This kit is intended for research use only, not for use in humans or clinical diagnosis.
This kit is shipped ambient. Upon receipt store the individual components as detailed in this leaflet.

Warning

This kit contains less than 74 kBq (2 µCi) ¹²⁵I. Although this limited amount of ¹²⁵I isotope may be exempt from licensing, in most countries of the world the receipt, possession, use, transfer and disposal of radioactive materials are subject to the regulations of appropriate governmental organizations. Responsibility for compliance with regulations concerning the procurement of radioactive materials is that of the purchaser.

Introduction

Arachidonic acid, released from the cell wall by phospholipase A₂, is converted to prostaglandin endoperoxides on the effect of cyclo-oxygenase (endoperoxide synthase (1, 2)). Endoperoxides are then common precursors to a variety of prostanoid compounds including prostaglandin F_2a (PGF_2a).

Prostaglandin F_2a can be detected in almost each animal and human tissue and has widespread biological effects. Since a general discussion on its occurence and significance in different physiological processes are beyond the scope of this booklet, users are kindly suggested to refer to general reviews (e.g. Ref 3).

Although in various biological tissues a large variation of the concentration of Prostaglandin F_2a is observed, its quantitation usually requires most sensitive procedures. Among possible experimental techniques, radioimmunoassay has become the most popular one, and in prostanoid field it is prostaglandin F_2a whose radioimmunological determination goes back to the longest past, for it has been known since as early as 1972 (4).

In this radioimmunoassay kit, the combination of a high specific activity iodinated derivative of Prostaglandin F_2a as tracer with rabbit anti-Prostaglandin F_2a antiplasma as specific antibody, provides an efficient tool for simple and sensitive determination of Prostaglandin F_2a in biological fluids.

Principle of the method

The principle of radioimmunoassay is competitive binding; the radioactive ligand (¹²⁵I-Prostaglandin F_2a -TME tracer) competes with the unlabelled ligand (Prostaglandin F_2a in standards or 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 precipitating the bound fraction with polyethylene-glycol. After centrifugation the supernatant is removed and 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

125I-Prostaglandin F_2a -TME Tracer

One vial contains approximately 2 µCi of ¹²⁵I-Prostaglandin F_2a -TME in 50 mM phosphate buffered saline, pH 7.3, containing 0.3% human gamma-globulin, 0.02% Triton X-100 and 0.01% merthiolate. This solution should be stored at 4°C, and is stable for at least two months from the date of receipt.

Prostaglandin F_2a antiserum

The antiserum was raised in rabbit againts a bovine serum albumin conjugate of Prostaglandin F_2a. Stored at 4°C, the lyophilized antiserum is stable for at least two months. For use in the assay, reconstitute the antiserum by adding 10 mL of distilled water with gentle mixing to avoid foaming. Make ensure that the lyophilized material is in solution. So as to make complete solution faster, the material can be equilibrated in water bath of 35°C for a few minutes. After resonstitution, the solution contains Prostaglandin F_2a antiserum of appropriate binding ability in 50mM phosphate buffer, pH 7.3, with 0.1% gelatin and 0.01% merthiolate. This solution should be stored at 4°C. Under these conditions the solution is stable for at least two months.

Prostaglandin F_2a standard

Reconstitute the lyophilized Prostaglandin F_2a standard by adding exactly 1.0 mL distilled water. Make ensure that the lyophilized material is in solution. The resulting solution contains 10 ng of Prostaglandin F_2a per mL in 50 mM phosphate buffer, pH 7.3 with 0.1% gelatin and 0.01% merthiolate. Stored at -20°C this stock solution is stable for at least two months. Immediately before use in the assay dilute an appropriate aliquot of the standard stock solution to prepare standards. A suggested dilution scheme is shown later. Store the remaining standard stock solution at -20°C. Do not store diluted standards.

Assay buffer

To prepare assay buffer for use in the assay, warm the bottle containing buffer concentrate to room temperature and add 80 mL water. The assay buffer thus obtained contains 50 mM phosphate buffer, pH 7.3 with 0.1% gelatin and 0.01% merthiolate. Stored at 4°C, it is stable for at least two months.

Polyethylene glycol solution

This reagent contains 20% PEG-6000 and 1% Tween-20 in distilled water, with 0.01% merthiolate as preservative. Stored at 4°C the solution is stable for at least two months.

