Low volume, highly sensitive immunoassays for detecting cytokines in animal fluids

Low volume, highly sensitive immunoassays for detecting cytokines in animal fluids
Jean-Francois Michaud, Nathalie Rouleau and Francesco Lipari
Cytokine levels are measured in many different experimental
models, with samples obtained in a variety of biological
matrices such as cell culture supernatant, serum, plasma,
saliva, cerebrospinal fluid, and tissue homogenate. The most
common method of cytokine analysis is enzyme-linked
immunosorbent assay (ELISA). However, ELISA often
requires high sample volume (at least 50 µL), which is
difficult to obtain when working with small animals such as
mice. Also, the ELISA method has a limited dynamic range
and low throughput due to its numerous wash steps.
Considering the limitations of ELISA technology, a faster
approach was developed for cytokine analysis in complex
biological samples. This novel analytical approach is a
chemiluminescent homogeneous bead-based immunoassay
that is performed without wash steps. The cytokine of
interest is sandwiched between two highly specific antibodies
bound to the beads. In presence of the analyte, the beads
come into proximity resulting in an emission of light upon
laser excitation. The amount of light emitted is directly
proportional to the amount of analyte present. Sample
volumes as low as 5 µL can be utilized in this sensitive
detection platform. In addition, upper quantitation limits can
extend to much higher concentrations than achievable using
traditional ELISA assay. For example, mouse IL6 can be
detected from 1 pg/mL to 100,000 pg/mL without dilution,
whereas a comparable ELISA has a range of 8 pg/mL to 500
pg/mL. The application of this immunoassay to different
types of biological samples was verified by performing spike
recovery experiments, and also by comparison of the levels
measured to the levels reported in the literature. Overall, this
assay technology will aid researchers in evaluating cytokines
from many different biological sources that, in some cases,
have been problematic to measure in the past.
3
Tested diluent
X
X
X
Assay buffer from kit
X
X
X
HiBlock buffer
X
X
X
PBS-0.1%BSA
X
X
X
PBS-0.01%BSA
X
X
X
Same fluid as sample fluid, but other species
X
FBS*
*For serum samples, FBS was the only diluent tested, as FBS has previously
been shown to be the best diluent for all serum samples (data not shown).
NaCl buffer
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
With step 1 results, select the two best diluents based on:
• Observed concentration X dilution factor (DF) as close as possible to spiked
value, for all dilution points
• Recoveries between 70-130% for all dilution points
Immunoassays – quick and simple
Spike neat and ½ diluted mouse/rat fluid with mouse cytokine
Use spiked diluent as spike control
Select 3-4 spikes and include a “no spike” control (to measure physiological level)
Recoveries between 70-130% are targeted
Evaluate other diluents if good recovery is not obtained
Assay protocol with 3 incubation steps (No wash steps)
5.0
5.0
5.0
5.0
3.0
3.0
3.0
3.0
3.0
2.0
2.0
2.0
2.0
2.0
1.0
1.0
1.0
1.0
1.0
0.0
0.0
0.0
0.0
r2 neat = 0.8832
2
4.0 r dil 1/2 = 0.9995
0.0
0.0
0.5
1.0
7
Example (no dilution required)
Step 1: Linearity of dilutions in 5 diluents for spiked mouse BALF
(3ng/mL mTNFα)
Add 10 L of AlphaLISA Anti-Analyte Acceptor beads
Add 10 L of Biotinylated Anti-Analyte Antibody
Incubate 60 minutes
AlphaLISA buffer
5.0
Mouse/rat sample fluids:
All mouse/rat sample fluids were supplied as pools of individuals, nonmedicated, non-immunized, from Bioreclamation LLC:
• Mouse bronchial lavage fluid (BALF): Strain CD-1, cat# MSE-BRONLEV
• Mouse serum: Strain BALB/C, cat# MSESRM-BALB
• Rat amniotic fluid (AF): Strain Sprague Dawley, cat# RATAMNFL
• Rat cerebrospinal fluid (CSF): Strain Sprague Dawley, cat# RATCSF
5
AlphaLISA Signal (counts)
Calibration curve
for mouse MCP-1
(LDL = Lower
Detection Limit)
1,000,000
LDL = 2.4 pg/mL
100,000
10,000
5.0
r2 neat = 0.9905
HiBlock buffer
r2 neat = 0.9906
PBS - 0.1%BSA
5.0
r2 neat = 0.9993
PBS - 0.01%BSA
5.0
r2 neat = 0.9998
Beagle BALF
5.0
r2 neat = 0.9977
4.0
4.0
4.0
4.0
4.0
3.0
3.0
3.0
3.0
3.0
2.0
2.0
2.0
2.0
2.0
1.0
1.0
1.0
1.0
1.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.5
1.0
Dilution
Incubate 30 minutes
Read at 615 nm using Envision® or EnSpireTM Reader and
analyze data
100
-
-12 -11 -10 -9 -8 -7 -6
Log [mouse MCP-1] (g/mL)
Fluids and cytokines chosen
0.5
1.0
Dilution
0.5
1.0
PBS-0.1%BSA
0.5
1.0
Dilution
Dilution
AlphaLISA buffer HiBlock buffer
PBS-0.01%BSA
0.5
Physiological range found in literature (pg/mL)
Cytokine Mouse serum Mouse BALF Rat CSF
Rat AF
IL6
5 - 70
20
Not found
10 - 60
TNFα
5 - 100
30-90
Not found
10 - 60
MCP-1
30 - 1500
40
Not found
1500
1
2
4
8
16
32
100
179
187
180
170
192
0.5
1.0
PBS-0.01%BSA
Recovery
from 1/2 dil.
