AlphaLISA Assays are Homogeneous Sensitive Immunoassays for Detection of Analytes in a Variety of Biological Matrices Gregory Cosentino, Zaava Ravid, Jean-Francois Michaud, Marie-Claude Loiselle, Julie Bédard, Philippe Bourgeois, Alexandre Marcil, Josée Frappier, Claire Normand, Véronique Brechler & Francesco Lipari 2 Materials and Methods Analyte detection AlphaLISA kits and buffers are all available from PerkinElmer. All calibration curves were done using analyte and reagents supplied in the kits, and using optimal protocols available in the Technical Data Sheets. Biological samples or fluids All biological samples or fluids were supplied as pools of individuals (for assay development), or separate samples from 10 to 20 individuals, non-medicated, nonimmunized, from Bioreclamation LLC: • Mouse bronchial lavage fluid (BALF): Strain CD-1, cat# MSE-BRONLEV • Rat bronchial lavage fluid (BALF): Strain Sprague Dawley, cat# RAT-BROLAV • Human CSF: cat# HMCSF • Mouse lung homogenates: Strain CD-1, cat# MSE-LUNG-HOMOG Diluents tested AlphaLISA Immunoassay Buffer: PerkinElmer, cat# AL000C AlphaLISA HiBlock Buffer: PerkinElmer, cat# AL004C AlphaLISA NaCl Buffer: PerkinElmer, cat# AL007C Fluids tested as diluents were supplied as pools of individuals, non-medicated, nonimmunized, from Bioreclamation LLC: • Beagle bronchial lavage fluid (BALF): cat# BGL-BROLAV Biological samples or fluids pre-treatment and storage Prior to being used in AlphaLISA assays: • BALF was centrifuged at 650xg for 15 min at 4˚C to remove cells and cell debris, and supernatants were used in assays. • CSF was not pre-treated. • Mouse lung homogenates were generated by rinsing the entire lung (right and left) with PBS 1X, by homogenizing each entire lung on ice in 2 mL PBS 1X (containing protease inhibitors) using a polytron, and then centrifuging 10 min at 1,500xg at 4°C (to remove insoluble debris). Supernatants were used in assays. Storage: BALF and CSF fluids were kept at -80˚C. Mouse lung homogenates were kept at -20˚C. Active caspase-3 cellular assay Jurkat cells (ATCC TIB-152™) were grown in RPMI medium supplemented with 10% FBS. 2.5 µL of staurosporine (LC labs, Cat# S-9300) prepared in serum-free medium at a 2X concentration was disposed into a CulturPlate®-384. 15,000 cells/well were seeded in a volume of 2.5 µL in serum-free medium and the plate was incubated at 37˚C, 5% CO2 for 2-4 hours. 5 µL of 3X Lysis Buffer (1.5X final) supplemented with protease inhibitors were added to each well and the plate was gently agitated on a plate shaker for 10 minutes at room temperature. Then an AlphaLISA assay was performed by adding 5 µL of a 10X MIX (freshly prepared) AlphaLISA Anti-Caspase-3 Acceptor beads (10 µg/mL final) and Biotinylated Antibody Anti-Caspase-3 (1 nM final), and by incubating for 60 minutes at 23˚C. 35 µL of 1.43X Streptavidin Donor beads (40 µg/mL final) were added and plates were incubated 30 minutes at 23˚C in the dark. Reading was performed using an EnVision Multilabel Plate Reader with Alpha HTS option. 6 Alpha Technology Assay Principle 8 Active Caspase-3 Assay Precision A. Intra-assay precision. B. Inter-assay precision The intra-assay variability was evaluated by performing a Z’ factor determination. Inter-assay precision was determined using a total of two independent determinations with 48 measurements for each control sample. 15,000 cells + 30 μM staurosporine Assay Z’ CV max S/B 15,000 cells + 30 µM staurosporine CV min 1 0.69 16 9.3% 8.3% 2 0.71 17 8.8% 6.3% Counts Max SD Max 22842 CV Max Counts Min SD Min CV Min 1395 103 7.4% 2057 9.0% Mouse VEGF A in Lung Homogenates Step 1: Linearity of dilutions in PBS-1%BSA for non-spiked or spiked mouse lung homogenates (10 ng/mL mVEGF A) Concentration (ng/mL) The AlphaLISA® assay is a homogeneous immunoassay alternative to classical ELISA. AlphaLISA assays were originally utilized to detect analytes in cell culture supernatants or serum/plasma samples. More recently, AlphaLISA has been applied to a wider