Description
The
bicyclic-prostaglandin E2 [125I] assay system
provides the quantitative determination of
13,14-dihydro-15-keto-prostaglandin E2 (PGEM). PGEM can be
assayed in the range 0.6-50 pg/tube. Each kit contains materials
sufficient for 100 assay tubes, permitting the construction of one
standard curve and assay of 25 unknowns in triplicate.
Introduction
Prostaglandin E2 (PGE2) plays an important role in
various biological functions and in pathological processes. Because of
this, there are efforts in research to obtain some correlation between
PGE2 concentration and the normal or pathological functions
investigated. Like other primary prostaglandins, however, PGE2
is a substrate for the dehydrogenase enzyme that converts it into
13,14-dihydro-15-keto-PGE2 (PGEM), the main metabolite found
in the circulation. It is accepted generally that the only reliable way
for monitoring the endogeneous production of prostanoids is to measure
metabolites, rather than parent compounds.
However, PGEM is a subject to further chemical reactions, it readily
dehydrates into 13,14-dihydro-15-keto-11ß,16e-cyclo-PGE2
(bicyclic-PGEM). In order to overcome the likelihood for primarily
produced PGEM to escape in another metabolite pool, the stable bicyclic
end-product produced by chemical treatment is monitored.
The
current bicyclic-PGEM RIA kit combines the advantages of radioiodine
labelled tracer 11-deoxy-13,14-dihydro-15-keto-11ß,16e-cyclo-PGE2-[125I]tyrosine-methyl
ester (bicyclic-PGEM-[125I]TME) with a highly sensitive and
specific antibody to provide a rapid, simple and sensitive method for
the determination of PGEM concentration with about ten times as high a
sensitivity as that obtained with the most sensitive assays using
tritium-labelled tracer.
Principle of the method
The
assay is based on the conversion of PGEM in samples and standard
solution into 11-deoxy-13,14-dihydro-15-keto-11ß,16e-cyclo-prostaglandin
E2 on the action of sodium carbonate. Then a radioactive
ligand (bicyclic-PGEM-[125I]TME tracer) competes with the
unlabelled ligand (bicyclic-PGEM in standards or in samples) for binding
to a specific antibody. Allowing to react a fixed amount of tracer and
antibody with different amounts of unlabelled ligand the amount of
radioligand bound by the antibody will be inversely proportional to the
concentration of unlabelled ligand.
In this
kit, separation of bound from free tracer is achieved by absorption of
the free ligand onto dextran coated charcoal. After centrifugation an
aliquot of supernatant containing bound radioligand is counted in a
gamma-counter equipped with a well-type NaI (Tl) scintillation crystal.
Results obtained from the standards are used to construct a standard
dose - response curve that enables the amount of unlabelled ligand in
the sample to be calculated.
Contents of the kit
|
1 vial |
TRACER
0.3 ml per vial, containing about 75 kBq bicyclic-PGEM-[125I]TME
in ethanolic solution |
|
1 vial |
STANDARD, lyophilised, containing 7.5 ng/ml PGEM in
buffer with 0.01% thimerosal |
|
1 vial |
ANTISERUM, lyophilised, containing polyclonal PGEM
antiserum (rabbit) in buffer with 0.01% thimerosal. |
|
1 bottle |
ASSAY BUFFER CONCENTRATE
15 ml per bottle, containing 0.01% thimerosal |
|
1 bottle |
CHARCOAL SUSPENSION
Ready to use.
55 ml per bottle, containing 1% Norit A and 0.5% Dextran T-70 in
buffer with 0.01% thimerosal |
| |
Quality certificate |
| |
Pack leaflet |
Materials and equipment required
Round
bottom polystyrene or polypropylene assay tubes, about 12 x 75 mm
Test tube racks
Plastic film to cover tubes
Precision pipettes (100 µl and 500 µl)
Vortex mixer
Incubator to maintain a 37°C temperature
Magnetic stirring base and stir bars
Refrigerated centrifuge
Gamma counter
Recommended
tools and equipment
repeating
pipettes
Preparation of reagents
TRACER
One vial of the tracer concentrate contains about 75 kBq of
bicyclic-PGEM-[125I]TME in ethanolic solution and is stable
for at least two month, if stored at -20°C. Dilute the tracer with 10 ml
assay buffer prediluted with water from the assay buffer concentrate.
The resulting solution contains about 75 kBq of the tracer in 50 mM
phosphate buffer, pH 7.3, with 0.1% gelatin and 0.01% thimerosal. The
diluted solution is stable until expiry date if stored at 2-8°C.
