cAMP [125-I] Radioimmunoassay kit (Adenosine 3',5' cyclic monophosphate)

PerkinElmer Life and Analytical Sciences, Inc.
cAMP [125I]
RADIOIMMUNOASSAY KIT
(ADENOSINE 3', 5'
CYCLIC MONOPHOSPHATE)
CATALOG NUMBER NEK033
For Laboratory Use
CAUTION: A research chemical for research purposes only.
1
TABLE OF CONTENTS
I.
Proprietary Name
3
II.
Intended Use
3
III.
Explanation of Test
3
IV.
Principle of the Method
3
V.
Reagents
4
VI.
Sample Collection, Processing and Storage
8
VII.
Non-Acetylated Procedure (Urine)
10
VIII.
Acetylated Procedure (Plasma)
14
IX.
Procedure for Calculating Unknowns in Urine
and Plasma Samples
18
X.
Precautions
22
XI.
Performance Characteristics for the Non-Acetylated
(Urine) Procedure
22
Performance Characteristics for Acetylated (Plasma)
Procedure
25
XIII.
Correlation
27
XIV.
References
27
XII.
2
I.
PROPRIETARY NAME
cAMP [125I] RIA Kit
PerkinElmer Life Sciences, Inc. Catalog Number: NEK033, 200 Tubes
II.
INTENDED USE
This kit is designed to measure cAMP levels in plasma, urine and
tissues.
III.
EXPLANATION OF TEST
Adenosine 3', 5' cyclic monophosphate (cAMP) plays a critical role in
the transmission of hormonal signals by functioning as a “second
messenger”1,2. The binding of a hormone to its receptor can either
enhance or inhibit the rate at which cAMP is produced. This is
accomplished by altering the enzymatic activity of adenyl cyclase, the
membrane associated enzyme which catalyzes the production of cAMP
from ATP3. By this mechanism, intracellular levels of cAMP are
altered in response to hormonal stimulation. In turn, the intracellular
level of cAMP regulates the enzymatic activity of a protein kinase
which phosphorylates other substances setting off a cascade of cellular
events which leads to the expression of the hormone4.
The cAMP Assay is a double-antibody RIA which utilizes a prereacted
antibody complex. The assay is performed using acetylated or nonacetylated standards and samples. Acetylation of the sample allows for
a more sensitive assay of cAMP5,6. The assay is accurate over a wide
range of values and has a high degree of specificity.
IV.
PRINCIPLE OF THE METHOD
The basic principle of radioimmunoassay is the competition between
a radioactive and a non-radioactive antigen for a fixed number of
antibody binding sites. This interaction is illustrated in Figure 17.
3
Figure 1
Labeled Antigen
Ag*
+
Specific Antibody
Ab
→
←
Labeled Antigen
Antibody Complex
Ag*Ab
+
Unlabeled Antigen
Ag
(In standard solutions
↑↓
or unknown samples)
Unlabeled Antigen-Antibody Complex
AgAb
When unlabeled antigen from standards or samples and a fixed amount
of the labeled antigen are allowed to react with a constant and limiting
amount of antibody, decreasing amounts of the labeled antigen are
bound to the antibody as the amount of unlabeled antigen is increased.
In the cAMP [125I] Kit, separation of the bound from free antigen is
achieved by using a prereacted primary antibody/secondary antibody
complex. The second antibody is derived from an animal species
different from that used to generate the primary antibody.
After incubation and centrifugation, the supernatant is discarded and
the antigen-antibody complex is counted to quantitate the bound tracer.
The data are used to construct a standard curve from which the values
of the unknowns may be obtained by interpolation.
V.
REAGENTS
All necessary reagents are supplied and are intended FOR
LABORATORY USE. Kits are shipped at ambient temperatures and
must be stored upon receipt at refrigerator temperature (2-8°C). The
reagents are stable for the times indicated if the specific precautions
given below are followed. Sodium azide has been added as an
antibacterial agent where appropriate.
4
NOTE:
The National Institute for Occupational Safety and Health
has issued a bulletin citing the potentially explosive hazard
due to the reaction of sodium azide with copper, lead, brass,
or solder in plumbing systems. Although sodium azide is
added at a minimal concentration, it is still recommended
that copious amounts of water be used to flush the drain
pipeline after disposal of these reagents in the plumbing
system. Copper-free and lead-free discharge lines should be
used whenever possible. Decontamination procedures
should be followed prior to maintenance on drain lines
which have been used for azide-containing reagents.
Recommended decontamination procedures are available
from RIA Technical Services.
NOTE:
An excess of each reagent beyond the labeled content is
added to each container in order to allow the withdrawal of
the number of aliquots required to fill the stated quantity of
tubes. Any reagent remaining after the stated quantity of
tubes have been prepared should be discarded.
