A DELFIA® Assay for Measuring cAMP in Plasma - PerkinElmer

A DELFIA Assay for Measuring
cAMP in Plasma
The DELFIA cAMP kit
(PerkinElmer 4003-0010) is
intended for the quantitative
determination of cAMP in cell
culture samples. The assay has
been developed to standardize
cAMP determination when high
sensitivity is needed. In this
study, we show that the DELFIA
cAMP kit can be used for measurement
of cAMP in plasma.
To detect cAMP in plasma
samples, we followed a high
sensitivity protocol, that includes
an acetylation step. Samples and
standards were assayed in
duplicate on a 96-well plate.
All tips used for pipetting were
chilled to +4°C.
Preparation of Samples
Blood samples were drawn from
healthy volunteers into glass
tubes containing ethylenediamine
tetraacetic acid (EDTA) as anticoagulant.
The plasma was
separated by centrifugation for
10 minutes at 2210 g. The plasma
samples were then stored at -75°C
until analysis. Before the assay,
the samples were thawed on ice
for approximately 1 hour, then
centrifuged for 20 minutes at
2210 g and diluted 1:5 in the
cAMP Buffer for Standards. 50 µL
of the diluted sample was used in
the acetylation procedure.
Figure 1. DELFIA cAMP assay principle.
Protocol
Anti-cAMP serum solution was
added to all wells (50 µL/well)
reserved for standards and
samples on the 96-well plate, and
Assay Buffer was added to wells
reserved as blanks. The plate was
incubated for 1 hour and 40
minutes at room temperature on a
DELFIA Plateshake (PerkinElmer
1296-001/002 or 1296-003/004).
During the incubation, standards
were prepared. A 50 µL aliquot
of cAMP standards was acetylated
with 3 µL of the Acetylating
Reagent, vortexed and incubated
for 10 minutes and then diluted
1:10 in the cAMP Buffer for
Standards.
A 50 µL aliquot of the centrifuged
and diluted plasma sample was
acetylated with 3 µL of the
Acetylating Reagent, vortexed,
incubated for 10 minutes, and
then diluted 1:12 in the cAMP
Buffer for Standards. The plasma
samples were kept on ice during
the assay.
Authors
Susann Björk
Department of Pharmacology
and Clinical Pharmacology,
University of Turku, Finland
Sofia Vikström
PerkinElmer Life and Analytical
Sciences, Wallac OY, Turku, Finland
DELFIA C AMP KIT
A P P L I C A T I O N N O T E
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50 µL of the acetylated and diluted
standards and samples were added
to the 96-well plate, which had
been pre-incubated with anticAMP
serum solution, followed by
addition of 100 µL Eu-cAMP Tracer
Solution. No Eu-cAMP Tracer
Solution was added to the wells
reserved as blanks.
The competition reaction was
performed at room temperature for
1 hour on the DELFIA Plateshake.
The plate was subsequently
washed 4 times with Wash
Solution using an automated
DELFIA Platewash (PerkinElmer
1296-026). Finally, 200 µL of
Enhancement Solution was added
to all wells. The Enhancement
Solution dissociates Europium ions
from the labelled antigen into
solution where they form highly
fluorescent chelates with components
of the Enhancement Solution.
The plate was incubated for 8
minutes on the DELFIA Plateshake
at room temperature. The plate was
then incubated in the dark, at room
temperature for 35 minutes before
measuring. The Eu-fluorescence
was measured using a plate reader
with time-resolved fluorescence
capability (PerkinElmer VICTOR 3
Multilabel Plate Reader).
Note: We do not recommend the use
of EDTA as anticoagulant for blood
samples and recommend that
alternative anticoagulants be used.
The DELFIA chelate is not stable at
high concentrations of EDTA.
However, when preparing samples
according to this protocol, having
too high EDTA concentration in the
samples is avoided. The EDTA
concentration in this assay was
approximately 70 µM.
Figure 2. DELFIA cAMP
Assay flow chart
2

