Description

The prostaglandin E₂ [¹²⁵I] assay system provides the quantitative determination of prostaglandin E₂ (PGE₂). PGE₂ can be assayed in the range 3.1-200 pg/tube. Each kit contains materials sufficient for 100 assay tubes, permitting the construction of one standard curve and assay of 23 unknowns in triplicate.

Introduction

Arachidonic acid, released from the cell wall by phospholipase A₂, is converted to prostaglandin endoperoxides on the effect of cyclo-oxygenase / endoperoxyde synthase. Endoperoxydes are then converted to a series of prostanoids including prostaglandin E₂ (PGE₂). 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 contrentration and the normal or pathological functions investigated.

PGE₂ has been detected in a variety of human tissues. Because of low concentration, its quantitation requires sensitive procedures. With the use of a high specific activity iodinated derivative of PGE₂ as tracer and rabbit anti-PGE₂ antiplasma as specific antibody, sensitive determination of PGE₂ in biological fluids can be achieved with high sample capacity.

Principle of the method

This assay is based on the competition between unlabelled PGE₂ and a fixed quantity of ¹²⁵I-labelled PGE₂ for a limited number of binding sites on PGE₂ specific antibody. Allowing to react a fixed amount of tracer and antibody with different amounts of unlabelled ligand the amount of tracer bound by the antibody will be inversely proportional to the concentration of unlabelled ligand. Upon addition of magnetizable immunosorbent the antigen-antibody complex is bound on solid particles which are then separated by either magnetic sedimentation or centrifugation. Counting the radioactivity of solid phase enables a standard curve to be constructed and samples to be quantitated.

Contents of the kit

Materials and equipment required

Round bottom polystyrene or polypropylene assay tubes, about 12 x 75 mm
Plastic film to cover tubes
Precision pipettes (100 µl and 500 µl)
Vortex mixer
Magnetic separator, or, alternatively, centrifuge
Decanting racks
Gamma counter

Recommended tools and equipment

orbital shaker
repeating pipettes

Preparation of reagents

TRACER
One vial of the tracer concentrate contains about 75 kBq of PGE₂-[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 -20°C.

ANTISERUM
The antiserum was raised in rabbit agains a bovine serum albumin conjugate of PGE₂. 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 PGE₂ 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 10 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 80 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.

MAGNETIC IMMUNOSORBENT
This reagent contains paramagnetic particles coated with anti-rabbit immunoglobulin suspended in 50 mM phosphate buffer, pH 7.3 with 0.1% sodium azide and 0.05% Triton X-100. Stored at 4°C, it is stable until expiry date.

Sample handling

A) Collection and storage

Because of "de novo" prostanoid synthesis brought about by tissue injury, a special care should be taken to inhibit artifactual production during collection. In addition, PGE₂ can be converted to PGA₂ as an artifact of sample handling. Pitfalls encountered with artifactual formation of prostanoids necessitate the sample handling being done under strictly controlled conditions.

Blood samples should be collected in pre-chilled plastic or siliconized glass tubes containing anticoagulant and cyclo-oxygenase inhibitor. In our laboratories blood is drawn in polypropylene tube containing 10% (v/v) of 2% EDTA (pH 7.3) with 1mM indomethacin. At this concentration we have found no interference of indomethacin in the assay. The plasma is separated by centrifugation and assayed as soon as possible. If storage of plasma samples is necessary, -70°C or lower is recommended, since during a long-time storage at -20°C an increased PGE₂-like immunoreactivity was reported.

Urine samples should be stored at -20°C after pooling the single samples collected from one patient.
For tissue sample storage at -70oC or lower until assay is recommended.

B) Preparation of samples prior to assay

Plasma
Solid-phase extraction of human plasma

The low concentration of analyte on one hand and the non-specific interference with tracer binding obtained with unextraced plasma on the other, will necessitate extraction of plasma prior to assay. At our laboratories solid-phase extraction on Amprep C₂ (Amersham International plc) minicolumns has been applied successfully according to the following procedure.

This procedure usually results in a neglibigle loss of PGE₂ with a recovery over 90%, as checked by [³H]-PGE₂ as recovery marker. In spite of high recovery expected, it is suggested that user follow extraction efficiency throughout procedure employed in his laboratory. To achieve this 1000 - 2000 cpm of [³H]-PGE₂ should be added to samples prior to extraction and procedure recovery is calculated from radioactivity measured from an aliquot of assay sample.

