Preparation of Tritium Labeled Compounds and Their Scintillation Proximity Assays

Transcription

1 Preparation of Tritium Labeled Compounds and Their Scintillation Proximity Assays Michael P. O'Malley*, Yui S. Tang, and Nadine M. Gan PerkinElmer Health Sciences, Inc. * Main author: Mike.O Malley@Perkinelmer.com PerkinElmer

2 Contents A: Introduction : Tritium labeled ligands and Scintillation Proximity Assay and Microplate assays for receptor bindings B: Methods for preparing tritium labeled ligands C: Scintillation Proximity Assay results D: Conclusion 2

3 The Uses of Tritium Labeled Ligands Convenience : half life years Easily detectable: weak beta-emitter, LSC counting High specific activity obtained: from Ci/mmol Large catalog : thousands of radio-ligands available from PKI [ 3 H]Ligands can be left chemically unaltered, and thus, biologically indistinguishable from the unlabeled compound. 3

4 Ligand Receptor Bindings Receptor binding assays The assay can be in a wide variety of formats. The receptors can be on whole cells, pieces of membrane or purified. The ligands are typically bound to a marker that can be visualized or quantified such as dyes, and fluorescent or radioactive labels. Diagnostic uses Analyze breast tumors for estrogen receptors because estrogen stimulates tumor growth. Other examples: Insulin in Diabetes; Dopamine for depression. In drug development These assays can be modified to look at which cells have particular receptors, the affinity of new drugs for receptors or ways to block receptors. 4

5 Uses for Tritiated Compounds for SPA Receptor-ligands Binding Radio-ligand binding assays measure the interactions between specifically ligand molecules and the protein receptors. Tritium is ideally suited for Scintillation Proximity Assay, Filtration LSC, and Microplate because its β-particle has an extremely short path length through aqueous buffers of only 1.5 μm. Can be applied to SPA beads, Flashplates and Cytostar-T plates. The non-specific binding obtained from unbound tritium molecules is low. 5

6 Using Tritium Labeled radio-ligands Radio-ligands are easy to use and readily available from PerkinElmer. Useful in two common assay formats: scintillation proximity assay and heterogeneous assays such as filtration. Low non-specific binding. Reproducible 6

7 What is SPA (Scintillation Proximity Assay)? Homogeneous and versatile technology for the rapid and sensitive assay of a wide range of biological processes. Uses include enzyme assays, RIA, and protein molecular interaction assays. 7

8 The Scope of SPA SPAs using either SPA scintillation beads or SPA imaging beads can be read on scintillation counters and CCD imagers respectively. SPA reagents are suitable for the detection of a range of isotopes including 3 H, 125 I, 33 P and 14 C, when used as radiolabels. PerkinElmer SPA assays and Microplate techniques offer convenient cost-effective and safer technology for a range of applications 8

9 Receptor Binding Assays: First method- SPA Advantages: No separation required Reduced handling steps Multiple bead types 9

10 Bead Method Step 1: Radio-ligand + unlabeled ligand or sample + receptor are added and incubated. Step 2: Unbound radio-ligand is not detected. Radio-ligand bound to target is detected. 10

11 Receptor Binding Assays: Second method- Filtration Advantage: Liquid Scintillation Counter is the only instrument needed Disadvantage: Quenched by colored compounds 11

12 Filtration Method- LSC Counting PKI s DELFIA TRF Assays 12

13 Standard Curve is Created to Compare Competitive Bindings Standard curve of [ 3 H]Kainic acid and structurally similar Domoic acid 13

14 State-of-the-arts instrument :MicroBeta 2 MicroBeta 2 is ideal for assays using 3H, 14C, 32P, 33P, 35S, 51Cr, 125I and glow-type luminescence labels as well as for ScintiPlate, FlashPlate and other Scintillation Proximity Assays. It counts filters and tubes in addition to microtitration plates 14

