AlphaScreen : A Straightforward and Powerful Alternative to ELISA Martina Bielefeld-Sévigny Ph.D., R&D Director
Overview AlphaScreen - an alternative to ELISA Why an alternative to ELISA? Assay principle Conjugation Essay examples Non-serum / plasma samples NEW : Insulin detection in serum / plasma 1 O 2 A B Page 2
AlphaScreen - an alternative to ELISA Why an alternative to ELISA? Assay principle Conjugation Essay examples Non-serum / plasma NEW : Insulin detection in serum / plasma 1 O 2 A B Page 3
ELISA Alternatives PerkinElmer Assay Platforms AlphaScreen LANCE DELFIA Detection Luminescence Proximity TR-FRET TRF Homogenous Yes Yes No Throughput High Ultra-High Medium Ease to automate *** *** * Relative Sensitivity ***** *** ***** Dynamic range 2.5-5 logs 2-3 logs 2.5-5 logs Micro-plate formats 96, 384, 1536 96, 384, 1536 96, 384 Multiplexing No No up to 4-plex Substrate sizes Small molecules to viruses Small molecules to peptides Small molecules to proteins Use of low affinity antibodies Yes Yes (lower signal) No Use of polyclonal antibodies Yes No Yes Recommended Instrumentation EnVision EnVision, Victor, ViewLux EnVision, Victor, ViewLux Advanced Luminescent Proximity Homogenous Assay Lanthanide Chelate Excitation Dissociation-Enhanced nhanced Lanthanide Fluorescent Immuo Assay Page 4
Analyte Detection and Quantification DELFIA DELFIA LANCE Eu 3+ Eu 3+ LANCE Page 5
Steps: ELISA AlphaScreen ELISA* AlphaScreen** Add assay buffer, matrix solution, standard (or sample) ; Add antibody detection solution Add assay buffer, matrix solution, standard (or sample) ; Add biotin antibody and EasyLISA acceptor beads Incubate 1 hour on orbital shaker Incubate 30 60 minutes (RT) Remove solution Add donor beads wash wash wash Incubate 30 60 minutes (RT) Add enzyme Read Incubate 30 minutes on orbital shaker $ wash wash wash wash wash Remove solution Add substrate Incubate 30 minutes on orbital shaker Read * insulin ELISA kit $$$ ** insulin test using EasyLISA acceptor beads Page 6
AlphaScreen - an alternative to ELISA Why an alternative to ELISA? Assay principle Conjugation Essay examples Non-serum / plasma NEW : Insulin detection in serum / plasma 1 O 2 A B Page 7
AlphaScreen principle Excitation 680 nm 1 O 2 Emission 520-620 nm AlphaScreen Donor Bead S B A B AlphaScreen Acceptor Bead Page 8
AlphaScreen beads Donor beads Acceptor beads 1 O 2 N N N S 1 g Ο 2 S O O S O O O O O N N N Si N N OSi 340-350 nm energy transfer SiO N N N 450-500 nm 680 nm O 1 2 g O 2 Donor: Phthalocyanine Signal Amplification Rubrene emission : 540-680 nm Detection: 520-620 nm EasyLisa emission: 605-620 620 nm Page 9
AlphaScreen Immunoassays Sandwich Assay Analyte 0 0 250 500 7501000 Biotinylated antibody Antibody coupled to bead Competition Assay Streptavidin bead Biotinylated Analyte Antibody coupled to bead -12-11 -10-9 -8-7 Competing analyte Page 10
AlphaScreen - an alternative to ELISA Why an alternative to ELISA? Assay principle Conjugation Assay Examples Non-serum / plasma NEW : Insulin detection in serum / plasma 1 O 2 A B Page 11
AlphaScreen conjugation: simple and easy Reductive amination: O H 2 0 H H H 2 N R N R (unstable) Easy & clean Reproducible Mild conditions NaBH 3 CN CH 2 NH R (stable) conjugation 1.25 µl 10% Tween-20 50 µg Protein (ex. IgG) 50 µl Aldehyde Acceptor beads (20 mg/ml) 10 µl NaHCNBH 4 0.3 M complete to 200 µl with 0.1 M MES ph 6.0 (add 20 µl 0.5 M MES for [protein] < 2mg/ml) Incubate 48 hour at 37C blocking purification Add 10 µl blocking solution Incubate 1 hour at 37C Centrifugation or TFF. Page 12
AlphaScreen biotinylation: simple and easy N-hydroxysuccinimide (NHS)-ester labeling is the simplest method for labeling proteins and antibodies. NHS ester react efficiently with w primary amino groups (-NH2)( to form stable amide bonds. NH2 + biotin-peg PEG-NHS ph>7 PEG-biotin + N-Hydroxysuccinimide Conjugation: -100 µg Antibody -20 µg biotin-peg-nhs (biotin-polyethyleneglycol- N- Hydroxysuccinimide) -complete to 100 µl with PBS Incubate 2 hour at 37C Ready to use Page 13
AlphaScreen - an alternative to ELISA Why an alternative to ELISA? Assay principle Conjugation Assay examples Non-serum / plasma NEW : Insulin detection in serum / plasma 1 O 2 A B Page 14