A.
cAMP Sodium Acetate Buffer
One vial of concentrated buffer is supplied. Dilute to 500 mL
with distilled water. The final solution will contain sodium
acetate buffer, pH 6.2, and a stabilizer. The diluted buffer is
stable for at least two months when stored at 2-8°C. Refer to
vial label for exact expiration date of the concentrated reagent.
B.
cAMP Standard
One vial of liquid standard is supplied. The solution contains:
cAMP at a concentration of 50,000 pmol/mL, sodium acetate
buffer, and 0.09% sodium azide. The cAMP Standard has
been calibrated spectrophotometrically using the molar
absorption coefficient, Π= 14.6 x 103l mol-1 cm-1 at 259 nm,
pH 6.9. The standard is stable for at least one year when stored
at 2-8°C. Refer to vial label for expiration date of reagent.
5
C.
cAMP Antiserum Complex
One vial of lyophilized, prereacted, first and second antibody
is supplied. Reconstitute with 21 mL of distilled water. The
resulting solution will contain the prereacted antibody
complex, and an inert ingredient, in sodium phosphate buffer,
pH 6.0. This solution may appear cloudy and will settle upon
standing. Before use and during prolonged use, mix
thoroughly.
The reconstituted antiserum complex is stable for at least two
months when stored at 2-8°C. Refer to vial label for expiration
date of lyophilized reagent.
D.
cAMP [125I] Tracer (Succinyl cAMP Tyrosine Methyl Ester
[125I])
Two vials of concentrated tracer are supplied. Each vial
contains less than 74KBq (2 µCi) on calibration date in 1 mL
of a 1:1 n-propanol water solution. Use one vial at a time as
directed. Add 5.0 mL of distilled water to each vial as
required. The concentrate and diluted tracer are stable for at
least two months when stored at 2-8°C. This material is
radioactive and the user should follow the precautions listed
on the following page.
6
INSTRUCTIONS RELATING TO THE HANDLING, USE, STORAGE,
AND DISPOSAL OF THIS RADIOACTIVE MATERIAL
This radioactive material may be received, acquired, possessed, and used only
by research laboratories for in vitro laboratory tests not involving internal or
external administration of the material, or the radiation therefrom, to human
beings or animals. Its receipt, acquisition, possession, use and transfer are
subject to the regulations and a general license of the U. S. Nuclear Regulatory
Commission or of a State with which the Commission has entered into an
agreement for the exercise of regulatory authority.
1.
All radioactive materials must be labeled and secured in specifically
designated posted areas. Records of receipt and survey must be
maintained.
2.
All work with these materials must be carried out only in authorized
areas.
3.
Prohibit mouth pipetting of radioactive materials.
4.
There must be no smoking or eating within the work area.
5.
Hands must be washed after handling radioactive materials.
6.
Any spilled material must be wiped up quickly and thoroughly and the
contaminated substances transferred to a suitable receptacle. The
surfaces involved must be washed thoroughly with an appropriate
decontaminant. Monitor to ensure the area has been effectively
decontaminated.
7.
When use of the Tracer reagent has been completed, empty and decontaminate the vial. This radioactive material can be discarded into the
sanitary sewerage system using copious amounts of water to ensure a
minimal discharge concentration.
8.
Prior to disposal of the empty, uncontaminated Kit and Tracer containers
to unrestricted areas, remove or deface the radioactive material labels
or otherwise clearly indicate that the containers no longer contain
radioactive material.
7
E.
cAMP Carrier Serum
Two vials of lyophilized carrier serum are supplied. Use one
vial at a time. Reconstitute the contents of each vial with
exactly 6.0 mL of distilled water. (One vial of reconstituted
carrier serum is sufficient for 100 tubes.) The resulting
solution will contain carrier serum, 0.1% sodium azide, a
stabilizer, and an inert ingredient in sodium acetate buffer,
pH 6.2. Refer to vial label for expiration date of lyophilized
reagent. Store at 2-8°C.
F.
cAMP Acetic Anhydride
One vial containing 1 mL is supplied. CAUTION:
FLAMMABLE, CORROSIVE, LACHRYMATOR. Store
tightly closed in the refrigerator. Allow vial to equilibrate to
room temperature before use. Protect from moisture. This
material is stable for at least two months under these
conditions. Refer to vial label for expiration date.
G.
cAMP Triethylamine
One vial containing 1 mL is supplied. CAUTION:
FLAMMABLE, VAPOR HARMFUL. Store tightly closed in
the refrigerator. Allow vial to equilibrate to room temperature
before use. Refer to vial label for expiration date.
H.
cAMP Precipitator
One bottle containing 100 mL is supplied ready to use and
contains a precipitation enhancer and sodium azide in sodium
acetate buffer. Refer to bottle label for expiration date. Store at
2-8°C. Shake well before using.
VI.
SAMPLE COLLECTION, PROCESSING AND STORAGE
A.
