AD0094P-1 (en) 1 DELFIA® Eu-labeled bombesin AD0227 - 150 pmol AD0228 - 600 pmol For Research Use Only INTENDED USE This DELFIA Eu-labeled bombesin is intended for use in ligand receptor binding assays. INTRODUCTION Bombesin is a 14 amino acid long peptide, isolated from the skin of the amphibian frog Bombina bombina. The mammalian homologues to bombesin consist of various forms of gastrin-releasing peptide and neuromedin B. So far, four subtypes of bombesin receptors have been cloned; BB1, BB2, BB3 and BB4. Upon receptor ligand binding, the receptor transmit extracellular signals across membranes by activating specific signal-transducing heterotrimeric G-proteins, which in turn regulate a variety of intracellular effectors. The mammalian bombesin-like peptides contribute to diverse biological functions in the central nervous system and peripheral tissues, which include e.g. thermoregulation, satiety, smooth muscle contraction, stimulation of pancreatic secretion and gastrointestinal hormone release. DELFIA europium (Eu)-labeled bombesin is a synthetic peptide, similar to amphibian bombesin with a europium chelate coupled to the amino end of the peptide. The sequence of DELFIA Eu-labeled bombesin is Ac-Eu3+-Ahx-PQRLGNQWAVGHLM-NH2. DELFIA Eulabeled bombesin binds with high affinity to mammalian BB1 and BB2 receptors and is a useful tool to study the function of these receptors. PRODUCT USE The DELFIA Eu-labeled bombesin binding assay is based on dissociation-enhanced timeresolved fluorescence. DELFIA Eu-labeled bombesin and the ligand specific receptor are incubated on an AcroWell1 filter plate (when using e.g. membrane preparations or whole cells), on a coated plate (e.g. soluble receptors, membrane preparations or whole cells) or on a cell culture plate (e.g. adherent cells), after which unbound labeled ligand is removed. Eu is dissociated from the bound ligand by using DELFIA Enhancement Solution. Dissociated Eu creates highly fluorescent complexes, which are measured in a multilabel counter with TRF option. During the development phase, the biological activity of the DELFIA Eu-labeled bombesin has been demonstrated on AcroWell filtration based assays. DELFIA is a registered trademark of PerkinElmer, Inc. 1 AcroWell is a trademark of Pall Corporation. AD0094P-1 (en) 2 CONTENTS Figures in parentheses refer to the 600 pmol package. Component Quantity Storage DELFIA Eu-labeled bombesin 150 pmol (600 pmol) 1 vial, lyophilized +2 - +8°C The lyophilized DELFIA Eu-labeled bombesin contains Tris-HCl buffered salt solution, bovine serum albumin (BSA) and Dextran T-40. NOTE: The powder contains sodium azide (< 1 %) as preservative and it is harmful by inhalation, in contact with skin and if swallowed. PREPARATION OF REAGENT Reagent Storage and reconstituted stability DELFIA Eu-labeled bombesin Keep the vial on ice. Stable for 5 days at +2 - +8°C. For longer periods, aliquot and store at -20°C. Avoid freezing and thawing. Stable for at least one month at -20°C. Reconstitute the lyophilized DELFIA Eu-labeled bombesin by adding 150 µL (600 µL) of distilled water to yield a DELFIA Eu-labeled bombesin concentration of 1000 nmol/L and mix gently. Allow to stand for at least 30 minutes on ice before use to ensure that all solid material is dissolved. NOTE: The powder contains sodium azide (< 1 %) as preservative and it is harmful by inhalation, in contact with skin and if swallowed. The dissolved ligand contains < 0.1 % sodium azide and is not considered harmful. MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE REAGENT The DELFIA Eu-labeled bombesin filtration based assay requires the following items, which are available from PerkinElmer Life and Analytical Sciences or its distributors. 