variety of biological matrices including lysates from cultured cells, or fluids and tissue homogenates from animals. We report the development of an assay to measure active caspase-3 in cell lysates from both suspension and adherent cell models (Jurkat and HeLa, respectively). For compound screening, cells are treated with test material and subsequently lysis buffer is added. After a short incubation, targetspecific AlphaLISA reagents are directly added, providing a highly efficient all-in-one-well assay format. Under optimal conditions, signal to background (S/B) values up to 17 and Z’ values up to 0.8 were obtained with staurosporine-treated Jurkat cells. Assays were also developed for biological samples derived from rodents or humans. Quantitation of analytes in animal tissue extracts or biological fluids requires an appropriate diluent so that samples can be accurately extrapolated from the calibration curve. Following optimization of the assay, recovery of spiked analytes was generally in the range of 70 to 130%. As examples, mouse interleukin 6 was measured in bronchioalveolar lavage fluid (BALF) at a level of 20 pg/mL, human amyloid beta 1-42 peptide was measured in cerebrospinal fluid (CSF) at a level of 0.3 ng/mL, and mouse vascular endothelial growth factor (VEGF) was detected in lung homogenates at a level of 1 ng/mL. In general, a major advantage of using AlphaLISA for analysis of biological samples is that sample volumes as low as 2.5 µL can be utilized. Also, the absence of wash-steps greatly reduces the assay time and improves the reproducibility of the data. 3 Spike 10 ng/mL From neat 12.0 10.0 8.0 r2 neat = 0.9130 8.0 6.0 4.0 2.0 0.0 0.0 1.0 0.5 Spike 10 ng/mL From 1/2 dilution r2 dil 1/2 = 0.9913 The biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads resulting in a sharp peak of light emission at 615 nm. 4 15,000 cells + 1 µM staurosporine Assay Expanding Applications Cat. no. Analyte Species AL254 AL255 AL271 AL274 AL275 AL276 AL278 AL288 AL504 AL505 AL509 AL516 AL517 AL518 AL520 sAPPα sAPPß Tau Aß 1-15 / 16 Aß 1-40 Aß 1-42 Caspase-3 active Amyloid ß 1-x IL6 TNFα MCP-1 CCL5/RANTES GM-CSF IL1-ß VEGF A Human Human Human Human Human Human Human Human Mouse Mouse Mouse Mouse Mouse Rat Mouse BALF Cell Lysate CSF Lung tissue √ √ √ √ √ √ CV max 1 0.55 8 12.0% 8.3% 2 0.46 9 15.3% 6.3% Counts SD Max Max 11607 Z’ S/B CV max CV min 1 0.69 9 8.2% 7.4% 2 0.76 8 6.3% 7.0% CV Max Counts SD Max Max 4.0 0.6 0.4 2.0 0.3 0.2 0.0 0.00 0.0 0.0 0.0 0.00 5407 573 √ √ 17 kDa 30,000 Jurkat cells MCF-7 cells HiBlock buffer 5.0 r2 neat = 0.9906 r2 neat = 0.9905 Recovery from neat (%) 1395 103 7.4% 1 2 4 8 16 32 100 161 190 203 203 212 Counts Min SD Min CV Min 10.6% 636 56 8.8% PBS + 0.1%BSA PBS - 0.01%BSA r2 neat = 0.9993 r2 neat = 0.9998 r2 neat = 0.9977 2.0 1.0 1.0 0.0 0.5 1.0 0.0 Dilution Dilution 0.5 0.0 1.0 0.5 1.0 Dilution Dilution 0.0 0.5 1.0 Dilution AlphaLISA buffer HiBlock buffer PBS-0.1%BSA PBS-0.01%BSA Beagle BALF Dilution factor (DF) Recovery from neat (%) Recovery from neat (%) Recovery from neat (%) Recovery from neat (%) Recovery from neat (%) 1 2 4 8 16 32 100 119 128 137 139 149 100 113 135 150 146 145 100 95 93 90 92 94 100 97 96 96 95 99 100 92 85 82 83 83 Diluents not chosen: Linearity not good from neat 1,000 Choose 2 good diluents (PBS-0.1%BSA and Beagle BALF): • Good linearity from neat • Observed concentration X DF close to spiked value (3 ng/mL) for all dilutions Step 3: Chosen diluent showing the best spike and recovery results -9 -8 -7 0.25 0.50 Dilution Dilution PBS 1%BSA (no spike) Recovery from 1/2 dil. (%) Recovery from neat (%) Recovery from 1/2 dil. (%) 100 118 126 126 132 100 148 176 188 183 170 100 119 127 124 115 PBS-1%BSA is a good diluent: - Good linearity starting at dilution 1/2 (so, 1/2 dilution required) - Observed X DF close to spiked value (10 ng/mL) starting at dilution 1/2 Spike (ng/mL) No spike 1 3 10 Diluent: PBS-1%BSA Spiked diluent Spiked 1/2 mouse lung control homogenates Concentration