Plasma: EDTA-treated plasma should be used. Collect blood
by venipuncture in a 5 or 10 mL glass blood collection tube
containing EDTA (lavender top). The usual precautions for
venipuncture apply. Samples should be kept on ice after
drawing. Separation of cells from plasma should be carried out
as soon as possible after collection in a refrigerated centrifuge
(15 minutes, approximately 760 x g). Plasma may be stored at
-20°C for at least four weeks. Repeated freezing and thawing
should be avoided.
B.
Urine: Voided urine specimens are required for the assay.
Urine samples should be centrifuged to remove any particulate
8
matter present. Random, timed, or 24 hour urine collections
can be used. For twenty-four hour specimens, it may be
necessary to prevent bacterial growth by collecting urine into
acid (2 mL of 6 N HCl per 100 mL urine). Urine collected
utilizing antibacterial agents should be validated as a suitable
specimen by the operator. Urine samples can be stored
undiluted at 2-8°C for 24 hours, but should be frozen at -20°C
for longer storage.
C.
Tissue Samples: Precipitation of proteins from plasma or
tissues has been accomplished with trichloroacetic acid
(TCA), perchloric acid, or ethanol followed, in some cases,
by ion exchange or alumina column chromatography. The
decision as to which procedure to use depends on the nature
of the sample and is left up to the individual investigator.
This protocol is applicable to tissue culture cells as well as
solid tissue; however, it is the responsibility of the investigator
to validate this procedure for each specific application. As part
of this validation, we recommend adding phosphodiesterase to
some samples and noting the loss of immunoreactivity.
1.
Homogenize the frozen tissue sample at 4°C with
6% TCA to make a 1 mL 10% (w/v) homogenate.
Add an equal volume of cold 10% TCA to cell
culture preparations or supernatants.
2.
To determine the recovery of cyclic AMP during
extraction, add to each sample extract approximately
4,000 cpm of 3 H-cyclic AMP marker
(NET275 [2,8 3H] Cyclic AMP, diluted 1:200 in
50% Ethanol / 50% H2O solution, available from
PerkinElmer Life Sciences) to the TCA extract. At a
specific activity of approximately 37 Ci/mmol and at
a 50% counting efficiency, this represents
approximately 0.1 pmol (100 fmol) of cyclic AMP.
This amount must be taken into account when
calculating the cyclic AMP content of the tissue.
3.
Centrifuge TCA extracts at 2,500 x g at 4°C for
15 minutes.
4.
Collect the supernatant and extract 4 times with
5x volume of water-saturated ether. Discard the ether
phase.
5.
Place sample in a water bath at 70-80°C and
evaporate to dryness under a stream of air.
9
6.
Dissolve the residue in Assay Buffer (volume
depends on the amount of c-AMP in the sample) and
use 100 µL directly in the Assay or the sample may
be diluted. The decision as to which procedure to use
for the sample depends on expected levels of cyclic
AMP in the sample.
If necessary, the TCA extract may be purified further
by ion exchange column chromatography. In this
example, it is not necessary to remove the TCA with
ether as the sample may be applied directly to the
column.
a.
Prepare a 0.6 x 5.0 cm column of Dowex
50w - X8 (H+), 200-400 mesh in water. This
is conveniently done in a disposable Pasteur
capillary pipet.
b.
Prior to the sample additions to the column,
it should be characterized to locate which
fraction contains 3H-cyclic AMP marker.
Use water as the eluate.
c.
After the elution volume containing
3
H-cyclic AMP has been determined, allow
the water to drain into the resin bed, pipet
1 mL of the TCA extract onto the column
and start collecting 1 mL samples.
d.
After the TCA extract has drained into the
column, add water and continue to collect
the effluent.
e.
Combine the fractions previously
determined to contain 3H-cyclic AMP
marker and continue at Step 5. We have
found recoveries > 90% with this procedure.
If an excess of cyclic AMP is suspected in
the sample, dilute with Assay Buffer.
VII.
NON-ACETYLATED PROCEDURE (URINE)
A.
Reagents Supplied - sufficient for 200 tubes
1.
1 vial cAMP Antiserum Complex, lyophilized
2.
2 vials cAMP [125I]-Tracer, 1 mL (concentrate)
10
B.
3.
2 vials cAMP Carrier Serum, lyophilized
4.
1 vial cAMP Standard, liquid
5.
1 vial cAMP Buffer, 25 mL (concentrate)
6.
1 bottle cAMP Precipitator, 100 mL
Equipment and Reagents Required
In addition to the reagents supplied with the kit, the
following materials are required:
C.
1.
Pipettors and/or pipets that can accurately and
precisely deliver the required volumes.
2.
Gamma scintillation counter.
3.
Laboratory vortex mixer.
4.
Test tube rack.
5.
Distilled water.
6.
Centrifuge - refrigerated (swing bucket head).
7.
