HU209849B - Process for radioimmunological determination of content of 5-hydroxi- 6,8,11,14-eicosatetra-en acid in biological samples - Google Patents

Abstract

Radio immuno-assaying based on antigen - antibody reactions of 5-(S)-hydroxy-6-trans, 8 cis, 11 cis, -14 cis, -eicosatetranoic acid, (5-(S)-HETE), present in samples of biological media, is improved by application of 5-HETE-mono: iodo-tyrozine -methyl-ester derivs., marked by iodo 125 isotope, as radio ligands or tracers of high specific activity.

Description

FIELD OF THE INVENTION This invention relates to radioimmunoassay based on the antigen-antibody reaction of 5- (S) hydroxy-6 (trans), 8 (cis), 11 (cis), 14 (cis) -ecatetetraenoic acid [5- (S) -HETE] in biological samples. determination. The tracer method of the new type (I) indicated in formula Iizotóppal ^125, high specific activity of 5 HETEmonojód-tyrosine methyl ester derivative is used.

HU 209 849 B

Scope of the description: 6 pages (including 1 page figure)

HU 209,849 Β

The present invention relates to a method for radioimmunoassay of 5- (S) -hydroxy-6 (trans), 8 (cis), 11 (cis), 14 (cis) -eciodetraene av (hereinafter referred to as 5- (S) -HETE) biological samples, by measuring the binding of ¹²⁵ I-labeled high specific activity 5-HETE derivative as a tracer (hereinafter radioligand or tracer) to a specific antibody in the presence of known concentrations of standard solutions and samples to be determined, and calculating their drug content from the binding changes.

According to the invention a new type of formula (I), ¹²⁵ I-labeled radiopharmaceuticals with high specific activity 5 (±) -Het-monojódtirozin ester derivative (hereinafter referred to as ¹²⁵ I-5ΗΕΊΈ ΤΜΕ-) used as a tracer in the immunochemical reaction.

It is known that arachidonic acid is a so-called. The 5-lipoxygenase cascade produces highly structured, high-potency bioactive substances. A common intermediate of these is 5-hydroperoxy-6,8,11,14-ejectetraenoic acid, one of its stable derivatives being 5- (S) -HETE. Knowledge of its amount is important in determining the activity of 5-lipoxygenase in various organs and tissues. 5 (S) -HETE, like other arachidonic acid metabolites, is found in biological systems at very low concentrations (less than ^10-12 mol / L), so that only the most sensitive methods are suitable for measurement.

The antigen-antibody-based radioimmunoassay (RIA) method is the most suitable for the rapid, inexpensive, sufficiently sensitive and accurate determination of low concentrations of bioactive substances. The method requires a radioactively labeled form of the molecule to be measured which has a sufficiently high specific activity. For this purpose, various literature sources (e.g., Prostaglandins 29; 481, 1985; Prostaglandins, Leukotrienes and Medicine 18: 272, 1985; J. Immunology 136: 3455, 1986; Prostaglandins 32: 857, 1986; J. Immunology 144: 2696). , 1990) have so far used only a commercial RIA kit (Advanced Magnetics, Inc., USA) that uses tritium-labeled 5- (S) -HETE as a tracer. The tritium-labeled tracer essentially meets the above requirements of the specification, and has the advantage that the isotope is considered stable due to its relatively long half-life (about 12 years) and the accuracy and reproducibility of such tracer-based RIAs is generally very good. However, the drawback is that the preparation of the tracer requires a unique, multistep chemo-radiochemical synthesis in which the specific activity available is theoretically maximal at 220 Ci / mmol based on the number of tritium atoms incorporated, but in practice above 150 Ci / mmol It matters. A further disadvantage is that the measurement of tritium is costly and laborious. It is an object of the present invention to provide an RIA method for determining the concentration of 5-HETE simply, accurately, and sensitively by replacing 5- (S) HETE with tritium with a new type of ¹²⁵ I-isotope instead of 5- (±) -HETE. is used as a tracer. Instead of tritium labeled tracers, many small molecule bioactive materials have been successfully used ^{, including 25} I-labeled tracers. They are obtained by pre-forming a derivative of the parent molecule, usually containing an aromatic group, on which the ¹²⁵ I isotope can be incorporated in a directed manner. This type of compound is

5 - (+) - HETE-tyrosine methyl ester, from which the preparation of ¹²⁵ I-labeled high activity mono-iodine was described (J. Chromatography 543: 307, 1991).