Concentration Recovery (%)** (ng/mL)* (ng/mL)* 0.00 0.63 NA 0.94 0.76 81 2.77 2.23 81 9.47 7.01 74 Excellent recovery for all 3 spikes tested in 1/2 mouse lung homogenates. Measured VEGF A physiological level in mouse lung homogenate (No spike) = 0.63 ng/mL X 2 = 1.26 ng/mL * Concentration for 1 to 10 ng/mL spikes = measured concentration – no spike value ** Recovery (%) = recovery compared to spiked diluent control Step 4: Test biological samples from individuals Analyte VEGF A (mouse) Biological sample Mouse Lung Homogenates LDL (pg/mL) Number of individuals tested 1 Average concentration Range (AlphaLISA) 0.96 ng/mL 0.5 - 1.3 ng/mL 240 pg/mg 110 - 350 pg/mg ptn 10 Expected range (literature) 350 - 400 pg/mg ptn* * Mori H et al. Am J Physiol Lung Cell Mol Physiol. 2008. Feb;294(2):L196-204. Step 2: 3,000 -∞ -10 1.0 Step 2: Beagle BALF 3.0 0.5 0.5 Step 3: Spike and recovery results 4.0 0.0 0.0 0.50 Dilution Dilution factor (DF) Mouse TNFα in BALF AlphaLISA buffer 0.25 CV Min CV Max r2 dil 1/2 = 0.9895 0.6 SD Min 2,500 cells + 30 µM staurosporine 0.8 r2 neat = 0.9391 0.9 Counts Min 1812 15.6% 1.2 PBS 1%BSA (spiked 10 ng/mL) Step 1: Linearity of dilutions in 5 diluents for spiked mouse BALF (3 ng/mL mTNFα) √ √ √ √ √ √ √ √ Assay 7 Active Caspase-3 Cellular Assay 10,000 S/B CV min 2,500 cells + 30 µM staurosporine Data can be found in the corresponding Technical Data Sheets for these kits (website), except for AL504, AL505, and AL509, which were presented in a poster at the Cytokines 2010 conference, held in Chicago on October 03-07, 2010 (Michaud et al., 2010). 5 Z’ 15,000 cells + 1 µM staurosporine No spike From 1/2 dilution No spike From neat 6.0 Dilution Concentration (ng/mL) Abstract AlphaLISA Signal (counts) 1 -6 -5 Diluent: PBS-0.1%BSA -4 Spiked diluent control Log [STS] M Dose-response curve of caspase-3 activation by increasing concentrations of staurosporine in Jurkat and MCF-7 cells. A Western blot for active caspase-3 (p17 subunit), which uses a rabbit monoclonal antibody against amino acids 171-175 of human caspase-3, is illustrated above the dose-response curve. MCF-7 cells were included in the test as a negative control, since they do not express caspase-3. The AlphaLISA results correlate well with the relative amounts of cleaved caspase-3 detected by Western blot. Spiked neat Mouse BALF Spike (pg/mL) Concentration (pg/mL)* Concentration (pg/mL)* Recovery (%)** No spike 10 30 300 3000 0.0 12.0 35.2 300.2 3141.2 17.1 9.2 32.3 292.6 2952.1 NA 76 92 97 94 Excellent linearity of dilutions from neat (Step 1) and excellent recovery for all 4 spikes tested. Measured TNFα physiological level in mouse BALF (No spike) = 17.1 pg/mL * Concentration for 10 to 3000 pg/mL spikes = measured concentration – no spike value ** Recovery (%) = recovery compared to spiked diluent control 9 Conclusion AlphaLISA technology has been applied to a wide variety of biological matrices from different species. Different types of analytes such as small peptides, cytokines, or proteases can be detected in cell lysate, BALF, CSF, or lung homogenate. A caspase-3 assay was validated for both HeLa and Jurkat cells by showing comparable results to Western blotting. The assay was shown to be appropriate for HTS since good Z’ values and inter-assay precision can be achieved. Quantitative assays for detecting mouse TNFα in BALF or mouse VEGFA in lung homogenate were optimized by choosing the correct matrix for the calibration curve and shown to detect endogenous level of cytokine. These assays are fast and easy no-wash assays. Moreover, they are extremely sensitive, as we obtained good recoveries from 10 pg/mL spikes. Since these assays can use volumes as low as 2.5 µL, the AlphaLISA technology offers the possibility to accurately detect physiological levels of analytes even if very low volume of fluid is collected per animal. Next step: evaluate sample to sample variation from different mouse/rat individuals. PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com