cell biology assays

product code:

RPN 222

New direct PGE EIA assay for quick and simple measurement of
intracellular, secreted and total cellular PGE
2

The measurement of Prostaglandin E2 is important

in many disease and research areas, particularly the
immune response and inflammation. The new
improved direct enzyme immunoassay offers the
researcher a more accurate approach by allowing
intracellular measurement of PGE2.
A diverse range of mammalian cells and tissues enzymatically
oxidise arachidonic acid to physiologically active compounds
including prostaglandins.
Prostaglandin E2 (PGE2) is the predominant arachidonate metabolite
from the kidney of many species. It plays an important role in
regulating vasopressin stimulated osmotic flow and its
immunosuppressive effects in seminal plasma have important
implications in reproduction. PGE2 is also produced in macrophages
affecting neighbouring cells and its possible role in a variety of other
physiological processes is the subject of much research.
The BiotrakTM PGE2 enzymeimmunoassay (EIA) system from
Amersham Biosciences is specifically designed for research
purposes. The kit includes protocols for use with novel lysis reagents*
which facilitate simple and rapid extraction of PGE2 from cell
cultures and plasma samples. Removal of extraction reagents is not
required prior to measurement; therefore time consuming sample
preparation procedures required for the tradional measurement of
PGE2 in plasma are not required.

Choice of assay procedure

Construction of the kit allows the assay to be carried out in one of
5 ways:
traditional enzyme immunoassay procedure: allows PGE2 to be
measured within the range 2.5-320pg/well at room temperature.
From urine and cell culture medium without extraction, or from
tissues with traditional sample extraction methods.
reduced temperature enzyme immunoassay: the above assay
performed at 2-8C allows PGE2 to be measured within the range
1-32pg/well.
direct intracellular method: measures PGE2 in the range
2.5-320pg/well in tissue culture lysates where cells are grown in
flasks, vessels or on plates. Cells are lysed and an aliquot transferred
to a second plate for assay.

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total cellular: combines the amount of intracellular and cell

supernatant PGE2. Decantation of the cell culture supernatant is not
required and levels within the range 1-32 pg/well can be measured.
plasma assay: a direct procedure where lysis reagent is added to
plasma samples and aliquots removed for subsequent analysis.
Obviates the need for tedious sample extraction procedures and
measures PGE2 in the range 2.5-320pg/well.

Direct PGE2 EIA assay procedure

1. Culture cells

2. Decant & wash

3. Lyse cells

4. Transfer to
new plate

5. Perform assay

Fig 1 Cell lysis-intracellular method.

Lysis reagent 1 is added to cultured cells or plasma samples, followed

by a 5 minute incubation.
Cell membranes are hydrolysed by the addition of lysis reagent 1, or
if using a plasma sample, the lysis reagent 1 causes dissociation of
PGE2 from soluble receptors and interfering binding proteins.
The antisera and PGE2 peroxidase conjugate are reconstituted in lysis
reagent 2 and added to the culture/plasma sample. Lysis reagent 2
sequesters the key component in reagent 1 and ensures PGE2 is free
for subsequent analysis. The detergent/ sequestrant complex does not
interfere with antibody:antigen building.
The assay is based on competition between unlabelled PGE2 and a
fixed quantity of peroxidase labelled PGE2 for a limited number of
binding sites on a PGE2 specific antibody. The amount of
peroxidase labelled ligand bound by the antibody is inversely
proportional to the concentration of added unlabelled ligand. The
peroxidase labelled ligand bound to the antibody is immobilised to
the walls of a microtitre plate coated with second antibody. Any
unbound ligand is removed by washing and the amount of
peroxidase labelled PGE2 bound to the antibody is determined by
the addition of tetramethylbenzidine (TMB)/hydrogen peroxide
substrate.

substrate

standard or
unknown

solid phase assay reagent

inhibitor
(indomethacin)

stimulate
A23187
2000

Mouse
antiPGE2
Ab

PGE2-peroxidase

TMB

PGE2 (pg)