The great advantage of using the ¹²⁵ I-labeled tracer is that it does not require complicated, costly preparation of the tritium-labeled parent molecule form. The use of the ¹²⁵ I isotope also allows measurement in laboratories where the expensive equipment for tritium is not available.

The present invention is based on the discovery that the determination of 5-HETE concentration by a radioimmunoassay based on antigen-antibody binding is possible by a novel type of high specific activity labeled ¹²⁵ I-isotope in the immunochemical reaction.

A 5-HETE derivative was used. This finding is not self-evident, since the chemical structure of the tritium-labeled tracer is identical to that of the inactive ligand, whereas the production of the ¹²⁵ I-radioligand involves the chemical transformation of the parent molecule, so that the radioligand and the inactive ligand are chemically different. Accordingly, it is not necessary that the ^{125 I} -labeled derivative be recognized by the specific antibody as an antigen.

It has been found that the use of the 5-HETE monoiodo tyrosine methyl ester derivative as a specific tracer is possible despite the structural difference and thus the aforementioned advantages of using the ¹²⁵ I isotope can also be negatively exploited in the determination of 5-HETE radioimmunity.

It is further recognized that not only the structural change resulting from derivatization, but also the C ₅ chirality of the parent molecule itself does not affect the immunochemical reactivity of the iodine-labeled derivative. In this way, it is preferable to use radioligands which are substantially easier to synthesize instead of 5- (S) -HETE and thus can be based on cheaper racemic 5- (±) -HETE as a synthesis starting material.

The present invention therefore relates to a method for the reactive determination of the content of 5- (S) hydroxy-6 (trans), 8 (cis), 11 (cis), 14 (cis) -ecatetraenoic acid [5- (S) -HETE] in an biological sample. in the presence of a known amount of standard 5-HETE at different concentrations in the dilution series and measuring the binding of tracemes to a specific antibody, and a known amount of standard 5- (S) -HETE, or preferably 5- (±) according to the invention. ) -HETE, calculate the amount of active substance in the sample by the dilution series by subtracting the high specific activity of ¹²⁵ I-labeled tracer as a tracer.

5- (±) -HETE is used.

HU 209,849 Β

According to the invention, various known amounts of inactive are used according to the dilution series

Standard solutions of 5-HETE and unknown samples were incubated with specific antibody and ¹²⁵ I-5-HETE-TME tracer, and the bound and non-bound fractions of the antibody were separated and the radioactivity of the bound fraction was measured. From the obtained radioactivity values, concentrations corresponding to changes in binding caused by unknown samples are calculated.

Prior to radioimmunoassays, the active ingredient is extracted from the various types of biological samples to be measured and purified to the extent necessary for measurement.

Generally, a tracer of 10,000 to 60,000, preferably 20,000 to 40,000 cpm is used and is preferably dissolved in buffer.

The buffer solution is preferably 0.01-0.2 molar, preferably 0.05 molar, pH 7.4 aqueous phosphate buffer containing 0.1% gelatin.

Depending on the particular composition of the solution, the incubation temperature is between 0 and 40 ° C, preferably about 0 ° C, and the incubation time varies between 0.5 and 24 hours, preferably between 15 and 25 hours, depending on the temperature.

Preferably, the specific antibody required for the method can be produced in rabbits. For this, a suitable carrier protein for 5-HETE, preferably 5 - (+) - HETE, e.g. bovine serum albumin, and the rabbits are inoculated again. The concentration of anti-5-ΗΕΤΕ antiserum obtained from immunized rabbits is chosen such that 10-80%, preferably 30-60%, of the tracer is bound to the antibody in samples without the respective separation reagent and incubation time. .

The standard material required for the method may be 5- (S) -HETE or 5- (±) -HETE, the latter being preferably used. The equivalent use of the two chiral materials is made possible by the fact that both forms of the tracer of the invention are clamped equally from the binding sites of the specific antibody. Thus, although 5- (S) -HETE is always present in the biological sample, the substantially cheaper 5- (±) -HETE can be used as a standard.

Preferably, 0.01 M phosphate buffer (pH 7.4) containing 0.5% dextran and 1% charcoal, which binds the free radioligand, is used to separate the bound and free fractions of the radioligand, and the supernatant obtained by centrifugation contains the antibody-bound tracer, whose radioactivity is measured by scintillation.