ANTISERUM
The antiserum was raised in rabbit agains a bovine serum albumin
conjugate of bicyclic-PGEM. Stored at 2-8°C, the lyophilised antiserum
is stable until expiry date.
Reconstitute the antiserum by adding 10 ml of distilled water with
gentle mixing to avoid foaming. Make ensure that the lyophilised
material is in solution. After reconstitution, the solution contains
bicyclic-PGEM antiserum of appropriate binding ability in 50 mM
phosphate buffer, pH 7.3, with 0.1% gelatin and 0.01% thimerosal. This
solution should be stored at 2-8°C. Under these conditions the solution
is stable until at least the date of expiry.
STANDARD
Reconstitute the lyophilised standard by adding exactly 1.0 ml distilled
water. Make ensure that the lyophilised material is in solution. The
resulting solution contains 7.5 ng of PGEM per ml in 50 mM phosphate
buffer , pH 7.3 with 0.1% gelatin and 0.01% thimerosal.
Stored
at -20°C this stock solution is stable until the date of expiry.
An
appropriate aliquot of the standard concentrate is used to prepare a
series of standard dilutions according to the suggested dilution scheme
shown later. Diluted standard solutions must not be stored.
ASSAY BUFFER CONCENTRATE
To prepare assay buffer for use in the assay system add 60 ml distilled
water to the bottle after warming it to room temperature and mix
thoroughly. The diluted assay buffer contains 50 mM phosphate buffer, pH
7.3 with 0.1% gelatin and 0.01% thimerosal.
Stored
at 2-8°C diluted buffer is stable until the date of expiry.
CHARCOAL SUSPENSION
This suspension contains 1% Norit A in 10 mM phosphate buffer, and 0.5%
Dextran T-70. Stored at 2-8°C the suspension is stable until the date of
expiry.
Preparation of samples prior to assay
Contrary to
primary prostaglandins whose concentrations increase rapidly when
tissues are injured, „de novo” prostanoid synthesis does not usually
alter the physiological levels of prostaglandin metabolites therefore
sampling procedure does not need any special precaution. However the
general problem of radioimmunoassay, i. e. that the inhibition of
binding to the antibody can be affected by factors other than the
analyte concentration itself holds for PGEM RIA, too. Because of this,
preparation of samples prior to assay is a prerequisite to the
radioimmunological determination of prostaglandins. Out of the various
useful sample preparation methods, the most reliable solid-phase
extraction technique using Sep-Pak C18 Cartridges (Waters
Ass.) has been applied succesfully in our laboratory with a slight
modification of the Powell’s method for preparation of human plasma
samples prior to radioimmunoassay. This procedure is provided for
guidance only, and it remains the investigator’s responsibility to
validate his experimental method.
Extraction on Sep-Pak C18 Cartridges
|
1 |
Pretreat the cartridge according to manufacturer’s
instructions. |
|
2 |
Dilute 1 ml plasma with 4 ml water, then acidify to pH
3.0 with the addition of about 100 microliter 2 M citric acid
(Check pH individually, if needed). |
|
3 |
Apply sample to the cartridge with a syringe and pass
through with gentle pressure (about 1 ml/min). |
|
4 |
Wash with 10 ml water. |
|
5 |
Wash with 10 ml 10% ethanol. |
|
6 |
Wash with 10 ml petroleum ether or n-hexane. |
|
7 |
Elute PG-s with 10 ml ethyl-acetate. |
|
8 |
Add 1 ml water to ethyl-acetate, shake,and separate the
water phase. Repeat this procedure once again. |
|
9 |
Dry the ethyl-acetate extract under vacuum at room
temperature. |
|
10 |
Dissolve the residue in 0.9% saline solution.
Warning! Never use the cartridges more than once with plasma
samples. |
Assay procedure
Preparation of
reagent solutions
These reagents
are required but not provided.
|
1: |
1 M sodium-carbonate:
Dissolve 10.6 g of anhydrous sodium-carbonate (Na2CO3)
in distilled water to a final volume of 100 ml. |
|
2: |
1 M potassium phosphate buffer:
Dissolve 13.61 g of anhydrous potassium-dihydrogen-phospate (KH2PO4)
in 70-80 ml of distilled water and adjust the pH to 7.4 with a
potassium-hydroxide solution. Make the final volume 100 ml with
the addition of distilled water. |
|
3: |
0.9% saline solution:
Weigh 0.9 g of sodium-chloride (NaCl) and add distilled water to
a final volume of 100 ml |
Day 1
Preparation of
PGEM working standards and conversion of PGEM to bicyclic-PGEM.