AlphaScreen Sandwich Assay: General Steps 1 + + Biotin-anti-analyte antibody Anti-analyte coupled beads Analyte = Immune complex 2 incubate 3 + = Streptavidin Donor 4 Read Page 15
Straightforward ELISA Conversion...from large to small antigens large molecule: ex. human IgG AlphaScreen signal (cps) 125000 100000 75000 50000 25000 S:B=3 S:B>120 higg migg rigg gigg anti-higg (λ/κ specific) anti-higg (polyclonal) 0 - -12-11 -10-9 -8 Log [IgG](M) high sensitivity: 1 pm = 0.15 ng/ml biotinylated anti-igg commercially available Page 16
Straightforward ELISA Conversion... medium size molecule: ex. TNFα TNFα detection assay 5000000 AlphaScreen signal (cps) 4000000 3000000 2000000 1000000 anti-tnfα (polyclonal) anti-tnfα (monoclonal) 0-16 -15-14 -13-12 -11-10 -9-8 -7-6 log [TNFa] M high sensitivity: 0,3 pm = 6 pg/ml biotinylated anti-tnf TNFα commercially available Page 17
Straightforward ELISA Conversion... 300000 AlphaScreen Signal (CPS) 250000 200000 150000 100000 50000 HIV-1 1 p24 protein proof of concept 0 - -12-11 -10-9 -8-7 -6-5 Log [HIV-1 p24] (M) 20000 Commercial antibodies used Detection limit of 100 pm HIV-1 1 p24 Assay range = 3 logs - up to 0.1 µm An even wider dynamic range and sensitivity can be obtained by through optimization AlphaScreen Signal (CPS) 17500 15000 12500 10000 7500 5000 2500 0 - -12-11 -10-9 Log [HIV-1 p24] (M) Page 18
Straightforward ELISA Conversion... small molecule: ex. Aβ40/42A 300000 AlphaScreen Signal (cps) 200000 100000 0 200000 150000 100000 50000 0 250 500 750 1000 Aβ 40 (pg/well) linear part 0 0 100 200 300 biotin IgG IgG coupled to bead high sensitivity: 0.5 ng/ml monoclonal antibodies developed by customer Page 19
Straightforward ELISA Conversion... very small molecule: ex. estradiol 30000 AlphaScreen Signal (cps) 25000 20000 15000 10000 5000 0 IC 50 = 0.4 nm -12-11 -10-9 -8-7 log [Estradiol] (M) biotin-estradiol anti-estradiol high sensitivity: 40 pm = 10 pg/ml commercially available antibody indirect capture possible Page 20
AlphaScreen - an alternative to ELISA Why an alternative to ELISA? Assay principle Conjugation Assay examples Non-serum / plasma NEW : Insulin detection in serum / plasma 1 O 2 A B Page 21
New EasyLISA acceptor beads EasyLisa Emission Allows for high sensitivity in serum samples Page 22
Insulin determination in serum/plasma samples Streptavidin Donor beads EasyLISA Acceptor beads Biotinylated anti-insulin (Ab1) Anti-insulin (Ab2) Insulin Page 23
Materials and assay set-up Materials: : commercial anti-insulin insulin antibody pair, commercial human insulin calibrator (LINCO), matrix solution = insulin-depleted serum/plasma sample prepared in house, Human insulin ELISA kit (LINCO). Three addition-step assay performed in either 96- or 384-well microplates (50 µl total assay volume): 1- Add serum/plasma sample 2- Add biotinylated Ab1 antibody and Acceptor beads coated with Ab2 A Incubate 30-60 min at RT 3- Add donor beads coated with streptavidin Incubate 30-60 min at RT 4- Read the plate on Envision-alpha Page 24
Human Insulin Calibration Curve in Assay Buffer A calibration curve was generated with insulin calibrators prepared red in assay buffer (96 format, 50 µl). AlphaScreen signal (counts) 1200000 1000000 800000 600000 400000 200000 150000 100000 50000 0 0 4 8 12 16 20 0 0 25 50 75 100 125 [Human Insulin] (µiu/ml) Detection limit : 0.7 µiu/ml (30 pg/ml ml) ) using a 5 µl sample size Page 25
Human Insulin Calibration Curve in serum A calibration curve was generated with insulin calibrators prepared red in matrix solution (96 or 384, 50 µl). AlphaScreen Signal (counts) 700000 600000 500000 400000 300000 200000 100000 120000 110000 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 0 10 20 30 40 50 60 70 80 90 100110120 0 0 100 200 300 400 500 600 700 800 900 10001100 [Human Insulin] (µiu/ml) Detection limit : 2 µiu/ml (85 pg/ml ml) ) using a 5 µl sample size (comparable to LINCO human insulin ELISA Kit, 20 µl sample size) Dynamic range: 2-10002 µiu/ml Page 26
Impact of Antibody Selection Detection Limit Customer Antibody Pair Human Porcine Insulin Insulin Commercial Antibody Pair Human Porcine Insulin Insulin µui/ml 0.03 0.07 2.75 0.25 pm 0.25 0.55 20.5 1.9 pg/ml 1.47 1.99 119.1 68.4 1 µui = 43.3 pg = 7.45 fmol Page 27