1. Time-resolved fluorometer, e.g. 2100 EnVision™ , 1420 VICTOR™ or Fusion™ Multilabel Reader 2. Automatic shaker - DELFIA Plateshake (prod. no. 1296-003/004) EnVision, VICTOR and Fusion are trademarks of PerkinElmer, Inc. AD0094P-1 (en) 3 3. Pipette for dispensing the DELFIA Enhancement Solution - Eppendorf Multipette (prod. no. 1296-014) with 5 mL Combitips (prod. no. 1296-016) or alternatively the DELFIA Plate Dispense (prod. no. 1296-041) 4. DELFIA L*R binding buffer concentrate (10x) (prod. no. CR134-250) 5. DELFIA L*R wash concentrate (25x) (prod. no. CR135-250) 6. DELFIA Enhancement Solution (prod. no. 1244-104 for 50 mL; 1244-105 for 250 mL and 4001-0010 for 1000 mL) 7. AcroWell Filter Plate (prod. no. P5020) 8. Membrane preparation from bombesin receptor expressing cell line, e.g. human BB1 (prod. no. RBHBS1M) or BB2 (prod. no. RBHBS2M) In addition to the DELFIA system the following are required: - Unlabeled bombesin (e.g. Bachem, prod. no. H-2155) Saponin (e.g. Calbiochem, prod. no. 558255) Distilled water Filterplate washing manifold: Multiscreen Vacuum Manifold, Millipore Precision pipettes for dispensing microliter volumes Pipettes for dispensing milliliter volumes Glass or polypropylene tubes PROCEDURAL NOTES 1. The binding buffer composition in ligand receptor assays is usually receptor specific. To achieve the best results in a DELFIA assay, the buffer should contain BSA to avoid non-specific binding, and low concentrations of a chelator, e.g. EDTA (ethylenediaminetetraacetic acid), to keep the fluorescence background low. The recommended binding buffer (see section "EXAMPLE OF FILTRATION BASED ASSAY CONDITIONS") is optimized to be used with human BB2 receptor in DELFIA assay. 2. Some receptor preparations may require an addition of a protease inhibitor, e.g. phenylmethylsulfonyl fluoride (PMSF), in the binding buffer. 3. When filtering the plate, ensure that each well is washed thoroughly, e.g. four times with 300 µL of recommended wash solution (see section "EXAMPLE OF FILTRATION BASED ASSAY CONDITIONS"). After washing the plate, check that the wells are dry. Remove any remaining moisture by blotting the plate on absorbent paper. 4. When using the Millipore manifolder/vacuum unit, the AcroWell Filter Plate may not fit tightly on the manifolder/vacuum frame. The plate will fit better on the frame if the metal grid is removed from the top of the frame. The black rubber part should, however, be left on the frame in order to prevent problems with vacuum leakage. 5. For optimal results, flush the pipette tips or the dispenser tips and tubing thoroughly with DELFIA Enhancement Solution. We recommend using plastic vials instead of glass vials. AD0094P-1 (en) 4 EXAMPLE OF FILTRATION BASED ASSAY CONDITIONS Binding buffer: DELFIA L*R binding buffer concentrate (10x), diluted 1:10 containing the following reagents as final concentrations: 50 mmol/L Tris-HCl, pH 7.5 5 mmol/L MgCl2 25 µmol/L EDTA 0.2 % BSA supplemented with 50 µg/mL saponin Wash solution: DELFIA L*R wash concentrate (25x), diluted 1:25 containing the following reagents as final concentrations: 50 mmol/L Tris-HCl, pH 7.5 5 mmol/L MgCl2 Binding protocol: Binding assays are performed in 100 µL total volume according to the following conditions. All the dilutions are prepared in binding buffer. 1. Pipetting: 25 µL binding buffer (total binding) OR unlabeled ligand (non-specific binding) 25 µL Eu-labeled ligand 50 µL diluted receptor 2. Incubation: shake the plate slowly for 15 seconds, then incubate the plate for 90 minutes at room temperature 3. Washing: four times with 300 µL of wash solution 4. Eu enhancement: 200 µL DELFIA Enhancement Solution/well, incubate for 15 minutes at room temperature with slow shaking 5. Measurement: time-resolved fluorometer with Eu-filter CALCULATION OF RESULTS S/B = Z' = 1 - Total values Non-specific values 3 x SD total + 3 x SD non-specific Mean signal total - Mean signal non-specific SD = standard deviation Kd and Ki values are calculated using GraphPad Prism® 2 software. 2 GraphPad Prism is a registered trademark of GraphPad Software Inc. AD0094P-1 (en) 5 RESULTS Saturation curve Figure 1 shows typical data for measuring the saturation binding. The saturation experiment was performed with increasing amounts of DELFIA Eu-labeled bombesin in the presence of 0.2 µg of human BB2 receptor (Bmax 9.3 pmol/mg protein) per well. Nonspecific binding was determined in the presence of 500 nmol/L unlabeled bombesin. A typical Kd value for DELFIA Eu-labeled bombesin is around 0.5 nmol/L. See Product Information Sheet for lot specific Kd value. Counts 20000 Total binding 10000 Specific binding Non-specific binding 0 0 1 2 3 4 5 DELFIA