A C i f H B d B d A lifi d L i T h l A Comparison of a Homogenous Bead Based Amplified Luminescence Technology A Comparison of a Homogenous Bead-Based Amplified Luminescence Technology A Comparison of a Homogenous Bead Based Amplified Luminescence Technology p g p gyAl h LISA d C l i t i ELISA f D t ti f H M t i M t ll t i 9 i AlphaLISA and a Colorimetric ELISA for Detection of Human Matrix Metalloproteinase 9 in AlphaLISA and a Colorimetric ELISA for Detection of Human Matrix Metalloproteinase 9 in AlphaLISA and a Colorimetric ELISA for Detection of Human Matrix Metalloproteinase 9 in p pC l S l M t i Complex Sample Matrices Complex Sample Matrices Complex Sample Matrices p pAnuradha Prasad Stephen Hurt Carol Luchette & David TitusAnuradha Prasad, Stephen Hurt, Carol Luchette & David Titus, p ,

ProtocolsProtocolsAssay Precision6Ab t t I t d ti Assay Precision6Abstract1 Introduction2 AlphaLISA:Assay PrecisionAbstract1 Introduction2 AlphaLISA:

1 Add 5 µL MMP9 standard 1. Add 5 µL MMP9 standard AlphaLISA ELISA

E li k d I b t (ELISA) AlphaLISA Technology 2 Add 20 µL mixture of anti-MMP9 acceptor beads and biotinylated P i iAlphaLISA ELISA

Enzyme-linked Immunosorbent assay (ELISA) AlphaLISA Technology 2. Add 20 µL mixture of anti MMP9 acceptor beads and biotinylated PrecisionEnzyme linked Immunosorbent assay (ELISA) AlphaLISA Technologyanti-MMP9 antibody # replicates %CV # replicates %CV

is a well known technique in the area ofanti MMP9 antibody

3 b 60 i# replicates %CV # replicates %CV

is a well known technique in the area of 3. Incubate 60 minutes at room temperature Intra Assay Low 9 2 3 9 9 1qi ELISA i l b f

3. Incubate 60 minutes at room temperature4 Add 25 L t t idi d b d

Intra Assay Low 9 2.3 9 9.1immunoassays ELISA involves a number of 4. Add 25 µL streptavidin donor beads (1 plate) Med 9 2 3 9 6 9immunoassays. ELISA involves a number of 4. Add 25 µL streptavidin donor beads

5 I b t 30 i t t RT i th d k(1 plate) Med 9 2.3 9 6.9

wash steps which makes it a laborious and 5. Incubate 30 minute at RT in the dark High 9 5 9 9 5 2wash steps, which makes it a laborious and 6 Read on EnVision ReaderHigh 9 5.9 9 5.2wash steps, which makes it a laborious and

h f ld f d6. Read on EnVision Reader

Inter Assay Low 3x9 7 2 3x9 16 9time-consuming process In the field of drug Inter Assay Low 3x9 7.2 3x9 16.9time-consuming process. In the field of drug (3 distinct M d 3 9 4 6 3 9 6 5g p g

di t h l i hi h i b t ELISA:(3 distinct

l t )Med 3x9 4.6 3x9 6.5

discovery, technologies which can give robust, ELISA: plates)Hi h 3 9 2 4 3 9 5 4discovery, technologies which can give robust,

1 Add 100 µL of Assay diluentp )

High 3x9 2.4 3x9 5.4sensitive automatable and reproducible

1. Add 100 µL of Assay diluent gsensitive, automatable and reproducible 2 Add 100 µL of MMP9 standard, pi t l t hi hl

2. Add 100 µL of MMP9 standardd h l h ll fimmunoassays at a lower cost are highly 3. Incubate 120 minutes on horizontal orbital microplate shaker Compared to ELISA the AlphaLISA assay allows for immunoassays at a lower cost are highly 3. Incubate 120 minutes on horizontal orbital microplate shaker

( 500±50 )Compared to ELISA, the AlphaLISA assay allows for

desirable Amplified Luminescent Proximity ( 500±50rpm) better intra- and inter- assay reproducibilitydesirable. Amplified Luminescent Proximity- ( 500 50rpm)4 7 W h l t 4X ith 400 L h b ff

better intra- and inter- assay reproducibility.des ab e p ed u esce t o tyb d ( l h S ®) i