Assay Validation Results
Effect of Anticoagulants
The effect of different anticoagulants
was tested. cAMP standards
were mixed with plasma samples
that had been drawn in serum
tubes, serum-gel tubes, lithiumheparin
tubes or EDTA tubes. The
spiked samples were then assayed
using the DELFIA cAMP kit using
the protocol for acetylated samples.
Results indicate that the chelate
cannot tolerate more than 70 µM
EDTA. If the EDTA concentration
is higher than this in the assay, the
signal will be reduced. Note that
when using the acetylated standards,
the analytical sensitivity for the
DELFIA cAMP kit is typically better
than 0.2 pg/well (2.8 pmol/L).
Intra-assay Precision
Three pools of plasma sample were
prepared from human plasma
samples containing different
amounts of cAMP. The sample
pools were stored at -75°C. The
intra-assay precision was determined
by running each sample
six times in triplicate. Results are
shown in Table 1.
Inter-assay Precision
Three pools of human plasma
samples containing different levels
of cAMP (different from the ones
used for the intra-assay) were
prepared. The pools were divided
into aliquots, and for each analysis
one aliquot was thawed and used.
The inter-assay precision was
determined according to the same
scheme as the intra-assay. All
analyses were performed on separate
days as individual experiments.
Results are shown in Table 2.
Figure 3. Effect of different anticoagulants on assay performance.
Table 1. Results from Intra-assay Precision Study
Sample Pool AVG SD CV%
Pool 1 47.86 1.99 4.15
Pool 2 32.10 3.28 10.19
Pool 3 20.37 1.65 8.10
Table 2. Results from Inter-assay Precision Study
Sample Pool AVG SD CV%
Pool 1 32.42 3.98 12.28
Pool 2 20.98 2.64 12.57
Pool 3 14.15 2.02 14.26
Comparison between the
DELFIA cAMP assay and a
standard RIA cAMP assay
Blood samples were taken from a
healthy volunteer, and a pool of
plasma was prepared. The pooled
plasma was spiked with 0–200
pmol/mL cAMP and prepared as
shown in Table 3. (The concentration
of the cAMP standard is 5000
pmol/mL)
Each 1 mL sample was divided into
4 tubes, so that separate aliquots
could be used for the RIA and the
DELFIA assay (to avoid the
effect of thawing, since both
analyses not could be performed
simultaneously). The standard
RIA cAMP assay was performed
with the FlashPlate ® cAMP
[ 125 I]- Radioimmunoassay Kit
(PerkinElmer SMP004) according
to the procedure protocol for
acetylated samples. The standards
and samples were assayed in
triplicate. The DELFIA cAMP assay
was performed according to the
protocol described earlier. Results
are shown in Figure 4.
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3

Conclusions
The DELFIA cAMP kit can be used
for measuring cAMP in plasma
samples. The DELFIA cAMP assay
shows excellent correlation with
a standard RIA cAMP assay. The
europium chelate is not stable at
high EDTA concentrations, so there
is reason to keep the EDTA concentration
as low as possible if using
EDTA tubes for blood samples.
This application note provides a
protocol for diluting the plasma
samples. The sensitivity of the
DELFIA assay allows for the plasma
samples to be diluted both prior to,
and after acetylation, so that a high
concentration of EDTA can be avoided
when performing the assay.
Table 3. Dilutions of samples for comparison between
RIA and DELFIA cAMP kits
Plasma Standard Buffer Final volume Spiked [cAMP]
(µL) (µL) (µL) (mL) (pmol/mL)
950 0 50 1000 0
950 2 48 1000 10
950 4 46 1000 20
950 7 43 1000 35
950 10 40 1000 50
950 12 38 1000 60
950 15 35 1000 75
950 18 32 1000 90
950 20 30 1000 100
950 24 26 1000 120
950 30 20 1000 150
950 35 15 1000 175
950 40 10 1000 200
Products Available
Cat. No.
CR89-102
CR92-102
Product
DELFIA cAMP kit,
2 plates (96-wells)
DELFIA cAMP 384 kit,
2 plates (384-wells)
4003-0010 DELFIA cAMP kit,
10 plates (96-wells)
4004-0010 DELFIA cAMP 384 kit,
10 plates (384-wells)
4005-0010 DELFIA Lysis Buffer,
30 mL
Complementary PerkinElmer
Multilabel Plate Readers
and Imagers:
VICTOR 3 Multilabel Reader
Envision Multilabel Plate Reader
ViewLux ultraHTS Microplate Imager
Figure 4. Comparison of the DELFIA cAMP assay and a [ 125 I]-cAMP RIA.
The spiked samples were acetylated. The DELFIA cAMP assay shows
excellent correlation with a standard RIA cAMP assay.
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Analytical Sciences
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Shelton, CT 06484-4794 USA
Phone: (800) 762-4000 or
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