Assay Precision (within and between assay variation) Assay precision was measured using 5 samples spiked with various concentrations of insulin. Intra-assay variation (insulin content in uiu/ml) replicate [spiked insulin] 1 2 3 4 5 6 Average %CV 85 74 76 84 85 86 81 81 6 48 41 44 46 52 51 48 47 9 27 23 23 24 29 29 26 26 11 18 15 17 17 21 20 20 18 12 3 3 5 7 5 4 5 5 23 Inter-assay variation (insulin content in uiu/ml) Experiment [spiked insulin] n=1 n=2 n=3 n=4 n=5 n=6 Average %CV 85 90 84 77 98 96 82 88 9 48 49 51 43 54 50 58 51 10 27 26 28 24 26 26 27 26 6 18 17 20 17 19 17 18 18 7 3 4 5 6 4 4 4 4 17 Page 28
Linearity (effect of serum dilution) A human serum sample spiked with 100 µiu/ml was diluted in matrix solution. Linearity (insulin content in uiu/ml) Experiment [expected Dilution insulin] n=1 n=2 n=3 Average % of expected 1 100 114 124 112 117 117 1/2 50 48 53 52 51 102 1/3 33.3 40 34 36 37 112 1/4 25 30 23 29 27 109 0 0 1 1 1 1 n/a Page 29
Analyte recovery matrix effect A-LISA Insulin assay Recovery rate of spiked samples ELISA Insulin assay Recovery rate of spiked samples 150 150 % recovery 100 50 serum 2 serum 5 serum 6 serum 7 serum 8 % recovery 100 50 serum 2 serum 5 serum 6 serum 7 serum 8 0 0 25 50 75 100 0 0 25 50 75 100 Insulin added Insulin added Five different insulin-depleted human serums were spiked with 4 insulin concentrations and recovery was calculated (observed insulin level / spiked level of insulin). Page 30
Correlation Graph (AlphaScreen versus ELISA) 25 serum or plasma samples were assayed for insulin content in both b AlphaScreen human insulin assay and ELISA human insulin assay. Insulin Level measured by AlphaScreen (µiu/ml) 100 75 50 25 0 0 25 50 75 100 125 Insulin Level measured by ELISA* (µiu/ml) r 2 : 0.943 Y=0.8 (x) - 1.1 Page 31
Conclusions ELISA assays could be converted to the AlphaScreen platform for different types of analytes. ELISA conversion using AlphaScreen can be applied in a sandwich or a competition binding format. A human insulin sandwich assay has been developed using the AlphaScreen technology platform to measure analyte in serum/plasma samples. The assay could measure insulin levels in biological samples (serum, plasma) a) at physiological concentration range (2.5-50 50 µiu/ml) ) and above (dynamic range of 3 logs) with high sensitivity, precision and accuracy. Main advantages of the assay over ELISA are: Homogeneous (no wash steps) Simple (few addition steps) and fast protocol Scalable (96 and 384 formats) and easily automatable Fast and less hands on time Lower costs Page 32
AlphaScreen product offering Fusion Tag Detection Kits C-MYC (6760611) DIG (6760604) FLAG M2 (6760613) GST (6760603) HA (6760612) Histidine (Ni2+ chelate, 6760619) FITC (6760605) GPCR Functional Assay Kits camp (6760625) IP 3 supplement (6760621) IgG Detection kits Protein A (6760617) Mouse IgG (6760606) Rabbit IgG (6760607) cgmp Detection Supplement (6760306) Streptavidin-coated Donor Beads (6760002) Unconjugated Beads Acceptor Beads (6762001) Donor Beads (6762011) New: EasyLISA Acceptor Beads TruHits Kit (6760627) Omnibeads (6760626) Labeling Services (ASLsvc) Conjugation Kit (6760000) Phosphotyrosine Assay Kits PT66 (6760602) PY20 (6760601) ptyr100 (6760620) TNFα Receptor Binding Kit (6760622) Page 33
Summary of Design and Development Identify optimal antibody pair Select anti-analyte antibody pairs Biotinylate all antibodies Conjugate all antibodies to Europium Acceptor beads Test all possible antibody combinations: Titrate the biotinylated antibodies in AlphaScreen Possible Backup- slide Optimize assay conditions & Determine assay characteristics Determine optimal: - Assay buffer - Conc. of Donor and Acceptor beads - Assay volume - Order of addition - Incubation time by generating biomarker calibration curves in Assay Buffer Determine assay characteristics from calibrations curves performed in Assay Buffer and in Matrix Solution: detection limit, dynamic range, reproducibility Determine biomarker level in unknown samples Perform an AlphaScreen biomarker calibration curve in Matrix Solution Extrapolate level of biomarker from calibration curve A full optimization guide (assay development guide) will be provided to help customers develop their own assay Page 34