AlphaLISA Assays are Homogeneous Sensitive Immunoassays for Detection of Analytesin a Variety of Biological Matrices

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.
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