15 ViewLux ultrahts Microplate Imager The ViewLux ultrahts Microplate Imager is an ultra high throughput microplate imager for high sensitivity and fast measurement of light from fluorescence polarization (FP), fluorescence intensity, timeresolved fluorescence (TRF), luminescence and absorbance assays. 15

16 Part B: Tritium Labeled Ligands: Preparation Three common methods for preparing tritium labeled ligands Catalytic exchange with tritium gas (no need for precursor general labels) Radio-chemistry (precursor is necessary, specific labels) Radioactive reagents (most suitable for macro-molecules) 16

17 17 [ 3 H]N,N,N Triacetylchitroise NET1202

18 [ 3 H]-Atorvastatin NET

19 [G- 3 H]-Biotin NET

20 [6,7,8-3 H]-Biotin NET721 20

21 Ubiquitin, Propionylated [N-propionyl-(2,3-3 H)]- NET

22 Glutathione-S-Transferase, Propionylated [N-propionyl-(2,3-3 H)]- NET

23 PART 3: Scintillation Proximity Assay Goal: Develop quantitative binding assay for analyte of interest Select most appropriate bead type Determine binding capacity of bead by saturation assay Determine and minimize non-specific binding (signal/ background) by adjusting bead and labeled ligand concentration Perform competitive assay using labeled ligand, unlabeled ligand, receptor binder and/or SPA bead. Quantitate analyte in sample of interest. 23

24 Popular SPA Bead Types Available Through PerkinElmer Wheatgerm Agglutin beads (WGA)- binds to n-acety glucosamine residues on glycoproteins and glycolipids Streptavidin beads- binds to biotinylated proteins, peptides and oligonucleotides Glutamine coated beads- binds to GST fusion proteins either directly labeled or through indirect radiolabel binding partner Copper or Nickel His-Tag Beads- Bind to 6X-Histidine fusion proteins 24

25 Saturation Assay and Determination of Non Specific Binding This assay example uses [ 3 H] N,N,N Triacetylchitotriose NET1202 tracer and Wheatgerm Agglutinin (WGA) coated SPA beads. (Specific Activity= 40 uci/ug) WGA is a lectin which binds to sugar residues on the surface of cells. WGA SPA beads can be used to investigate receptor-ligand interactions, using either partially purified cell membrane preparations or glycosylated, solubilized receptors. 25

26 Assay Results and Interpretation.-Streptavidin Coated Beads Saturation Binding Assay: Use [ 3 H]Biotin (NET1215) and Streptavidin Coated SPA Beads to determine binding capacity and amount of beads required for biotinylated analyte competitive binding assay. 26

27 Glutathione Binding SPA Beads- Saturation Assay Bead enables the trapping and quantification of GST fusion proteins Ability to trap and quantify cloned tagged proteins is desirable in drug screening market. 27

28 Competitive Binding Assay Using Tritium Labeled Ubiquitin In the cell, ubiquitination marks a target protein for proteolytic degradation. Using ATP and a specific ubiquitin ligase, [ 3 H]ubiquitin NET1228 and unlabeled ubiquitin are competitively bound to a GST fusion protein of interest,and then captured using the Glutathione SPA bead. Calibration Standard Curve allows quantitation of ubiquitin in the sample. 28

29 Conclusions PerkinElmer SPA beads and their associated [ 3 H] ligands can be used to develop RIAs, receptor binding assays, enzyme mediated and molecular interaction assays. Perkinelmer s SPA, together with the Microplate techniques, including Cytostar-T Scintillating Microplates and UniFilter greatly facilitate receptor binding assays studies and are well suited to high throughput screening assays. 29

30 Acknowledgements PKI Life Sciences & Technology Linda Lacy, Linda Meehan, Anthony Catalano PKI LST Research Reagents Kathryn Sobey, Barbara Killian, Pat Mayer 30

31 Acknowledgements Visit PerkinElmer at LSC Barcelona, March PerkinElmer continues to be committed to future of radiometric detection innovation and technology. Stop by our booth at LSC 2013 and learn about new cocktails and see our latest in instruments and software. 31