ab136949 – PGE2 ELISA Kit (Fluorescent)

Instructions for Use

For fluorescent and quantitative determination of PGE2 in cell supernatants.

This product is for research use only and is not intended for diagnostic use.

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Table of Contents

1. Introduction 3

2. Principle of the Assay 4

3. Assay Summary 5

4. Kit Contents 5

5. Storage and Handling 6

6. Additional Materials Required 6

7. Protocol 7

8. Calculation of Results 10

9. Performance Characteristics 13

10. Troubleshooting 20

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1. Introduction

ab136949 is a complete kit for the quantitative determination of

PGE2 in buffers and culture media. Please read the entire kit insert

before performing this assay.

Prostaglandin E2 (PGE2) is formed in a variety of cells from PGH2,

which itself is synthesized from arachidonic acid by the enzyme

prostaglandin synthetas. PGE2 has been shown to have a number of

biological actions, including vasodilation, both anti and

proinflammatory action, modulation of sleep/wake cycles, and

facilitation of the replication of human immunodeficiency virus. It

elevates cAMP levels, stimulates bone resorption, and has

thermoregulatory effects. It has been shown to be a regulator of

sodium excretion and renal hemodynamics.

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2. Principle of the Assay

1. Samples and standards are added to uncoated wells.

2. A solution of PGE2 covalently conjugated to fluorescein is then

added to the wells.

3. A solution of monoclonal antibody to PGE2 is next added. This

binds, in a competitive manner, the PGE2 in the standard,

sample or conjugate.

4. The plate is incubated at room temperature for at least 30

minutes. The FP signal is stable for at least 20 hours.

5. The plate is then read at 520-535 nm, with excitation at 485 nm.

The amount of signal is inversely proportional to the

concentration of PGE2 in the standards or samples.

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3. Assay Summary

Samples, conjugate, and antibody are added to each well

Incubate

Read Plate (measure at 520-535 nm emission with excitation at 485 nm)

4. Kit Contents

Item Description Quantity

PGE2 FPIA Antibody A solution of monoclonal antibody to PGE2.

8 ml

Assay Buffer 1 Concentrate

Tris buffered saline containing proteins and

sodium azide as preservative.

125 ml

PGE2 FPIA Conjugate Concentrate (fluorescein)

A solution of fluorescein conjugated to PGE2.

0.1 ml

PGE2 Standard A solution of 1,000,000 pg/ml PGE2.

0.5 ml

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5. Storage and Handling

All components of this kit, except the PGE2 FPIA Conjugate

Concentrate and PGE2 Standard, are stable at 4°C. The PGE2 FPIA

Conjugate Concentrate (fluorescein) and PGE2 Standard must be

stored at -20°C.

6. Additional Materials Required

Solid black uncoated low-binding microtiter plate.

Foil microtiter plate sealer.

Fluorescence polarization detector capable of reading

emissions at 520-535 nm, with excitation at 485 nm.

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7. Protocol

A. Sample Handling

This assay is compatible with PGE2 samples in defined buffers and

cell culture media.

Samples diluted sufficiently into the assay buffer can be read directly

from the standard curve. Samples containing some organic solvents

or inherently fluorescing materials may interfere with the assay.

Please refer to the interferences section for details.

B. Reagent Preparation

1. Assay Buffer

Prepare the assay buffer by diluting 100 ml of the supplied

Assay Buffer 1 Concentrate with 900 ml of deionized water.

This can be stored at room temperature for 3 months.

2. PGE2 ConjugateCount the total number of wells that will receive conjugate.

Use the following formula to calculate the volume of PGE2

conjugate Concentrate and the assay buffer needed to

prepare PGE2 conjugate.

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A. (Number of wells + 1) x 0.05 ml/ well = Volume of assay

buffer needed. Increase the calculated volume to the

next whole milliliter.

B. (Volume from part A) x 10 µl / ml = Volume of PGE2

Conjugate Concentrate needed.