Solvent residues, impurities, as well as biological matrix itself may often introduce considerable degree of non-specific immunoreactivity. It is important that contribution of this non-specific interference to real PGE₂ concentration be evaluated. To achieve this prostaglandin-free biological sample (to estimate matrix contribution) and/or buffer (to determine method-blank only) should be subjected to the procedure strictly indentical to that used for unknowns. Concentration of unknowns should be corrected accordingly.

Urine

Urine contains high level of immunoreactive PGE₂, generally regarded as being of renal origin. Its concentration is high enough to allow for such a dilution prior to assay that minimizes the interference of non-specific factors. At our laboratories urine is successfully assayed directly with dilutions up to 1:100.

Tissues

Tissue samples after homogenization and separation of aqueous phase can be extracted according to procedure outlined above for plasma. Because of difficulties encountered with determination of both recovery and matrix contribution, it remains the investigator's responsibility to validate his own procedure.

Assay procedure

Day 1

Table 1. Dilution scheme (all volumes in microliters)

Note: vial "s" is prepared by reconstituting the lyophilised standard with 1.0 ml distilled water
To prepare standard dilution and to dissolve or dilute assay buffer must be used.

Day 2

Table 2. Assay Protocol, Pipetting Guide (all volumes in microliters)

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)

Calculate the normalized percent binding for each standard, control and sample respectively by using the following equation: (See Note 2)

Using semi-logarithmic graph paper plot B / B₀ % for each standard versus the corresponding concentration of PGE₂. Figure 1 shows a typical standard curve. (See Note 2)
Determine the PGE₂ concentration of the unknown samples by interpolation from the standard curve. Do not extrapolate values beyond the standard curve range.

Notes on procedure

Table 3.    Typical Assay Data

PGE₂ concentration pg/tube

Figure 1.
A typical standard curve
(Do not use to calculate sample values)

Characterization of the assay

Assay parameters

Specificity

Cross reactivity was defined in per cent by weight at the 50% displacement level.

+ main urinary metabolite of acetyl-salycic acid

Reproducibility of the assay

Plasma

Table IV.  Reproducibility obtained with plasma sample after solid-phase extraction.

5 samples were measured in 5 separate assays with triplicates. Mean values in pg/ml are indicated.

Urine

Table V.  Reproducibility obtained with unextracted urine.

4 samples were measured in 5 separate assays with triplicates. Mean values in pg/ml are indicated.

Recovery of standard materia


pg added

Figure 2.  Recovery of known amount of PGE₂ with direct assay of human urine

Samples were spiked with different amounts of PGE₂ and assayed in 1-10 dilution. For measured (y) vs added (x) amount y = 1.053x + 9.185 regression line with an highly significant correlation (correlation coefficient 0.9992) was obtained. Standard curve set-up with prostaglandin-free urine as matrix was used.


Linearity with dilution

 

Figure 3.  Linearity of dilution of plasma samples after extraction

Plasma sample spiked with PGE₂ was extracted according to suggested method on Amprep C₂ and assayed at a number of dilutions.

Sample values

Table VI.  Comparison of PGE₂ concentrations of human urine measured with and without extraction

4 samples were measured in 5 separate assays with triplicates. Extraction was carried out on Amprep C₂ according to suggested method.

Table VII.  Comparison of solid-phase materials

Aliquots of the same plasma sample were extracted on different column materials and assayed in 5 separate assays with triplicates. Concentrations in pg/ml are indicated.

Evaluation of immunoreactive purity by immuno-chromatography

Figure 4.  Immunoreactivity profiles obtained with urine and plasma after solid-phase extraction

Pooled human plasma and normal 24-hours urine of healthy male volunteer, both containing tritiated PGE₂ were subjected to solid-phase extraction on Amprep C₂ and the extracts separated on Kieselgel-60 silica plate using ethyl-acetate: acetic acid:iso-octane:water (110:20:50:100,v/v/v/v, upper phase) as solvent system. Fractions were eluted and measured in PGE₂ assay. Homogenous immunoreactivity, co-migrating with tritiated PGE₂ in both urine (open bars) and plasma (solid bars), was obtained.

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.

Chemical and other hazard

Magnetic immunosorbent contains sodium azide (0.1% w/v) as an antimicrobial agent. Dispose the waste by flushing it with copious amounts of water to avoid build up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 55 mg.