(%)
Recovery
from neat
(%)
100
104
100
95
107
100
127
131
129
129
129
0.5
1.0
r2 neat = 0.9496
4.0 r2 dil 1/2 = 0.9998
0.0
0.0
0.5
Dilution
Dilution
Recovery
from 1/2 dil.
(%)
Recovery
from neat
(%)
100
103
101
101
101
100
144
157
154
163
169
1.0
r2 neat = 0.9935
4.0 r2 dil 1/2 = 0.9974
0.0
0.0
0.5
PBS-0.1%BSA
Recovery
from 1/2 dil.
(%)
Recovery
from neat
(%)
100
109
107
113
117
100
149
152
145
150
148
1.0
Dilution
Dilution
HiBlock buffer
Diluents not chosen:
observed concentration X DF not
close to spiked value (3ng/mL) (too
low or too high).
Rabbit AF
Recovery
from 1/2 dil.
(%)
Recovery
from neat
(%)
Recovery
from 1/2 dil.
(%)
100
102
98
100
100
100
114
126
123
116
119
100
110
108
102
104
Choose 2 good diluents: PBS-0.1%BSA and rabbit AF
• Excellent linearity from ½ dilution
• Observed concentration X DF close to spiked value
(3ng/mL) for all dilutions.
Step 3: Chosen diluent showing the best spike and recovery results:
Dilution
Step 3: Chosen diluent showing the best spike and recovery results
No spike
10
30
300
3000
Cytokine
Beagle BALF
Choose 2 good diluents: PBS-0.1%BSA and Beagle BALF
• Good linearity from neat
• Observed concentration X DF close to spiked value
(3ng/mL) for all dilutions
Diluent: PBS-0.1%BSA
Spiked diluent
Spiked neat Mouse BALF
control
Concentration
Concentration
Recovery (%)**
(pg/mL)*
(pg/mL)*
0.0
17.1
NA
12.0
9.2
76
35.2
32.3
92
300.2
292.6
97
3141.2
2952.1
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 10-3000 pg/mL spikes = measured concentration – no spike value.
** Recovery (%) = recovery compared to spiked diluent control
Spiked 1/2 Rat AF
Concentration
(pg/mL)*
575.6
317.2
750.4
2227.9
Recovery
(%)**
NA
119
84
85
½ dilution in diluent
required: excellent
recovery for all 3 spikes
tested in ½ rat AF, but not
in neat rat AF. Measured
MCP-1 physiological level in
rat AF (No spike) = 575.6
pg/mL X 2 = 1151pg/mL
* Concentration for 300-3000 pg/mL spikes = measured concentration – no spike value.
** Recovery (%) = recovery compared to spiked diluent control
IL6
TNFα
MCP-1
Diluents not chosen:
Linearity not good from neat
Spike (pg/mL)
Spiked
Spiked neat Rat AF
diluent
control
Concentration
Concentration
Recovery
Spike (pg/mL)
(pg/mL)*
(pg/mL)*
(%)**
No spike
0.0
910.5
NA
300
267.3
195.2
73
1000
890.5
442.6
50
3000
2627.6
1542.0
59
Summary table
1.0
Dilution Recovery from neat Recovery from neat Recovery from neat Recovery from neat Recovery from neat
factor (DF)
(%)
(%)
(%)
(%)
(%)
1
100
100
100
100
100
2
119
113
95
97
92
4
128
135
93
96
85
8
137
150
90
96
82
16
139
146
92
95
83
32
149
145
94
99
83
Cytokines and mouse/rat fluids were selected according to availability of
mouse/rat PerkinElmer AlphaLISA kits, as well as availability and cost of
fluids from suppliers.