Test tubes - 12 x 75 mm - glass.
8.
Test tube - 13 x 100 mm - glass.
9.
Absorbent paper for blotting.
10.
Radioactive waste container.
11.
2-8°C refrigerator or equivalent.
Urinary Radioimmunoassay Protocol
NOTE:
A full understanding of the Protocol and
Precautions is necessary for successful
completion of the RIA. Read these two sections
carefully before proceeding with the assay.
Seven levels of cAMP standards are recommended, each
point to be run in duplicate.
1.
Prepare standard solutions as follows, mixing each
solution thoroughly before adding it to the next
tube. Prepare the standards fresh each day.
11
Pipet mL
cAMP Std.
From
Tube
Add mL
Assay Buffer
Into
Labeled Tube
Concentration
(pmol/mL)
0.1
0.2
1.0
1.0
1.0
1.0
1.0
1.0
*
A
B
C
D
E
F
G
9.9
1.8
1.0
1.5
1.0
1.0
1.5
1.0
A
B
C
D
E
F
G
H
500
50
25
10
5
2.5
1.0
0.5
*50,000 pmol/mL (stock standard reagent)
2.
Prepare the urine sample as follows: First, dilute the
urine specimen tenfold by adding 100 µL of urine to
900 µL of Assay Buffer. Then, take 100 µL of the
tenfold dilution and add it to 4.9 mL of Assay
Buffer. This produces a 1/500 dilution of the
sample.
3.
Prepare Working Tracer Solution by adding one
volume of diluted cAMP [125I]-Tracer to one
volume of the reconstituted cAMP Carrier Serum.
Make enough of the Working Tracer Solution to run
the desired number of tubes (e.g., 1.0 mL of cAMP
Carrier Serum and 1.0 mL of cAMP [125I]-Tracer
will provide 2.0 mL of solution which is
theoretically sufficient for 20 tubes). Any remaining
volume is to be discarded appropriately. Do not
store and reuse this solution.
4.
Number a series of 20 tubes to be used for the
standard curve, plus two additional tubes for each
sample. The assay may be set up at room
temperature.
5.
Tubes 1 and 2 measure the total counts added and
receive only the Working Tracer Solution.
6.
Add 200 µL of Assay Buffer to tubes 3 and 4 (blank
tubes).
7.
Add 100 µL of Assay Buffer to tubes 5 and 6 (zero
standard tubes).
12
8.
Add 100 µL of each standard solution or sample to
the appropriate tubes (see Table I).
9.
Add 100 µL of the Working Tracer Solution to all
tubes (see Step 3).
10.
Thoroughly mix Antiserum Complex and add
100 µL to all tubes, except Total Count tubes
and Blank tubes.
11.
Mix all tubes, except 1 and 2, by using a vortex
mixer.
12.
Cover and incubate overnight (16-18 hours) at
2-8°C.
13.
Set tubes 1 and 2 aside, and add 500 µL of 2-8°C
cAMP Precipitator to all the other tubes. Mix well
with a vortex mixer and centrifuge 2-8°C for
15 minutes at approximately 1,200 x g.
14.
Decant by gently inverting all tubes once, preferably
at the same time, discarding the supernatant into a
radioactive waste container. Keeping the tubes
inverted, place them on absorbent paper for blotting.
To facilitate removal of remaining droplets, gently
tap the rims of the tubes on the paper. Allow tubes
to drain for 20-30 seconds.
15.
Count all tubes, including Tubes 1 and 2, in a
gamma counter. At the usual counting efficiency of
50-70%, a counting time of one minute should be
sufficient.
16.
Calculations are described in Section IX.
13
TABLE I - PROTOCOL FOR NON-ACETYLATED STANDARD CURVE
(All volumes are in microliters)
Tube
Total
Cts.
Blank
“0” Std.
0.5 Std.
1 Std.
2.5 Std.
5 Std.
10 Std.
25 Std.
50 Std.
Sample
1, 2
3, 4
5, 6
7, 8
9, 10
11, 12
13, 14
15, 16
17, 18
19, 20
21, 22
Assay Stds. SamBuffer
ple
200
100
-
100
100
100
100
100
100
100
-
100
Working
Tracer
Antiserum
Complex
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
Precipitator
Mix.
Cover
and
incubate
16-18
hours
at 2 8°C.
500
500
500
500
500
500
500
500
500
500
Centrifuge at 1,200 x g for 15 minutes at 2-8°C. Decant and blot tubes. Count pellets.
VIII.
ACETYLATED PROCEDURE (PLASMA)
A.
B.
Reagents Supplied - sufficient for 200 tubes
1.
1 vial cAMP Antiserum Complex, lyophilized
2.
2 vials cAMP [125I]-Tracer, 1 mL (concentrate)
3.
2 vials cAMP Carrier Serum, lyophilized
4.
1 vial cAMP Standard, liquid
5.