Pipet the volume of assay buffer from part A into an amber

container. From this volume, remove the volume calculated

in part B. Add the calculated PGE2 Conjugate Concentrate

to the assay buffer. Vortex thoroughly and use.

3. PGE2 Standard

Allow the 1,000,000 pg/ml standard stock to warm to room

temperature. Label seven 12 x 75 tubes #1 through #7. Pipet

900 µl of the assay buffer into tube #1. Pipet 500 µl of the

assay buffer into tubes #2 through #7. Add 100 µl of the

1,000,000 pg/ml PGE2 Standard into tube #1 and vortex

thoroughly. Add 500 µl of tube #1 to tube #2 and vortex

thoroughly. Add 500 µl of tube #2 to tube #3 and vortex

thoroughly. Continue this for tubes #4 through #7.

Diluted standards should be used within 60 minutes of

preparation. The concentrations of PGE2 in the tubes are

100,000, 50,000, 25,000, 12,500, 6,250, 3,125 and

1,562 pg/ml respectively.

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C. Assay Procedure

1. Pipet 150 µl of assay buffer into the Total Fluorescence

(TF) wells.

2. Pipet 100 µl of assay buffer into the Bo (0 pg/ml) wells.

3. Pipet 100 µl of Standards #1 through #7 to the bottom of

the appropriate wells.

4. Pipet 100 µl of the samples to the bottom of the

appropriate wells.

5. Add 50 µl of the conjugate into each well.

6. Add 50 µl of antibody into each well, except the TF

wells.

7. Seal the plate with a foil plate sealer. Incubate the plate

for at least 30 minutes at room temperature. The FP

signal is stable for at least 20 hours.

8. Read plate on a suitable fluorescence polarization

detector at 520-535 nm emission, with excitation at 485

nm, using the appropriate settings for that instrument

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8. Calculation of Results

Several options are available for the calculation of the concentration

of PGE2 in samples. We recommend that the data be handled by an

immunoassay software package utilizing a 4 parameter logistic curve

fitting program. If data reduction software is not readily available, the

concentrations can be calculated as follows:

1. Calculate the binding for each standard and sample as a

percentage of the maximum binding (Bo), using the following

formula:

Percent Bound=Average mPAverage Bo ×100

2. Using Logit-log paper, plot the Percent Bound for each standard

versus PGE2 concentration in each standard. Approximate a

straight line through the points. The concentration of the

unknowns can be determined by interpolation. Samples with

concentrations outside of the standard curve range will need to

be re-analyzed using a higher dilution

.

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Typical Results

The results shown below are for illustration only and should not be

used to calculate results.

Sample Net mP Percentage Bound (%)

PGE2 (pg/ml)

S1 25 10.7 100,000

S2 38 16.2 50,000

S3 56 23.8 25,000

S4 86 36.3 12,500

S5 146 61.5 6,250

S6 208 87.6 3,125

S7 224 94.7 1,562

Unknown 1 136 57.4 7,204

Unknown 2 48 20.3 27,803

Bo 237 100 0

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9. Performance Characteristics

A. Specificity

The cross reactivities for a number of related compounds were

determined by diluting the compounds in the kit assay buffer at

concentrations from 1,000,000 to1,000 pg/ml. These samples

were then measured in the PGE2 FPIA kit, and the measured

PGE2 concentration at 50% B/Bo calculated. The % cross

reactivity was calculated by comparison with the actual

concentration of the cross reactant in the sample and expressed

as a percentage.

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Compound Cross Reactivity (%)

PGE2 100

PGE1 100

PGD2 11

PGF2 2.5

6-keto-PGF1 2.3

PGB2 1.8

PGI2 0.9

TXB2 0.3

Arachidonic acid <0.1

Dihomo--linolenic

acid

<0.1

B. Sensitivity

The sensitivity of the assay, defined as the concentration of

PGE2 measured at 2 standard deviations from the mean of

24 zeros along the standard curve, was determined to be

684 pg/ml.