Expected physiological range:
Recovery
from neat
(%)
9
Step 2:
1,000
Dilution
factor
(DF)
r2 neat = 0.9552
4.0 r2 dil 1/2 = 0.9982
r2 neat = 0.9827
4.0 r2 dil 1/2 = 0.9998
Diluent: PBS-0.1%BSA
5 L Standard or animal fluid
Incubate 30 minutes
Rabbit AF
Step 2:
Step 3: Evaluate spike and recovery value:
•
•
•
•
•
PBS - 0.1%BSA
5.0
Dilution
Step 2: Diluent selection:
HiBlock buffer
PBS - 0.01%BSA
NACL buffer
Mouse and rat IL6, TNFα and MCP-1 detection:
AlphaLISA® kits available from PerkinElmer: IL6 (mouse) (AL504), TNFα
(mouse) (AL505) and CCL2/MCP-1 (mouse/rat) (AL509). Assay buffer is
AlphaLISA Immunoassay Buffer (AL000C) for mouse IL6 (mIL6) and mouse
TNFα (mTNFα), and NaCl Buffer (AL007C) for mouse MCP-1 (mMCP-1). All
calibration curves were done using mouse analyte supplied in the kit. All 3 kits
similarly detect mouse and rat cytokines.
Fluids pre-treatment and storage:
Prior to being used in AlphaLISA assays: FBS was triple filtered at 0.1µm by
Wisent; mouse serum was filtered at 0.2µm by Bioreclamation LLC; BALF and
AF fluids were centrifuged at 650xg for 15 min at 4˚C to remove cells and cell
debris, and supernatants were used in assays; CSF fluids were not pretreated.
Storage: Mouse serum and FBS were kept at -20˚C. All other fluids (BALF,
CSF, AF) were kept at -80˚C.
Example (½ dilution required)
Step 1: Linearity of dilutions in 5 diluents for spiked rat AF (3ng/mL mMCP-1)
Spiked sample fluid (3ng/mL or 10ng/mL mouse cytokine)
Mouse serum
Mouse BALF
Rat CSF
Rat AF
Materials and Methods
Diluents tested:
AlphaLISA HiBlock Buffer: PerkinElmer, cat# AL004C.
Foetal bovine serum Premium (FBS): Wisent, cat# 080150.
Fluids tested as diluents were supplied as pools of individuals, non-medicated,
non-immunized, from Bioreclamation LLC:
• Beagle bronchial lavage fluid (BALF): cat# BGL-BROLAV
• Beagle cerebrospinal fluid (CSF): cat# BGLCSF
• Rabbit amniotic fluid (AF): Strain New Zealand White, cat# RABAMNFL
8
Experimental approach
Step 1: Perform linearity experiment with the following diluents for all
3 cytokines:
Add 25 L of Streptavidin Alpha Donor beads
2
6
Alpha Technology Assay Principle
Concentration (ng/mL)
Abstract
Concentration (ng/mL)
1
10
Sample fluid
Best diluent
Mouse serum
FBS
Mouse BALF PBS-0.1%BSA
Rat CSF
Beagle CSF
Rat AF
PBS-0.01%BSA
Mouse serum
FBS
Mouse BALF PBS-0.1%BSA
Rat CSF
PBS-0.1%BSA
Rat AF
PBS-0.1%BSA
Mouse serum
FBS
Mouse BALF Beagle BALF
Rat CSF
PBS-0.1%BSA
Rat AF
PBS-0.1%BSA
Dilution of Physiological Spike range in
LDL in
sample fluid
level
sample fluid
diluent
required in
measured
giving good
(pg/mL)
diluent
(pg/mL)
recovery (pg/mL)
2.1
1/2
33
30 - 10000
2.4
Neat
22
10 - 3000
3.4
Neat
ND
10 - 3000
2.9
1/2
ND
10 - 3000
1.6
1/2
55
30 - 3000
1.6
Neat
17
10 - 3000
1.4
Neat
ND
3 - 3000
1.4
1/2
5
3 - 3000
5.4
1/2
444
300 - 3000
5.7
Neat
22
10 - 3000
2.9
Neat
501
100 - 3000
2.4
1/2
1151
300 - 3000
Conclusion
• Sensitive bead-based immunoassays were developed to detect IL6,
TNFα and MCP-1 from rodent serum, BALF, CSF and AF. Good
recoveries were obtained from fluids spiked as low as 3 pg/mL.
• This study highlights that the appropriate diluent is not easy to
predict. In some cases where a perfect match of diluent and fluid is
not found, sample dilution is required.
• These no wash rapid assays require only 5µL sample volumes.
This represents a substantial improvement over ELISA-type assays
in terms of speed of execution and sample consumption.
• Moreover,
results
presented
demonstrate
that
measured
physiological levels are also in agreement with the literature.
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com