4- 7. Wash plate 4X with 400 µL wash bufferbased Homogeneous Assay (AlphaLISA®) is a

p µ8 Add 200 µL of conjugatebased Homogeneous Assay (AlphaLISA®) is a 8. Add 200 µL of conjugateg y ( p )

bead based homogenous immunoassay whichµ j g

9 Incubate 60 minutes on horizontalbead-based homogenous immunoassay whichS h l

9. Incubate 60 minutes on horizontal hMMP9 ll l tit ti7bead based homogenous immunoassay whichELISA Technology 10 Shake microplate on orbital shaker (500±50rpm) hMMP9 cellular quantitation7

can give comparable or even better results in ELISA Technology 10. Shake microplate on orbital shaker (500±50rpm) hMMP9 cellular quantitation7can give comparable or even better results in gy

11- 14 Wash plate 4X with 400 µL wash bufferqg p

l ti t d l l I thi11- 14. Wash plate 4X with 400 µL wash buffer

less time, cost and sample volume. In this 15 Add 200 µL of TMB substrate solutionless time, cost and sample volume. In this 15. Add 200 µL of TMB substrate solution150

study we compared ELISA and AlphaLISA 16 Incubate 30 minutes at RT in the darkHRP labeled150

AlphaLISAstudy, we compared ELISA and AlphaLISA 16. Incubate 30 minutes at RT in the dark17 Add 50 L f l i

HRP labeledd t ti I G TMB S b t t

AlphaLISAy, p pl tf l d f i

17. Add 50 µL of stop solutiondetection IgG TMB Substrate ELISAplatforms commonly used for immunoassays

17. Add 50 µL of stop solution18 R d t 450 ithi 30 i t (λ ti 540 570 )

ELISA

L)platforms commonly used for immunoassays 18. Read at 450 nm within 30 minutes (λ correction 540 or 570nm) 100mL

and examined performance (sensitivity8 ead at 50 t 30 utes ( co ect o 5 0 o 5 0 ) 100

g/m

and examined performance (sensitivity, blue ngand examined performance (sensitivity,Assay conditions for hMMP9 cellular quantitation:

blue

9 (n

dynamic range variability) as well as assay Assay conditions for hMMP9 cellular quantitation:antigen P9dynamic range, variability) as well as assay y qantigen 50MPy g , y) y

l it d ti t fstop

50

MM

complexity and time to perform. • U937 cells (Suspension cell line ATCC CRL-1593 2) p Mcomplexity and time to perform. • U937 cells (Suspension cell line ATCC CRL-1593.2)

• Media: RPMI+ 10%FBSll 0• Media: RPMI+ 10%FBScapture IgG yellow 0

Th Al h LISA t i t ll t i 9RPMI 1640 (Life Technologies, 11835030)capture IgG y

-9.25 -9.00 -8.75 -8.50The AlphaLISA matrix metalloproteinase 9

RPMI 1640 (Life Technologies, 11835030)FBS (Hyclone Cat # SH30071)

9 5 9 00 8 5 8 50l PMA ( / L)The AlphaLISA matrix metalloproteinase 9 FBS (Hyclone Cat # SH30071) log PMA (g/mL)

(MMP9) kit EnVision® Multidetection Plate • PMA (Phorbol 12-Myristate 13 Acetate SIGMA-ALDRICH P8139)(MMP9) kit, EnVision® Multidetection Plate • PMA (Phorbol 12-Myristate 13 Acetate, SIGMA-ALDRICH, P8139)(MMP9) kit, EnVision Multidetection Plated d l l d b • 24 well Tissue Culture Plate (Corning 3542) PMA induced MMP9 secretion by U937 cellsReader and microplates were supplied by • 24 well Tissue Culture Plate (Corning, 3542)

5PMA-induced MMP9 secretion by U937 cells.Reader, and microplates were supplied by • Number of cells/mL = 3 5 X 105

yFollowing 24 hrs stimulation of U937 cells with PMA assay

, p pp yP ki El I Th l i t i ELISA MMP9

• Number of cells/mL 3.5 X 10V l ll 1 L

Following 24 hrs stimulation of U937 cells with PMA, assay PerkinElmer, Inc. The colorimetric ELISA MMP9 • Volume per well = 1mL

g , ysupernatants were diluted 100 fold with assay buffer and quantified Materials & Methods3