1 vial cAMP Buffer, 25 mL (concentrate)
6.
1 vial cAMP Triethylamine
7.
1 vial cAMP Acetic Anhydride
8.
1 bottle cAMP Precipitator, 100 mL
Equipment and Reagents Required
In addition to the reagents supplied with the kit, the
following materials are required:
14
C.
1.
Pipettors and/or pipets that can accurately and
precisely deliver the required volumes.
2.
Gamma scintillation counter.
3.
Laboratory vortex mixer.
4.
Test tube rack.
5.
Distilled water.
6.
Centrifuge - refrigerated (swing bucket head).
7.
Test tubes - 12 x 75 mm - glass.
8.
Test tube - 13 x 100 mm - glass.
9.
Absorbent paper for blotting.
10.
Radioactive waste container.
11.
2-8°C refrigerator or equivalent.
Acetylated (Plasma) Radioimmunoassay Protocol
NOTE: A full understanding of the Protocol and
Precautions is necessary for successful
completion of the RIA. Read these two sections
carefully before proceeding with the assay.
1.
Dilute the cAMP stock standard reagent
(50,000 pmol/mL) fifty-fold in a 13 x 100 mm test
tube by adding 100 µL of the 50,000 pmol/mL
solution to 4.9 mL of Assay Buffer. The resulting
solution will be 1,000 pmol/mL. Prepare a
40 pmol/mL solution by adding 0.1 mL of the
1,000 pmol/mL solution to 2.4 mL of Assay Buffer.
Place 200 µL of the 40 pmol/mL solution in a
marked glass tube.
2.
Prepare the plasma samples for acetylation as
follows: Pipet 100 µL of the plasma sample into
400 µL of Assay Buffer (1:5 dilution). Place 100 µL
of the 1:5 dilution in a marked glass test tube.
3.
Prepare 150 µL of acetylation reagent by mixing
together in a glass test tube 100 µL of Triethylamine and 50 µL of Acetic Anhydride. Vortex
before using. Make up a fresh solution each time.
15
4.
Prepare 10 mL of Modified Assay Buffer by adding 50 µL of
the acetylation reagent (Step 3) to 10 mL of Assay Buffer.
Mix well and let incubate at room temperature for at least
three minutes. Modified Assay Buffer is used for the
preparation of the cAMP standards and in the “blank”
and “zero standard” tubes (see Table II).
5.
Acetylate the 40 pmol/mL standard prepared in Step 1 (i.e.,
the 200 µL aliquot) by adding to it 10 µL of the freshly
prepared acetylation reagent. Allow the reaction to proceed
for at least three minutes at room temperature and then add
1.8 mL of Assay Buffer. Label this 4 pmol/mL standard as
tube A. The amount of acetylation reagent used is sufficient
to acetylate 25,000 pmols of cAMP.
6.
Acetylate the previously prepared 100 µL aliquot of diluted
sample (Step 2) by adding 5 µL of acetylation reagent to
each tube. Immediately vortex and let the sample incubate
for at least three minutes at room temperature. Then add
900 µL of Assay Buffer to each tube. This makes a 1:50
dilution of the sample.
7.
Dilute the 4 pmol/mL standard with Modified Assay Buffer
(see Step 4) as shown below. The standards should be
prepared fresh each day. Do not store for reuse.
Pipet mL
cAMP Std.
From
Tube
Add mL Modified
Assay Buffer
Into
Labeled Tube
Concentration
(pmol/mL)
1.0
1.0
1.0
1.0
1.0
1.0
A
B
C
D
E
F
1.0
1.0
1.0
1.0
1.5
1.0
A
B
C
D
E
F
G
4.0
2.0
1.0
0.5
0.25
0.10
0.05
8.
Prepare Working Tracer Solution by adding one volume of
diluted cAMP [125I]-Tracer to one volume of the
reconstituted cAMP Carrier Serum. Make enough of the
Working Tracer Solution to run the desired number of tubes
(e.g., 1.0 mL of cAMP Carrier Serum and 1.0 mL of cAMP
[125I]-Tracer will provide 2.0 mL of solution which is
theoretically sufficient for a maximum of 20 tubes). Any
16
remaining volume is to be discarded appropriately. Do not
store and reuse this solution.
9.
Number a series of 20 tubes to be used for the standard
curve, plus two additional tubes for each sample. The assay
may be set up at room temperature.
10.
Tubes 1 and 2 measure the total counts added and receive
only the Working Tracer Solution.
11.
Add 200 µL of Modified Assay Buffer to Tubes 3 and 4
(blank tubes).
12.
Add 100 µL of Modified Assay Buffer to Tubes 5 and 6
(zero standard tubes).
13.
Add 100 µL of each standard solution or sample to the
appropriate tubes (see Table II).
14.
Add 100 µL of Working Tracer Solution to all tubes.
