F A R R E D. Technical Manual. Transcreener UGT Far Red Assay

Transcription

1 F A R R E D Technical Manual Transcreener UGT Far Red Assay

2 Transcreener UGT Far Red Assay Kit Instructions for Part Number K 1.0 Introduction p Assay Principle p Instrumentation p Transcreener UGT Far Red Assay Kit Components p Additional Materials Required p Sample Protocol p Antibody Titration p UDP Detection Considerations p Standard Curves p User Notification p Introduction UDP-glycosyltransferases (UGTs) transfer a glycosyl group from an UDP-glycosyl donor substrate to a xenobiotic or endogenous acceptor substrate (carbohydrate, lipid, protein, or small molecule) generating a conjugated acceptor-substrate and uridine-diphosphate (UDP). The Transcreener UGT Far Red Assay Kit is a competitive fluorescence polarization immunoassay based on the detection of UDP. Enzyme reaction progress is indicated by a decrease in the fluorescence polarization. The Transcreener UGT Far Red Assay Kit is a simple two-step, endpoint assay (Fig. 1) that enables the interrogation of multiple UGT reactions with a single set of reagents. The assay provides an excellent signal under initial velocity conditions resulting in an overall Z > 0.6. This assay has been tested with concentrations of μm UDP- Glucuronic acid. Reduced development costs and accelerated drug discovery are achieved by utilizing this one simple streamlined screening method. Figure 1. Transcreener UGT Far Red Assay Overview Enzyme Reaction Add enzyme reaction to test compounds in assay plate. 15 µl reaction volume. UDP Detection Add 5 µl Transcreener UDP Detection Mix, incubate 90 minutes and measure fluorescence polarization. 2.0 Assay Principle The Transcreener UGT Far Red Assay detects UDP by competitive binding of UDP and UDP DyLight 632 Tracer with the UDP antibody. As UDP is enzymatically produced less tracer is bound to the UDP antibody leading to a reduction in fluorescence polarization (Figure 2). Reaction progress results in a proportional decrease in fluorescence polarization. p

3 Figure 2. Transcreener UGT Far Red Assay Principle 3.0 Instrumentation A microplate reader configured to read fluorescence polarization of DyLight 632 fluorophor is required. (Use standared Cy5 dye instrument configuration. Maximum excitation 637 nm and maximum emission 657 nm for DyLight 632 fluorophor.) During the development of this assay kit the following instruments were extensively used and validated: Tecan Ultra and Tecan Safire 2. Other instruments previously demonstrated effective for measuring far red fluorescence polarization include: BioTek s Synergy 2, BMG Labtech s PHERAstar and POLARstar, Molecular Devices Analyst (AD, HT, and GT), Perkin Elmer s EnVision, ViewLux, and Victor 3V and Tecan s F500 and GENios Pro. While BellBrook Labs continues to gather customer feedback regarding instrument set-up and assay performance it may be necessary to consult the manufacturer s instructions or technical service representative for instrument set-up to measure fluorescence polarization. 4.0 Transcreener UGT Far Red Assay Components Part Number K Kit Component Volume Storage Temp 2035 UDP DyLight 632 Tracer, 400 nm 100 μl -80 C 2036 UDP Antibody lot specific -20 C 2037* 500 mm Na 3 1 ml -20 C mm UDP 250 μl -20 C 2039* 500 mm EGTA 2 ml -20 C mm KPO 4, ph μl -20 C *The shaded reagents, EGTA and Na 3, are included specifically for non-purified preparations such as Human Liver Microsomes (HLMs) to stabilize UDP. 5.0 Additional Materials Required 5.1 Enzyme Reaction Mix The enzyme reaction components supplied by the end-user include enzyme, reaction buffer, UDP-glycosyl substrate, acceptor substrate and test compounds. p.3

4 5.2 Assay Plate Recommended plates are Corning 384 Plates, part number 3676 (black, round bottom, low volume, polystyrene, non-binding surface microplates). Note: It is important to use completely black plates with low to medium binding. 5.3 Liquid Handling Devices The user will need liquid handling devices capable of accurately dispensing volumes of 2.5 to 10 μl into 384-well plates. 6.0 Sample Protocol The Transcreener UGT Far Red Assay was designed as a simple two-step endpoint assay that consists of an enzyme reaction (15 μl) followed by the addition of the UDP detection Mix (5 μl) for a final volume of 20 μl. 6.1 Perform Enzyme Reaction Add enzyme reaction mix to test compounds. Start the reaction by adding an UDPglycosyl substrate, bringing total volume to 15 μl. Incubate at temperature and time ideal for enzyme target. 6.2 Detect UDP Add 5 μl of the UDP detection mixture containing the optimal UDP antibody concentration obtained from an antibody titration (section 7.0). Incubate at room temperature (20-25 C) for 90 minutes and measure fluorescence polarization. 7.0 Determine Antibody Concentration To maximize the assay window and sensitivity, an antibody titration is required in the buffer system ideal for your enzyme target. 7.1 Prepare UDP Antibody Titration Prepare your enzyme reaction mixture (include UDP-sugar, 2nM UDP DyLight 632 Tracer, exclude acceptor substrate) with and without UDP antibody. Dispense 30 μl of mixture with an appropriate starting concentration of antibody into wells in column 1. (A common starting [Ab] is 2 mg/ml.) Dispense 15 μl of the mixture without antibody across a 384-well plate (columns 2-24). Remove 15 μl from column 1 and serially titrate the contents across the plate (to column 24). Equilibrate at room temperature for at least 90 minutes, and measure fluorescence polarization. 7.2 Determine the EC 85 The concentration of antibody that is equal to the EC 85 value is recommended because it provides an excellent balance between maximum assay window and sensitivity. Fit data using a sigmoidal dose (variable slope) response algorithm. The equation for determining the EC 85 value is shown below (using the EC 50 and hillslope from the curve fit). EC 85 = ((85/(100-85))1/hillslope)* EC UDP Detection Considerations The Transcreener UGT Far Red Assay was optimized for initial velocity conditions (<20% conversion) with 20 mm KPO 4,, ph 7.5, and 5 mm MgCl 2 in the enzyme reaction (include 80 mm EGTA with insect cell and human liver microsomal enzyme p

