larson, and Dr. Kleman-Leyer are with BellBrook Labs, Madison, WI, U.S.A. Mr. Amouretti and Dr. Banks are with BioTek Instruments, Inc., P.O. Box 998, Highland Park, Winooski, VT 05404, U.S.A.; tel.: 888-451-5171; fax: 802-655-7941; e-mail: Applications@BioTek.com.