To prepare standard dilution and to dissolve or dilute 0.9% saline must
be used.
|
1 |
Label tubes for standards A-E and samples. |
|
2 |
Prepare standard dilution as shown in Table I. |
|
3 |
Pipette 0.5 ml from standard E and discard it. |
|
4 |
Pipette 1 ml samples into the appropriate tubes. |
|
5 |
Pipette 1 ml saline into the blank tubes. |
|
6 |
Pipette 40 microliter of 1 M sodium carbonate into each
tube. |
|
7 |
Vortex the contents of each tube, cover to prevent
evaporation and incubate the tubes at 37°C for 20-24 hours. |
|
8 |
Pipette 60 microliter of 1 M potassium-phospate buffer
(pH 7.4) into each tube and vortex for a few seconds. |
Table
1. Dilution scheme
|
Tube |
volume
of the standard dilution |
volume
of 0.9% NaCl |
Amount
of standard (pg/tube) |
|
s |
|
|
750 |
|
A |
100 of
sol. s |
1400 |
50 |
|
B |
500 of
sol. A |
1000 |
16.7 |
|
C |
500 of
sol. B |
1000 |
5.6 |
|
D |
500 of
sol. C |
1000 |
1.9 |
|
E |
500 of
sol. D |
1000 |
0.6 |
Vial
"s" is prepared by reconstituting the lyophilised standard with 1.0 ml
distilled water.
Note: All volumes are in microliter. Volume of standards, blank es
can be decreased to 0.5 ml, if necessary. In this case, the volume of
other reagent solutions should be decreased proportionally.
Radioimmunoassay protocol
For a quick
guide refer to Table 2.
Day 2
|
9 |
Prepare tracer and antiserum as described previously. |
|
10 |
Equilibrate all reagents (except charcoal but including
standards, blank and samples) to room temperature and mix before
use. (Refer to Table 2. for steps 11-27.) |
|
11 |
Label triplicate tubes according to Table 2.
(Determinations can equally be performed using duplicates.) |
|
12 |
Pipette 200 µl of assay buffer into tubes 4-6
(non-specific binding tubes) |
|
13 |
Pipette 100 µl of assay buffer into all remaining tubes. |
|
14 |
Pipette 100 µl of blank into tubes 1-9. |
|
15 |
Pipette 100 µl of each diluted standard in triplicate (A
through E into tubes 10-24). |
|
16 |
Pipette 100 µl of each sample in triplicate into tubes
25-100. |
|
17 |
Pipette 100 µl of tracer solution into each tube. |
|
18 |
Pipette 100 µl of antiserum into all tubes except 4-6 and
vortex throughly for 2-5 seconds. (Note: If drops remained on
the wall of tubes after vortexing, centrifuge the tubes at 1000
x g for a few seconds.) |
|
19 |
Incubate the tubes at 4°C overnight (16-20 hours). |
Day 3
|
20 |
Place the stir bars into the cold charcoal suspension and
mix the suspension thoroughly. |
|
21 |
Pipette 500 µl of assay buffer into tubes 1-3. |
|
22 |
While stirring pipette 500 µl charcoal suspension into
all remaining tubes and vortex each tube for 2-5 seconds. |
|
23 |
Allow the test tubes to incubate at 4°C (preferably in
the refrigerated centrifuge) for 10 minutes. |
|
24 |
Centrifuge the tubes at 4°C and 2000 x g for 10 minutes.
Note: g = 1.118 x 10-5(rpm)2 x (arm-length
of the centrifuge in cm). |
|
25 |
During centrifugation label tubes in the same manner as
the original series. |
|
26 |
After centrifugation pipette 500 µl from the supernatants
of each tube. |
|
27 |
Count the radioactivity of all tubes preferably not less
than 60 seconds. |
|
28 |
Calculate the concentrations as described in calculation
of results. |
Table 2.
Assay Protocol, Pipetting Guide (all volumes in microliters)
|
Tubes
Reagents |
Total count
1-3 |
Non specific binding
4-6 |
Zero
standard
7-9 |
Standard
A-E
10-24 |
Sample
25-100 |
|
Buffer |
100 |
200 |
100 |
100 |
100 |
|
Blank |
100 |
100 |
100 |
|
|
|
Standard |
|
|
|
100 |
|
|
Sample |
|
|
|
|
100 |
|
Tracer |
100 |
100 |
100 |
100 |
100 |
|
Antiserum |
100 |
|
100 |
100 |
100 |
|
Vortex mix
Incubate overnight (16-20 hours) at 4oC |
|
Buffer |
500 |
|
|
|
|
|
Charcoal |
|
500 |
500 |
500 |
500 |
|
Vortex mix
Incubate for 10 minutes at 4 C |
|
Centrifuge for 10 minutes at 4 oC and 2000 xg |
|
Pipette 500 µl from the supernatant of each tube into another
series of tubes |
|
Count
all tubes |
Calculation of results
The
calculation is illustrated using representative data. The assay data
collected should be similar to those shown in Table 3.