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C. Linearity

A buffer sample containing PGE2 was serially diluted 1:2 in

Assay Buffer 1 and measured in the assay. The results are

shown in the table below.

DilutionExpected

(pg/ml)Observed

(pg/ml)Recovery

(%)

Neat - 76,035 -

1:2 38,017 31,610 83

1:4 19,009 19,701 104

1:8 9,504 11,713 123

1:16 4,752 4,228 89

1:32 2,376 2,329 98

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D. Z Factor

The Z Factor is a dimensionless statistic that reflects the

dynamic signal range and variation of an assay. This provides

a useful parameter to evaluate the robust quality of a given

assay. The following formula was used:

Z Factor=1[(3×1SD of Positive)+(3×1SD of negative)Positive -Negative ]

The Z-Factor of the assay was determined to be 0.88.

E. Precision

Intra-assay precision was determined by assaying 24 replicates

of three buffer controls containing PGE2 in a single assay.

PGE2 pg/ml % CV

24,142 21

16,629 13

5,392 8.3

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Inter-assay precision was determined by measuring buffer

controls of varying PGE2 concentrations in multiple assays over

several days.

PGE2 pg/ml % CV

24,103 11.4

15,598 10.7

6,574 12.7

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D. Sample RecoveriesPGE2 standard was spiked into the following buffers, which

were already diluted with the assay buffer, and measured in

the kit. The results are shown below. These are the

minimum dilutions required to remove the matrix interference

of these solutions.

SampleRecovery

(%)Recommended

dilution

Hanks Buffered Saline

solution 108 ≥neat

RPMI-1640 without

phenol red 95 ≥1:4

RPMI-1640 with phenol red,

sodium pyruvate and

Essential Amino Acids

100 ≥1:16

RPMI-1640 with phenol red,

10% FBS and antibiotics105 ≥1:32

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E. InterferencesPGE2 standard was spiked into the following solutions,

which were already diluted with Assay Buffer 1, and

measured in the kit. The results were as follows:

SampleRecovery

(%)Recommended percentage (%)

Methanol 107 ≤10

Acetonitrile 109 ≤5

2-propanol 98 ≤2.5

Ethanol 106 ≤2.5

DMF 104 ≤1.3

DMSO 102 ≤1.3

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10. Troubleshooting

Weak Color Development

How long was the substrate

incubation?

It is possible that Stop Solution was

added to the plate without allowing the

full substrate incubation.

What were the conditions of

the substrate incubation?

If a plate is left to incubate on a cold lab

bench or under a drafty area during

ambient incubations, signal values (e.g.

optical density) may be lower than

expected.

Were reagents brought to

room temperature prior to

use?

It is important to ensure that all reagents

are brought to room temperature prior to

use, or as mentioned in the product

specific instruction manual. Usually

leaving the kit out on the bench top at

ambient temperature for about half an

hour prior to setting up the assay will be

sufficient, when the reagents can be

stored at 4°C. Frozen volumes take a

little more time to come to room

temperature. Do not thaw frozen

reagents in a water bath. If a different

standard/sample diluent is used (such

as culture media) this must also be

warmed.

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What were the conditions of

the incubations?

If the incubation times and temperatures

are not observed, this can lead to lower

than expected signal values (e.g. optical

density). Pay attention that in air-

conditioned rooms the temperature does

not drop below 21°C.

How was the plate shaken

during incubations (if

required)?

If customers do not have a plate shaker,

they will often use an orbital flask shaker

or some other piece of equipment. This

is not a problem as long as the liquid is

vigorously displaced about 3/4 of the

way up the sides of the wells without

coming out. It is very important that the

plate is secured into place. If the plate is

not shaken and it is required in the

procedure, a longer incubation may be

necessary to bring the reagents to

equilibrium.

How long after the addition of

Stop Solution was the plate

read?

The plate needs to be read at the

correct wavelength as soon as possible

after the addition of the Stop Solution.

We generally recommend that the plate

be read within 10 minutes.

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High Background

How was the plate washed?