Sample handling

It is well known that prostaglandins are produced artificially, when tissues are collected (5). When working with blood samples, a special care should be taken to minimizing ex vivo biosynthesis and isolating samples under strictly identical conditions. With respect to special difficulties and pitfalls encountered with handling and preparation of samples, users are recommended to refer to more detailed methodological reviews (5, 6).

A) Collection and storage

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 buffer (pH 7.3) with 1mM indomethacin. At this concentration we have found no interference of indomethacin in the assay. If storage of plasma samples is necessary, -70°C or lower is recommended.

Urine samples should be stored at -20°C after pooling the single samples collected from one patient.

Methods for handling tissue samples are varying according to experimental setup. More generally, they are homogenized in organic solvent immediately after isolation and stored at -70°C or lower until assay.

B) Preparation of samples prior to assay

Depending on species, sex and status of blood, plasma level of prostaglandin F_2a may vary in a wide range, but, in majority of cases, it should be extracted from plasma samples. Even though the apparent endogenous concentration is high enough for a direct assay to be enabled, plasma should be extracted because of interference of plasma proteins with radioimmunoassay.

For the isolation of prostaglandin F_2a from plasma, C₂-silica cartridges (e.g. Bond-Elut C₂ from Varian Group Ltd, or Amprep C₂ from Amersham International plc) can be used according to the following procedure.

Solid-phase extraction of human plasma using Bond-Elut C₂ minicolumns

Solid-phase extraction of human urine using Bond-Elut C₂ minicolumns

The procedure above is used except that step 7 is ommitted.

Remarks

This procedure usually results in a recovery of 70 - 80%, as checked by ³H-labelled Prostaglandin F_2a as recovery marker. Because of potential errors encountered with individual factors, it is suggested that user follow extraction efficiency throughout procedure employed in his laboratory.

Quality requirements for tritiated prostanoids being suitable as recovery marker are high; specific activity, chemical and radiochemical purity should be as high as possible. Depending on the chemical structure of prostanoid, tritium is quickly exchanged by solvent hydrogen, a phenomenon resulting in serious underestimation of extraction efficiency. To overcome this source of error, special care should be taken of the chemical and radiochemical integrity of tritiated recovery marker. It is recommended to store it according to manufacturer's instruction, to check its radiochemical purity regularly, and to purify it, if needed, by chromatographic method before use.

Solvent residues, impurities as well as biological matrix itself may often introduce an astonishingly high non-specific immunoreactivity whose degree is a function of analyte, assay medium, quality of antibody and solvents, etc. This may give rise to a considerable overestimation of real concentration and the lower the concentration the higher the relative error due to method blank. In order for the blank contribution to be corrected, prostaglandin-free sample (to estimate matrix contribution and method blank simultaneously) and/or buffer (to determine method-blank only) should be subjected to the procedure strictly identical to that used for unknowns. Concentration of unknowns should be corrected accordingly.

It should be stressed that the same types of solid phase materials perform very differently and slight batch-to-batch variation can be observed frequently even with same type of cartridges from the same manufacturer.

These variations may give rise to changes of recovery as well as immunoreactive purity, when using such a "fine tuned" elution pattern as the one above. Preparation of solvent mixtures is also a critical step; freshly prepared mixtures should be used for each procedure and the concentrations should be strictly identical to that suggested above. In case of undesired loss of recovery, it is recommended to decrease the concentration of acetonitrile in wash step and/or n-hexane content in final eluent.

Because of pitfalls and difficulties encountered with determination of eicosanoids in general, it remains the investigator's responsibility to validate his own procedure.

As it can be seen on Figs 2 and 3, the immunoreactivity profiles obtained by using the suggested solid phase extraction procedure showed one major immunoreactive peak co-migrating with authentic PGF_2a. Percent ratio of the specific peak was much higher than that observed with traditional unoptimized elution scheme. The uniform immunoreactivity makes it unnecessary to apply any subsequent chromatographic purification step after extraction.

It has been reported that in plasma samples upon long-lasting storage epimeric PGF-ring compounds - 8-iso-PGF_2a above all - can be produced in large amount by non-enzymic oxidation processes (7, 8). Therefore plasma samples should be assayed freshly and, in critical cases, it should also be checked, whether or not the apparent PGF_2a-like immunoreactivity reflects, at least in part, the concentration of 8-iso-PGF_2a. This is ruled out by the complete separation of PGF_2a from the 8-iso-epimer in the HPLC system used in Figs 2, 3. Moreover, merely the low cross-reactivity of present antibody with the 8-iso-epimer makes it unlikely that PGF_2a-like immunoreactivity may result from the contribution, to a considerable degree, of 8-iso-PGF_2a.