Calculate the average counts per minute (CPM) for each triplicate of
assay tubes.
Calculate the percent B0 / T for zero standard (S0)
by using the following equation: (See Note 1)
| |
S0 cpm - NSB cpm |
|
|
% B0 / T = |
——————— |
x 100 |
| |
T cpm - NSB cpm |
|
Calculate the
normalized percent binding for each standard, control and sample
respectively by using the following equation: (See Note-2)
| |
SA-E / sample cpm - NSB cpm |
|
|
% B / B0 = |
———————————— |
x 100 |
| |
S0 cpm - NSB cpm |
|
Using
semi-logarithmic graph paper plot B / B0 % for each standard
versus the corresponding concentration of PGEM. Figure 1 shows a typical
standard curve. (See Note 2)
Determine the PGEM concentration of the unknown samples by interpolation
from the standard curve. Do not extrapolate values beyond the standard
curve range.
Notes on
procedure
|
1) |
B0 / T is an optional quality control
parameter unnecessary for determination of sample
concentrations. If this is ignored, one more sample can be
measured instead of total count. |
|
2) |
Note that PGEM amounts calculated in this way refer to
the quantity pg/tube before alkaline treatment. Calculation by
computing data using logit-log or other fitting programs may
also be applied but is not dealt with here. |
Table
3. Typical Assay Data
|
Tubes |
Tube
No |
Count
cpm |
Average
cpm |
Average
net cpm |
B / B0
% |
|
Total |
1
2
3 |
15520
15945
16351 |
15938 |
12489 |
|
|
NSB |
4
5
6 |
164
165
194 |
174 |
5928 |
|
|
S0
0 pg/tube |
7
8
9 |
7606
7114
7498 |
7406 |
7232 |
|
|
SA
0.6 pg/tube |
10
11
12 |
7097
6891
7079 |
7022 |
6848 |
94.7 |
|
SB
1.9 pg/tube |
13
14
15 |
6045
6294
5947 |
6095 |
5921 |
81.9 |
|
SC
5.6 pg/tube |
16
17
18 |
4783
4566
4490 |
4613 |
4439 |
61.4 |
|
SD
16.7 pg/tube |
19
20
21 |
2179
2230
2079 |
2163 |
1989 |
27.5 |
|
SE
50.0 pg/tube |
22
23
24 |
908
9647
1001 |
958 |
784 |
10.8 |
PGEM concentration pg/tube
Figure 1.
A typical standard curve
(Do not use to calculate sample values)
Characterization of the assay
Assay
parameters
|
NSB / T |
|
< 3% |
|
B0 / T |
|
45 ± 10% |
|
ED-50 |
|
7.5 ± 1 pg/tube |
Specificity
Cross
reactivity was defined in per cent by weight at the 50% displacement
level. Cross-reacting substances were subjected to the same alkaline
treatment as used with specific analyte.
|
13,14-dihydro-15-keto-PGE2 |
100.0% |
|
13,14-dihydro-15-keto-PGF2a |
0.15% |
|
PGA2 |
0.02% |
|
PGD2 |
<
0.001% |
|
PGE2 |
<
0.001% |
|
PGE1 |
<
0.001% |
|
PGF1a |
<
0.001% |
|
6-keto-PGF1a |
<
0.001% |
|
6-keto-PGE1 |
<
0.001% |
|
Thromboxane B2 |
<
0.001% |
|
13,14-dihydro-6,15-diketo-PGF1a |
<
0.001% |
|
9a,11ß
-PGF2a |
<
0.001% |
|
11-dehydro-TXB2 |
<
0.001% |
Additional information
Storage
Store
this kit between 2 and 8°C.
Availability
From stock.
Shelf life
The shelf life
of kit reagents is 8 weeks from the date of manufacturing. The actual
expiry date is given on package label and in the quality certificate.
Components from various lots or from kits of different manufacturers
should not be mixed or interchanged.
Precautions and warnings
This kit
should only be used for research purposes.
Radioactivity
This
kit contains radioactive material. Receipt, acquisition, possession, or
use of radioactive materials are subject to regulations, and a licence
of (inter)national authorizing bodies. It is the responsibility of the
user to ensure that local regulations or codes of practice are
satisfied.
|