PerkinElmer, Inc. The colorimetric ELISA MMP9 Volume per well 1mL supernatants were diluted 100 fold with assay buffer and quantified Materials & Methods3kit was from a well known supplier Thep y q

using AlphaLISA and ELISA kits Both AlphaLISA and ELISA kits Materials & Methods3kit was from a well-known supplier. The using AlphaLISA and ELISA kits. Both AlphaLISA and ELISA kits

Calibration c r es4pp

d i i t d i t i ti show equivalent resultsMicroplates Calibration curves4dynamic range inter- and intra-assay variation show equivalent results.Microplates Calibration curvesdynamic range, inter and intra assay variationAlphaLISA: 96-well ½ area white (PerkinElmer #6005560)was studied in spiked MEM + 10% FBS Both SAlphaLISA: 96 well ½ area white (PerkinElmer #6005560)

S l 9 l ( d d h h k )was studied in spiked MEM + 10% FBS. Both 10AlphaLISA ELISA

ELISA: Total MMP-9 Microplate (provided with the kit)as stud ed sp ed 0% S ot

ki h d d d ibili d1000000s)

10AlphaLISA ELISAELISA: Total MMP 9 Microplate (provided with the kit)

kits showed good reproducibility and accuracy ntskits showed good reproducibility and accuracy.

oun

M t i t d iti f Al h LISACell culture samples containing MMP9 secreted 100000(co

1Matrix components and compositions for AlphaLISACell culture samples containing MMP9 secreted al ( 1p p p

MEM (Invitrogen #11370021) S8Cell culture samples containing MMP9 secreted

na OD

• MEM (Invitrogen, #11370021) Summary8upon PMA stimulation of U937 cells also showed 10000sig O( g , )

• FBS (Hyclone # SH30071 lot # ARF26748)Summary8upon PMA stimulation of U937 cells also showed

A s 0.1• FBS (Hyclone # SH30071, lot # ARF26748)

ypi il lt i th t kit U i th SA

0.1

• For standard curve and unknowns used MEM + 10% FBSsimilar results using the two kits. Using the 1000

aLI

• For standard curve and unknowns, used MEM + 10% FBSIn comparison to ELISA for the detection of hMMP9 the

similar results using the two kits. Using the ha In comparison to ELISA for the detection of hMMP9, the AlphaLISA assay we can obtain a PMA dose 100Alp 0 01 p ,

Al h LISA kit h t i d bAlphaLISA assay, we can obtain a PMA dose- 100

12 11 10 9 8 7 6

A 0.01-10 -9 -8 -7

Matrix components and compositions for ELISA AlphaLISA kit was characterized by:p y,

f MMP9 i d i i h-∞ -12 -11 -10 -9 -8 -7 -6 -10 -9 -8 -7

log [MMP9] g/mLMatrix components and compositions for ELISA AlphaLISA kit was characterized by:response curve of MMP9 induction without any log [MMP9] (g/mL) log [MMP9] g/mL

• For standard curve supplier recommends diluent provided with kit • Larger assay window (linear dynamic range)response curve of MMP9 induction without any g [ ] (g )• For standard curve, supplier recommends diluent provided with kit

k l d 00 f ld d l f• Larger assay window (linear dynamic range),

sample dilution even in RPMI + 10% FBS The D i • For unknowns, supplier recommends a 100-fold dilution of MEM + g y ( y g ),

Similar sensitivity (based on LDL) sample dilution, even in RPMI + 10% FBS. The Dynamic range:For unknowns, supplier recommends a 100 fold dilution of MEM + 10% FBS

• Similar sensitivity (based on LDL), sample dilution, even in RPMI + 10% FBS. The Dynamic range:10% FBS

Similar sensitivity (based on LDL), B tt i t d i t i i (l %CV)AlphaLISA assay also requires twenty times less Range AlphaLISA ELISA10% FBS

• Better intra- and inter-assay precision (lower %CV)AlphaLISA assay also requires twenty times less Range AlphaLISA ELISA Better intra and inter assay precision (lower %CV)p y q yl l d i fi f ld id ( ) 30 / 0 /AlphaLISA Kit • Equivalent performance in cell based assayssample volume and gives a five-fold wider Minimum (LDL) 30 pg/mL 40 pg/mLAlphaLISA Kit • Equivalent performance in cell based assays.sample volume and gives a five fold wider ( ) pg/ pg/p