15.
Thoroughly mix Antiserum Complex and add 100 µL to
all tubes, except Total Count tubes and Blank tubes.
16.
Mix all tubes, except 1 and 2, by using a vortex mixer.
17.
Cover and incubate overnight (16-18 hours) at 2-8°C.
18.
Set Tubes 1 and 2 aside, and add 500 µL of 2-8°C cAMP
Precipitator to all the other tubes. Mix well with a vortex
mixer and centrifuge at 2-8°C for 15 minutes at
approximately 1,200 x g.
19.
Decant by gently inverting all tubes once, preferably at the
same time, discarding the supernatant into a radioactive
waste container. Keeping the tubes inverted, place them on
absorbent paper for blotting. To facilitate removal of
remaining droplets, gently tap the rims of the tubes on the
paper. Allow tubes to drain for 20-30 seconds.
20.
Count all tubes, including Tubes 1 and 2, in a gamma
counter. At the usual counting efficiency of 50-70%, a
counting time of one minute should be sufficient. Include
an instrument blank.
21.
Calculations are described in Section IX.
17
TABLE II - PROTOCOL FOR ACETYLATED STANDARD CURVE
(All volumes are in microliters)
Tube
Assay
Buffer
Stds.
Sample
Working
Tracer
Antiserum
Complex
Precipitator
Total
Cts.
1, 2
-
-
-
100
-
Blank
3, 4
200
-
-
100
-
“0” Std.
5, 6
100
-
-
100
100
0.05 Std.
7, 8
-
100
-
100
100
0.10 Std.
9, 10
-
100
-
100
100
500
0.25 Std.
11, 12
-
100
-
100
100
500
0.5 Std.
13, 14
-
100
-
100
100
500
1.0 Std.
15, 16
-
100
-
100
100
500
2.0 Std.
17, 18
-
100
-
100
100
500
4.0 Std.
19, 20
-
100
-
100
100
500
Sample
21, 22
-
-
100
100
100
500
Mix.
Cover
and
incubate
16-18
hours
at
2-8°C.
-
500
500
500
Centrifuge at 1,200 x q for 15 minutes at 2 - 8°C. Decant and blot tubes. Count pellets.
IX.
PROCEDURE FOR CALCULATING UNKNOWNS IN URINE
AND PLASMA SAMPLES
NOTE:
Urine and plasma sample values are determined in an
identical manner, i.e., by interpolation from their
respective standard curves. The range of standard
concentrations on a non-acetylated (urine) standard
curve is from 0-50 pmol/mL. The range of standard
concentrations on an acetylated (plasma) standard
curve is from 0-4 pmol/mL.
18
A.
If all tubes have been counted for the same period of time,
use the total accumulated counts, otherwise, correct all
counts to a common count rate.
B.
Average the counts for each set of duplicates.
C.
Calculate the average Net Counts for all standards and
samples by subtracting from each the average blank counts.
D.
Express the average Net Counts for each standard and
sample as a percentage of the average Net Counts for the
zero standard. (This is termed “normalized” percent bound or
% B/Bo.)
% B/Bo = Average Net Counts of Standard or Sample x 100
Average Net Counts of Zero Standard
E.
Using semi-logarithmic or log-logit graph paper, plot
% B/Bo for each standard against the corresponding
concentration of cAMP in pmol/mL.
F.
Determine the concentration of cAMP in the samples by
interpolation from the standard curve. Since identical
volumes are used for standards and samples, and the
standard curve is expressed as pmol/mL of cAMP, samples
can be read as pmol/mL and then multiplied by the
appropriate dilution factor (i.e., 50 for plasma samples, 500
for urine samples) to calculate the sample concentration. Any
samples with concentrations above the range of the standard
curve must be diluted and re-assayed. The values are then
multiplied by the appropriate dilution factor. See Tables III
and IV for sample calculations, and Figure 2 for typical
standard curve.
19
TABLE III - TYPICAL DATA FOR NON-ACETYLATED (URINE) ASSAY
Tube
Number
Counts
Bound
Average
Counts
Bound
Net
Counts
Bound
Normalized
%
Bound
Blank
1
2
1016
950
983
-
-
“0”
Standard
3
4
16892
17078
16985
16002
100
0.5 pmol/mL
5
6
15340
15662
15501
14518
91
1.0 pmol/mL
7
8
14305
14369
14337
13354
83
2.5 pmol/mL
9
10
12436
12461
12448
11466
72
5.0 pmol/mL
11
12
10385
10508
10446
9463
59
10 pmol/mL
13
14
7837
8034
7936
6952
43
25 pmol/mL
15
16
5287
5502
5394
4412
28
50 pmol/mL
17
18
3764
3844
3804
2812
18
20
TABLE IV - TYPICAL DATA FOR ACETYLATED (PLASMA) ASSAY
Tube
Number
Counts
Bound
Average
Counts
Bound
Net
Counts
Bound
Normalized
%
Bound
Blank
1
2
923
1003
963
-
-
“0”
Standard
3
4
15856
16227
16042
15079
100
0.05
pmol/mL
5
6
14942
15008
14975
14012
93
0.10
pmol/mL
7
8
13671
13452
13562
12598
84
0.25
pmol/mL
9
10
10839
11179
11009
10046
67
0.50
pmol/mL
11
12
8003
8050
8026
7064
47
1.0 pmol/mL
13
14
5412
5571
5492
4528
30
2.0 pmol/mL
15
16
3589
3539
3564
2601
17
4.0 pmol/mL
17
18
2493
2578
2536
1572
10
Figure 2
cAMP Typical Standard Curve
100
90
80
nonAcet
70
Acet
%B/B0
60
50
40
30
20
10
0
0.01
0.1
1
10
cAMP, pmol/mL
21
100
1000
X.