Recently the reference intervals for the excretion rates of various prostanoids were determined by authentic gas-chromatography / mass spectrometry technique (9). The mean value reported for daily excretion rate of urinary prostaglandin F_2a was about 1000 ng/24 h. From this value, an average of 500 - 1000 pg/mL for concentration range in normal human subjects can be expected. By the use of suggested optimized extraction method alone, without any further chromatographic purification, the mean value (900 pg/mL) for pooled human urine of male volunteers obtained in our laboratory was well within expected range.

Assay procedure

Materials required

Table I – Dilution scheme  (All volumes are in microliter. To prepare standard dilution and to dissolve or dilute assay buffer must be used.)

Vial "s" is prepared by reconstituting the lyophilized standard with 1.0 mL distilled water

Radioimmunoassay protocol

Day 1

Day 2

Calculation of results

To calculate results, refer to Table III.

Calculation by computing data using logit-log or other fitting programs may also be applied but is not dealt with here.

Table II – PGF_2a assay protocol    (Volumes in microliters)

Table III – Sample calculation

Figure 1
A typical standard curve
(Do not use to calculate unknowns)
 

Performance characteristics

A) Specificity of the antibody

Table IV – Typical cross reactions at 50 % displacement

B) Assay parameters

C) Reproducibility of the assay

Table V – Between-assay

Quality control samples containing low, medium and high concentration of Prostaglandin F_2a were measured in 15 independent assays using triplicates.

D) Evaluation of immunoreactive purity by immuno-chromatography
 

Figure 2.
Immunoreactivity profile obtained with urine after solid-phase extraction.

24-hour pooled urine collected from healthy male volunteers, containing tritiated Prostaglandin F_2a was subjected to solid-phase extraction on Bond-Elut C₂ according to suggested procedure and the extracts separated by reversed-phase HPLC using a combined gradient elution with water:acetonitrile (0.1% CH₃ COOH) as a mobile phase on Spheri-5 C₁₈ Microbore column at a flow-rate of 0.4 mL/min. Fractions eluted were measured in Prostaglandin F_2a assay. The main immunoreactive fraction co-migrated with tritiated Prostaglandin F_2a (marked by asterisks).
 

Figure 3.
Immunoreactivity profile obtained with plasma after solid-phase extraction

Pooled normal human plasma containing tritiated Prostaglandin F_2a was subjected to solid-phase extraction on Bond-Elut C₂ according to suggested procedure and the extracts separated by reversed-phase HPLC using a combined gradient elution with water:acetonitrile (0.1 % CH₃ COOH) as the mobile phase on Spheri-5 C₁₈ Microbore column at a flow-rate of 0.4 mL/min. Fractions eluted were measured in Prostaglandin F_2a assay. Homogeneous immunoreactivity, co-migrating with tritiated Prostaglandin F_2a (marked by asterisks) was obtained.

References

PROSTAGLANDIN F_2a ASSAY KIT WITH MAGNETIC IMMUNOSORBENT

Code: RK-15M

Apart from the insignificant differences detailed below, formulation, assay procedure and characteristics of magnetic version are identical to those of RK-15.

Contents of the kit

Except for PEG solution which is substituted for paramagnetic immunosorbent, reagents are identical to those of RK-15 assay kit.

Magnetic immunosorbent suspension (MIS)

This reagent contains paramagnetic particles coated with anti-rabbit immunoglobulin suspended in 50 mM phosphate buffer, pH 7.3 with 0.01% merthiolate and 0.05% Triton X-100. Stored at 4°C, it is stable for at least two months.

Assay procedure

Materials required

In addition to those listed with RK-15, magnetic separator comprising 50 tube rack and magnetic base may be necessary.

Assay Procedure

Until Day 2, steps 1–14, the procedure is identical to that used with RK-15.
For Day 2, steps 15–19 are modified as described below.

Option 1

Centrifuge the tubes at 4°C and 2000 x g for 20 minutes.[g = 1.118 * (rpm)² * (arm-lenght of the centrifuge in cm)]

Option 2

When working with magnetic separator, attach the tube rack on to the separator base. Ensure that all tubes are in contact with the base plate. Leave for 15 minutes, then invert the separator (with base plate kept attached!) and pour off the supernatant. Keeping the separator inverted, place the tubes on absorbent tissue, and allow to drain for 5 minutes.

Continue with step 20 of standard assay procedure.