AlphaLISA MMP9 Kits (#AL243C lot P10902)q p y

dynamic range A wider dynamic range allows Maximum 100,000 pg/mL 20,000 mIU/mLAlphaLISA MMP9 Kits (#AL243C, lot P10902)dynamic range. A wider dynamic range allows Maximum 100,000 pg/mL 20,000 mIU/mLp ( )y g y gth t t t l ith t

Total 3.5 Log 2.7 Logthe customer to measure most samples without

Total 3.5 Log 2.7 Log

EnVision® Plate Reader 2104 settingsthe customer to measure most samples withoutU i th i ti d d d t l Al h LISA d EnVision® Plate Reader 2104 settings

The AlphaLISA assay had these advantages over ELISA:any dilutions Because the AlphaLISA kit does Using their respective and recommended protocols: AlphaLISA and AlphaLISA The AlphaLISA assay had these advantages over ELISA:any dilutions. Because the AlphaLISA kit does Us g t e espect e a d eco e ded p otoco s p a S a d

ECL sho simila sensiti ities hilst AlphaLISA has a b oade AlphaLISA p y gL l l i t f i l t

any dilutions. Because the AlphaLISA kit doesh b h h l d

ECL show similar sensitivities whilst AlphaLISA has a broader • Distance between plate and detector: 0.15 mm • Lower sample volume requirement for equivalent not require any wash steps both the elapsed

pdynamic range • Distance between plate and detector: 0.15 mm

E i i i 35 Lower sample volume requirement for equivalent

fnot require any wash steps, both the elapsed dynamic range.

• Excitation time: 35 ms performancetime and hands on time required to perform Sensitivity: Lower detection limit (LDL = mean of background value + 3SD)Excitation time: 35 msE i i ti 100

performancetime and hands-on time required to perform Sensitivity: Lower detection limit (LDL = mean of background value + 3SD)• Emission time: 100 ms • Shorter total assay duration

time and hands on time required to perform• Aperture: 96 Plate HTS AlphaScreen aperture

• Shorter total assay durationthe assay was much less than for the ELISA kit • Aperture: 96 Plate HTS AlphaScreen aperturey

No ash stepsthe assay was much less than for the ELISA kit.

hMMP9 R5p p p

• Crosstalk correction: no • No wash stepsy

hMMP9 Recovery5• Crosstalk correction: no No wash stepsh k

hMMP9 Recovery5• No shaking

y• No shaking

Overall the AlphaLISA kit offered benefits over Spiking Concentrations AlphaLISA % ELISA %ELISA • No need for special platesOverall, the AlphaLISA kit offered benefits over Spiking Concentrations AlphaLISA % ELISA %ELISA • No need for special plates, p

th ELISA kit f id d i % recovery spike Low 95 100• ELISA Instrument settingsp p

the ELISA kit of wider dynamic range a % recovery spike‐ Low 95 100• ELISA Instrument settingsk l

the ELISA kit of wider dynamic range, a ins Medium 105 90• Instrument: PerkinElmer EnVisiony g

significantly faster and easier protocol andins Medium 105 90• Instrument: PerkinElmer EnVision

b b f l This comparative study demonstrates the excellent significantly faster and easier protocol and Hi h 110 90• Absorbance filter 1: 450 nm This comparative study demonstrates the excellent significantly faster and easier protocol and High 110 90Absorbance filter 1: 450 nmAb b filt 2 540 performance and markedly enhanced and ease of use comparable performance in other respects

g

• Absorbance filter 2: 540 nm performance and markedly enhanced and ease of use comparable performance in other respects. AlphaLISA and ELISA both gave good recoveriesAbsorbance filter 2: 540 nm p yf th Al h LISA t h l ELISA

p p p AlphaLISA and ELISA both gave good recoveries.of the AlphaLISA technology over ELISAAlphaLISA spike-in concentrations: 100 1000 and 10 000 pg/ml of the AlphaLISA technology over ELISA.AlphaLISA spike in concentrations: 100, 1000 and 10,000 pg/ml

ELISA ik i t ti 250 1500 d 5000 / l ELISA spike-in concentrations: 250, 1500 and 5000 pg/ml. p , pg/

PerkinElmer Inc 940 Winter Street Waltham MA USA (800) 762-4000 or (+1) 203 925-4602 www perkinelmer comPerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com