PRECAUTIONS
A.
Incubation conditions should be standardized for proper day
to day internal quality control.
B.
As with all radioimmunoassay procedures, pipetting is
crucial. It is essential that pipetting be accurate and
reproducible.
C.
Samples with concentrations above the range of the standard
curve may be reassayed after dilution with Assay Buffer
(urine samples) or Modified Assay Buffer (plasma samples).
The values obtained are then multiplied by the appropriate
dilution factor. During the preparation of plasma samples, it
is important to make an initial 1:50 dilution of the acetylated
sample as previously specified, and then to dilute further
with Modified Assay Buffer.
D.
The use of grossly hemolyzed or lipemic samples should be
avoided.
E.
The presence of exogenous radioactivity in clinical samples
may lead to erroneous results.
F.
Avoid using a centrifuge which may overheat due to
prolonged use.
G.
The reagents in this kit should be used as a unit. Do not mix
different lots of any component within a given assay.
H.
This product has not been tested for use with any methods
other than those stated in this Instruction Manual.
I.
XI.
The average Net Counts for the “0” Standard represents the
counts bound to the antibody in the absence of added cAMP. This
figure should fall between 40-60% of the average net total
counts. The acetylated procedure will generally have a slightly
lower Bo than the non-acetylated procedure.
PERFORMANCE CHARACTERISTICS
ACETYLATED (URINE) PROCEDURE
A.
FOR
THE
NON-
Recovery
To demonstrate the accuracy of the method, recovery studies
were performed by adding known quantities of cAMP to
22
aliquots of Assay Buffer and three urine samples. The
resulting solutions were re-assayed and the recovery of
added cAMP was determined. Typical results are given
below.
+ 5 nmol/mL cAMP
Sample
Assay
Buffer
A
B
C
B.
nmol/mL
Base
Value
nmol/mL
Measured
% Rec
0
2.4
1.4
3.0
5.4
7.6
6.2
8.5
108
104
96
110
+ 15 nmol/mL
cAMP
+45 nmol/mL
cAMP
nmol/mL % Rec nmol/mL % Rec
Measured
Measured
15.6
17.5
18.2
19.0
104
101
112
107
44.1
49.5
51.2
48.8
98
105
111
102
Reproducibility
Precision was determined by multiple duplicate analyses of
urine pools. Typical results are given in nmol/mL.
Within Assay Variation
Sample
n
Mean + 1
S.D.
A
B
C
D
10
10
9
10
5.4 + 0.19
2.4 + 0.07
1.3 + 0.03
4.0 + 0.07
Between Assay Variation
Coeff. Sample n
of
Var.
(%)
3.5
2.9
2.3
1.8
23
A
B
C
D
E
F
G
H
6
6
6
6
6
6
6
6
Mean + 1
S.D.
Coeff.
of
Var. (%)
9.5 + 0.89
3.4 + 0.16
7.1 + 0.69
3.2 + 0.22
3.8 + 0.25
3.5 + 0.2
5.0 + 0.56
1.9 + 0.18
9.0
4.7
9.7
6.8
6.5
5.7
11.0
9.4
C.
Linearity
To demonstrate the absence of non-specific urine
interferences, five samples were assayed by the standard
procedure and diluted to varying degrees with Assay Buffer.
The recommended 100 µL aliquot was used for all samples.
Typical results are given in nmol/mL, corrected for dilution
Sample
1:100
1:250
1:500
1:1000
A
B
C
D
2.5
1.4
6.0
2.5
5.8
1.4
5.8
2.4
5.8
1.4
5.8
2.5
5.2
1.3
D.
Sensitivity
The mean and standard deviation were determined for 8
duplicate measurements of the zero standard binding. The
sensitivity of the method, defined as the cAMP concentration
corresponding to the mean cpm minus twice the standard
deviation, is < 0.025 pmol/mL.
E.
Specificity
The following compounds have been checked for crossreactivity. The percentages are calculated at the 50% B/Bo
point.