5 preparations) using 100 μm UDP-glycosyl substrate. The Transcreener UGT Far Red Assay is optimized for the initial velocity region. If intended use is outside the initial velocity region please contact BellBrook Labs for reformatting recommendations. 8.1 UDP Antibody The amount of antibody in the final assay should be optimized in the target enzyme reaction conditions (section 7.0). 8.2 UDP DyLight 632 Tracer, 400nM The UDP DyLight 632 Tracer is provided at a concentration of 400 nm in 2 mm HEPES, ph 7.5 containing 0.01% Brij-35. This kit was optimized with 2nM UDP DyLight 632 Tracer in the final 20 μl reaction. 8.3 UDP Detection Mixture When using the UDP Detection Mix, dilute the antibody to 4x its optimal concentration in 10 mm KPO 4, ph 7.4 for the 5 μl Detection Mix. For impure enzyme reactions, like HLMs, include Na 3 to a final concentration of 25 mm in the final 20 μl reaction to stabilize the signal molecule, UDP. 8.4 UDP Detection Controls These controls are used to calibrate the fluorescence polarization plate reader No Antibody (free tracer) Control: This is the reference sample for the tracer and is set to 20 mp by G-factor calibration. Include everything in the enzyme reaction except UDP-Antibody No Tracer Control: This control is the reference blank for the No Antibody Control and the sample blank for all other wells. Include everything in the enzyme reaction except the UDP DyLight 632 Tracer. 8.5 Equilibration After addition of UDP Detection Mixture equilibrate at room temperature for 90 minutes prior to fluorescence polarization measurement. This signal is stable for 4.5 hours after equilibration. 8.6 UDP Stability Determination of UDP stability is recommended in the presence of your enzyme source. EGTA and Na 3 have been included with this kit to stabilize UDP in the presence of impure enzyme sources like HLMs (80 mm EGTA during an HLM reaction and 25 mm Na 3 during detection). A mock enzyme reaction, like one of the standard curve points from section 8, read over an extended time interval, can be used to determine UDP stability in the presence of your enzyme source. 8.7 UDP Glycosyl Substrate It is important to use as pure a UDP-glycosyl substrate as available to maximize the assay window. Contaminating UDP will effect results. p.5

6 9.0 UDP Standard Curve When creating a Standard Curve, the total nucleotide concentration should remain constant. The standard curve below mimics a UGT reaction (as UDP is produced, UDPsugar is depleted--for this example the depleted UDP-sugar is UDP-Glucuronic acid). 9.1 Prepare Stock Solutions Prepare UDP and UDP-glycosyl substrate stock solution in the target enzyme reaction buffer. 9.2 Prepare Standard Curve Add 15 μl of each standard to the wells of a 384 well plate. An example of a twelve point UDP/UDPGA standard curve mimicking a UGT reaction with 100 μm substrate follows. Table 1 12 Point Standard Curve % Substrate Conversion [UDP] µm [UDPGA] µm Prepare Detection Mixture Prepare 4X UDP Detection Mixture (5 μl/well) with an antibody concentration that is 4X the EC 85 concentration determined in Section 7.0. NOTE: After adding 5 μl of the 4X Detection Mixture to the enzyme reaction (15 μl/well), the concentration of the antibody will be at the EC 85 concentration in the final 20 μl reaction. 9.4 Measure FP Incubate for 90 minutes at room temperature, and measure fluorescence polarization. 9.5 Plot Standard Curve Below is sample data (Figures 3 & 4) of a standard curve with initial substrate (UDPGA) concentrations of 100 μm in the 15 μl enzyme reaction. These standard curves are designed to mimic the 2-step, end point assay procedure in which 5 µl of 4X Detection Mix is added to a 15 μl enzyme reaction. The nucleotide concentrations indicated for each point on the standard curve reflect those in the enzyme reaction not those at time of FP measurement (Table1). p

7 Figure μmudp/udpga Standard Curve of mp data Figure 3 plots the mp data from 24 replicates per percent conversion. To include zero in log scale format, 0.04 was used as the 0% conversion point. The assay was performed in 20 mm KPO 4 ph 7.5, 5 mm MgCl 2, 2 nm UDP DyLight 632 Tracer, and 62.5 μg/ml UDP Antibody. Figure μm UDP/UDPGA Standard Curve mp Data Figure 4 plots mp data obtained from Figure 3. mp = mp zero [UDP] - mp [UDP] μm Z =1- (3*((StdDev zero [UDP] +StdDev [UDP] μm )/(Abs(mP zero [UDP] - mp [UDP] μm ) ))) p.7

8 10.0 User Notification U.S. and foreign patents applied. The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. BellBrook Labs LLC will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use, or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, BellBrook Labs LLC is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, BellBrook Labs LLC, 5500 Nobel Drive, Suite 250, Madison, WI Fax Transcreener is a trademark of BellBrook Labs. DyLight 632 is a trademark of Thermal Fisher Scientific Inc. and its subsidiaries. Corning is a registered trademark of Corning Incorporated. Transcreener HTS Assay Platform technology is patent pending BellBrook Labs. All rights reserved. p