Goat
antimouse
Ab

well
PGE2

1000

Stop reaction,
measure OD
Incubate 30 min

Incubate 1 hr

10

15

intracellular

Typical results

60

120

supernatant

Benefits

10000

7500
PGE2 (pg/ml)

30

Time (min)

Fig 2 Principle of EIA

Fig 5 Effect of indomethacin on

PGE2 levels in A23187 stimulated
3T3 cells. Cells were stimulated for
5 minutes with A23187. Following
addition of the inhibitor,
indomethacin, intracellular levels of
PGE2 are shown to drop at a much
quicker rate than the supernatant
levels. This result shows how the
measurement of intracellular PGE2
gives a more sensitive and
accurate reflection of the cellular
response to inhibition.

5000

2500

0
intracellular supernatant

total

Fig 3 Measurement of
PGE2 from A23187stimulated 3T3 cells. Cells
were stimulated for 5
minutes with 100M
A23187, and measurements of intracellular,
supernatant and total PGE2
EIA kit. Intracellular levels
contributed 57% of the
supernatant value and
35% of the total value.

For cell cultures

measuring actual cell responsiveness.
accurate reflection of the action of a drug/stimulant/agonist/inhibitor on
cellular processes.
activity of a potential drug target can be masked by measuring PGE2
secreted into cell culture medium.
time saving.
ten fold increase in sensitivity with intracellular method over
measurements on supernatant alone.
changes in PGE2 levels over time can be measured, not possible when
measuring secreted PGE2.
kit is capable of measuring intracellular, total cellular and secreted PGE2.

6000

intracellular
supernatant

PGE2 (pg/ml)

4000

2000

0.1

0.2

0.5

1.0

5.0 10.0 50.0 100.0

A23187 (M)

Fig 4 Measurement of PGE2

from A23187 stimulated
3T3 cells. Cells were
stimulated for 5 minutes
with varying concentrations
of A23187, and measurements were made using the
Direct PGE2 EIA kit.
Intracellular levels of PGE2
were detected at lower
concentrations of stimulant
therefore demonstrating the
improved sensitivity of the
assay.

For plasma samples

no extraction process required.
PGE2 recovery values double with the use of lysis reagents.

References
1 Currie, MG and Needleman, P: Ann. Rev. Physiol., 46, pp 327-341, 1984.
2 Herbert, RL, Jacobson, HR and Breyer, MD: Review, Prostaglandins, Leukotrienes and
Essential Fatty Acids, 42, pp 143-148, 1991.
3 Bonney, RJ and Humes, JL, leukocyte Biol., 35, pp 1-10, 1984.
4 Chensue, SW and Kunkel, SL, Clin. Lab. Med., 3, pp 677-694, 1983.
5 Goodwin, JS and Ceuppens, JJ, Clin. Immunol., 3, pp 295-315, 1983.
6 Davies, P, Bailey, PJ and Goldenberg, MM, Ann. Rev. Immunol., 2, pp 335-357, 1984.
7 Ruzicka, T and Printz, MP, Rev. Physiol. Biochem. Pharmacol., 100, pp 122-160, 1984.
8 Kelly, RW, Editorial, International J. Andrology, 14 (4), pp 243-247.

ordering information
for further details se also the Amersham Biosciences BioDirectory 99
Prostaglandin E2 enzymeimmunoassay system
96 wells

RPN 222

to order:
US +1 800 234 5678

UK +44 (0)1727 890 1234

Europe +46 (1)923 45 67

www. apbiotech.com

Biotrak is a trademark of Amersham Biosciences Limited or its subsidiaries. Amersham Biosciences is a trademark of Amersham plc.
All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions of sale is available on request.
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Amersham Biosciences Inc, 800 Centennial Avenue, Piscataway, New Jersey 08855, USA
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Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany
Amersham Biosciences Inc. 1999All rights reserved.

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