Compound
% Cross Reactivity
CGMP
GMP
ATP
ADP
AMP
~ 0.02
< 0.01
< 0.01
< 0.01
< 0.01
24
XII.
PERFORMANCE CHARACTERISTICS FOR ACETYLATED
(PLASMA) PROCEDURE
A.
Recovery
To demonstrate the accuracy of the method, recovery studies
were performed by adding known quantities of cAMP to
aliquots of Modified Assay Buffer and three plasma samples.
The resulting solutions were re-assayed and the recovery of
added cAMP was determined. Typical results are given on
below.
+25 pmol/mL
cAMP
Sample
pmol/mL pmol/mL
Base Measured
Value
Modified
Assay
Buffer
A
B
0
10
7.5
B.
24
34
50
+50 pmol/mL
cAMP
+100 pmol/mL
cAMP
% Rec
pmol/mL
Measured
% Rec
pmol/mL
Measured
% Rec
96
94
90
44
57
54
88
94
93
94
96
100
94
86
92
Reproducibility
Precision was determined by multiple duplicate analyses of
plasma pools. Typical results are given in pmol/mL.
Within Assay Variation
Sample
n
A
B
C
D
E
10
10
10
10
10
Between Assay Variation
Mean + 1 S.D. Coeff. Of
Var. (%)
16 + 0.49
6.3 + 0.26
12.8 + 0.24
35.9 + 0.88
24.7 + 0.48
3
4.1
1.9
2.4
1.9
25
Sample
n
A
B
C
D
E
F
6
6
6
6
6
6
Mean + 1 S.D. Coeff. of
Var. (%)
16 + 0.98
5.6 + 0.51
27 + 3.2
16 + 2.4
11.6 + 1.6
11 + 1.5
6.1
9.1
11.8
15
14
14
C.
Linearity
To demonstrate the absence of non-specific serum interferences,
five plasma samples were assayed by the standard procedure and
diluted to varying degrees with Modified Assay Buffer. The
recommended 100 µL aliquot was used for all samples. Typical
results are given in pmol/mL, corrected for dilution.
Sample
1:50
1:100
1:200
A
B
C
D
E
13
16
18
9
20
11
14
18
8
20
12
13
16
17
D.
Sensitivity
The mean and standard deviation were determined for 8
duplicate measurements of the zero standard binding. The
sensitivity of the method, defined as the cAMP concentration
corresponding to the mean cpm minus twice the standard
deviation, is < 0.025 pmole/mL.
E.
Specificity
The following compounds have been checked for crossreactivity. The percentages are calculated at the 50% B/Bo
point.
Compound
% Cross Reactivity
CGMP
GMP
ATP
ADP
AMP
~ 0.02
< 0.01
< 0.01
< 0.01
< 0.01
26
XIII.
CORRELATION
The cAMP concentraion of plasma, urine, and cell culture samples
were measured. Summarized below is the correlation obtained
comparing the present with the previous antibody.
Sample
Type
N
Range
(pmole/mL)
Slope
Y-intercept
Correlation
Coefficient
Plasma
40
8 to 75
0.99
-0.01
0.98
Urine
16
60 to 9070
0.96
-141
0.99
Cell Culture
12
876 to 1942
1.04
-192
0.98
XIV.
REFERENCES
1.
Sutherland, E. W., Robison, G. A., and Butcher, R. W.
Some aspects of the biological role of adenosine 3', 5'monophosphate (cAMP). Circulation, 37 (1968), 279-306.
2.
Jost, J. P. and Rickenburg, H. A. cAMP. Annual Review of
Biochemistry, 40 (1971), 741-774.
3.
Perkins, J. P. (1973): Adenyl cyclase. In: Greengaard, P. and
Robison, G. A. (Eds.) Advances in Cyclic Nucleotide
Research, Vol. 3:1-64. Raven Press, NY.
4.
Langan, T. A. (1973): Protein kinase and protein kinase
substrates. In: Greengaard, P. and Robison, G. A. (Eds.).
Advances in Cyclic Nucleotide Research, Vol. 3:99-153,
Raven Press, NY.
5.
Harper, J. F. and Brooker, G. J. (1975): Femtomole Sensitive
Radioimmunoassay for cAMP and cGMP after 2'0
acetylation by Acetic Anhydride in Aqueous Solution. Cyclic
Nucleotide Research 1 (4) 207-218.
6.
Frandsen, E. K. and Krishna, G. (1976): A simple
ultrasensitive method for the assay of cyclic AMP and cyclic
GMP in tissues. Life Sciences, 18:529-541.
7.
Yallow, R. S. and Berson, S. A. in Principles of Competitive
Protein Binding Assays. Eds. Odell, W. D. and Daughaday,
W. H. J. B. Lippincott Co., Philadelphia. 1971, Ch. 1.
27
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