It is important that the plate is washed

thoroughly. If plate washing is

troublesome, a squirt bottle can be filled

with diluted Wash Buffer and all of the

wells completely filled from this. The

plate contents can be dumped into the

sink and shaken to remove excess

buffer. This should be repeated for the

number of times recommended in the

instruction manual. It is important to

remember that adding too little Wash

Buffer can result in high background,

while adding too much is not a problem.

The contents of the wells should be

aspirated and the plate tapped dry on

lint-free paper towels.

What were the incubation

times and temperatures?

If the plate was incubated for too long or

at a higher than recommended

temperature, high background could

result.

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Drift

Were reagents brought to

room temperature prior to

use?

If the reagents are not at a constant

temperature prior to their addition into

the wells, the results from one side of

the plate to the other can differ

depending on the temperatures at

addition.

Was the set-up of the assay

interrupted?

If the assay is interrupted at any point

during the addition of reagents, it is

possible that differing results will be

seen before the interruption versus after.

The wells that had reagents added

before the interruption will have been

incubating for longer than those after.

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Poor Precision

Were the wells washed

properly?

All wells receive the same treatment

during the wash step. If some are

washed less than others, this can

translate to poor precision. It is

important that the plate is washed

thoroughly. If plate washing is

troublesome, a squirt bottle can be filled

with diluted Wash Buffer and all of the

wells completely filled from this. The

plate contents can be dumped into the

sink and shaken to remove excess

buffer. This should be repeated for the

number of times recommended in the

instruction manual. It is important to

remember that adding too little Wash

Buffer can result in high background,

while adding too much is not a problem.

The contents of the wells should be

aspirated and the plate tapped dry on

lint-free paper towels.

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Were the wells aspirated

sufficiently after the wash

steps?

It is very important that as little Wash

Buffer as possible remains in the wells

after aspiration. Residual buffer can

cause dilution of subsequent reagents.

After the last wash step, it is a good idea

to hit the plate several times over a

piece of paper toweling to remove

excess buffer.

How were reagents pipetted

into wells?

In order to eliminate precision error,

customers need to remember to pre-

rinse all pipet tips used in the assay. We

usually recommend that the customer

draw up the liquid into the tip and

aspirate it three times prior to addition

into the well. Regular pipet calibration

and maintenance is also essential to

ensure that the tips fit properly and that

the correct volumes are dispensed. Be

sure reagents are not splashed between

wells or outside of the wells during

pipeting (especially if using repeater

pipets).

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Poor Standard Curve

What was used as the

standard diluent?

Diluents other than the supplied assay

buffer may contain interfering

substances that can affect the standard

curve.

How was the precision of the

standard curve?

If the %CV values for the standard curve

signal values (e.g. optical density) are

consistently above 5%. If the standard

curve signal values were acceptable but

the sample precision was not, the

problem relates to the sample. Also, see

recommendations under "Poor

Precision".

Were the Blank and NSB

values subtracted out?

If the net signal values (e.g. optical

density) are not used, the signal values

will appear higher than those presented

in the sample data in the instruction

manual.

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How were the standard

dilutions prepared?

It is important that test tubes of an

appropriate size and material are used.

Standard dilutions must be properly

mixed (e.g. vortexed) while preparing

the serial dilutions. It is also crucial that

the standard dilutions be prepared and

used within the time specified in the

product specific instruction manual.

Never store unused standard dilutions

for a later use.

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Edge Effects

Where was the plate

incubated?

Often times the conditions for ambient

incubations can be less than ideal. If

there is a draft in the area or the plate is

incubated on a cold lab bench, this can

lead to uneven color development.

If multiple plates were run,

were they stacked on top of

each other during incubation?

Multiple plates should only be incubated

in a single layer. This will assure that no

area of the plate is at a different

temperature than any other.

If a non-ambient incubation

was required, was the plate

properly sealed?

Making sure that the plate sealer is

tightly covering all of the wells will help

to discourage uneven evaporation of the

well contents, or condensation for colder

incubation conditions.

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