Eu-labeled bombesin (nmol/L) Figure 1. A Kd value of 0.47 nmol/L was obtained using the human BB2 receptor. The assay was performed as described in "EXAMPLE OF FILTRATION BASED ASSAY CONDITIONS". The fluorescence was measured with VICTOR2. The values represent the mean ±SD from triplicate wells. In addition, a Kd value of 0.56 nmol/L was obtained using 0.4 µg of human BB1 receptor (Bmax 3.4 pmol/mg protein); data not shown. Competition curve The competition between the DELFIA Eu-labeled bombesin and unlabeled bombesin is shown in Figure 2. The displacement curve was performed with 0.5 nmol/L of DELFIA Eulabeled bombesin and increasing amounts of unlabeled bombesin in the presence of 0.2 µg of human BB2 receptor (Bmax 9.3 pmol/mg protein) per well. A typical Ki value is around 0.2 nmol/L. AD0094P-1 (en) 6 % of maximum signal 150 100 50 0 -7.5 -5.0 -2.5 0.0 2.5 5.0 Log unlabeled bombesin (nmol/L) Figure 2. A Ki value of 0.19 nmol/L was obtained using the human BB2 receptor. The assay was performed as described in "EXAMPLE OF FILTRATION BASED ASSAY CONDITIONS". The fluorescence was measured with VICTOR2. The values represent the mean ±SD from triplicate wells. In addition, a Ki value of 0.24 nmol/L was obtained using 0.4 µg of human BB1 receptor (Bmax 3.4 pmol/mg protein) in the presence of 0.5 nmol/L DELFIA Eu-labeled bombesin (data not shown). S/B ratio and Z' value Typical readings, S/B ratios and Z' values using VICTOR2 and EnVision (n = 12) are shown in Table 1. S/B and Z' were calculated using a concentration of DELFIA Eu-labeled bombesin close to the Kd value. Decreasing the number of flashes in EnVision shortens the measurement time at the expense of counts. AD0094P-1 (en) 7 Table 1. S/B and Z' were calculated using a concentration of 0.5 nmol/L DELFIA Eulabeled bombesin. VICTOR EnVision EnVision EnVision 100 50 10 1 min 7 sec 55 sec 45 sec 8530 6466 3202 623 7.9 7.1 6.3 6.3 Non-specific binding 1103 760 369 73 CV% 12.8 17.3 17.7 21.4 8 9 9 8 0.67 0.69 0.72 0.70 Flash Time / plate Total binding CV% S/B Z' WARNINGS AND PRECAUTIONS DELFIA Eu-labeled bombesin is intended for research use only. Lyophilized DELFIA Eu-labeled bombesin contains sodium azide (NaN3) as a preservative. The powder is harmful by inhalation, in contact with skin and if swallowed. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide build-up. To avoid Eu-contamination that can result in a high fluorescence background in assays, high standard pipetting and washing techniques are required. Avoid contaminating pipettes with Eu-labeled reagents. Disposal of all waste should be in accordance with local regulations. WARRANTY Purchase of the product gives the purchaser the right to use this material in his own research, development, and investigational work. The product is not to be injected into humans or used for diagnostic procedures. PerkinElmer Life and Analytical Sciences, Wallac Oy reserves the right to discontinue or refuse orders to any customer who plans to use these products for any other purposes. PerkinElmer Life and Analytical Sciences, Wallac Oy does not warrant or guarantee that the product is merchantable or satisfactory for any particular purpose, nor free from any claim of foreign or domestic patent infringement by a third party, and there are no warranties, expressed or implied, to such effect. PerkinElmer Life and Analytical Sciences, Wallac Oy will not be liable for any incidental, consequential or contingent damages involving their use including damages to the property or personal injuries. AD0094P-1 (en) 8 All information supplied with the product and technical assistance given is believed to be accurate, but it remains the responsibility of the investigator to confirm all technical aspects of the application. We appreciate receiving any additions, corrections, or updates to information supplied to the customer. AD0094P-1 (en) 9 LITERATURE Zhang, J.H., Chung, T.D.Y. and Oldenburg, K.R. (1999): A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol Screen 4, 67-73. DELFIA® Ligands Guide 006783 To download pdf file: www.perkinelmer.com/delfia June 2003 Manufactured by: PerkinElmer Life and Analytical Sciences, Wallac Oy P.O. Box 10 FIN-20101 Turku FINLAND