The advantages of the process of the invention are summarized as follows:

The method determines 5-HETE content using a radioimmunoassay method based on the derivative of racemic 5-HETE ¹²⁵ I-labeled monoiodine tyrosine methyl ester as a tracer. This eliminates the need for the multi-step, costly and laborious synthesis route for tritium-labeled 5 (S) -HETE, and the overall benefits of using ¹²⁵ I-isotope, ie high sensitivity, simple, fast and inexpensive measurement technology - 5-HETE radioimmune definition can also be exploited.

It is also an advantage that 5- (±) -HETE reacts with the radioligand used in the process of the invention in the same way as 5- (S) -HETE in the samples, so that the more expensive 5- (S) -HETE is not required as a standard.

The invention will be described in more detail by reference to specific examples, which are, of course, not limited to the scope of the invention.

Example 1

Determination of 5- (S) -HETE in Human Plasma after Ionophoric Stimulation Previously stimulated with calcium ionophore (e.g., A-23187, Sigma, USA, e.g. Prostaglandins 42: 279, 1991). Plasma blood samples were assayed for solid phase extraction prior to determination by adding 100 μΐ 2 M citric acid solution to 1 ml plasma, pipetting into a syringe and passing through a syringe. Plastic containing "reverse phase" silica cartridge, or miniature column (Sep-Pak _C18, Waters Ass., USA, and Bond-Elut C _18, Varian, U.S.A.) which had been prepared as described by the manufacturer's directions. The cartridge was then pressed through the same syringe with 10 mL of distilled water and 10 mL of 5% aqueous ethyl alcohol. These liquids were discarded and the cartridge was flushed through 10 mL of ethyl acetate, collected in a plastic test tube, added with 1 mL of distilled water, stirred in a tube mixer for a few seconds, then suctioned off with a pipette and discarded. This operation was repeated once more and the ethyl acetate solution was evaporated to dryness under vacuum at 30-35 ° C on a Rotavapor rotary evaporator (Büchi, Switzerland) and dissolved in RIA buffer (see below). In parallel to the unknown samples, a plasma sample derived from non-stimulated blood ("blank") is processed in exactly the same manner as above. Samples prepared as described above are diluted 100-fold with RIA buffer immediately prior to RIA measurement.

For RIA measurement, buffer dilutions were made from standard 5 (±) -HETE stock solutions at 10 ng / ml to obtain 1 ml samples containing 0.12-0.37-1.11-1.3310 ng of active ingredient. / ml. Weigh out 100 to 100 μból of the standard solutions and specimens to be determined into serially placed plastic test tubes, then add 100 μΐ of antiserum in dilution capable of binding 30 to 40% of the tracemes. Subsequently, 100 μ tr of the tracer (I) with 30,000-40,000 cpm / 100 μΐ is pipetted into each tube. For the determination of nonspecific binding (NSB), 100 μΐ pure buffer solution was added to additional test tubes instead of the antiserum, while pure buffer solution was added to the antibody instead of the standard solutions to determine specific binding (Βθ / Τ) and total radioactivity (TC).

HU 209,849 Β

Three replicates (three tubes of the same reagent composition) are used for each measuring point and the volume of reaction mixture in each tube is made up to 400 μΐ with the required volume of buffer. 0.05 M aqueous phosphate buffer (RIA), pH 7.4, containing 0.1% gelatin, was used to dissolve and volume up all reagents (tracer, antibody, standards and samples).

After weighing the reagents, the contents of the test tubes are mixed with the test tube mixer for a few seconds, then sealed and allowed to stand in the refrigerator at 0-4 ° C overnight. Then, 500 μΐ of cold RIA buffer was added to the TC tubes and 500 μΐ of the charcoal suspension was added to the rest of the tubes, which was stirred vigorously with magnetic stirrer in an ice-water bath. The carbon suspension consisted of 0.01 M phosphate buffer, pH 7.4, 0.5% Dextran T-70 (Pharmacia, Finland) and 1% Norit-A (Serva, Germany). The test tubes were centrifuged at 0 ° C for 10 minutes. 0.5 ml of the supernatants are then pipetted into suitable measuring tubes and their radioactivity is auto-sampled with Nal (TI) scintillation crystals, e.g. Minigamma (LKB, Sweden) equipment was weighed. Performing the calculation as usual for radioimmunoassays, correcting for the dilution factor, and subtracting the blank value from the values obtained for the unknown samples, gives a concentration of 5-HETE in plasma samples which ranges from 100-300 ng / mg.

Example 2

Determination of 5-HETE biosynthesis of isolated cells

All proceed as in Example 1, with the exception that aqueous buffered media of isolated cells or cell cultures are used as a sample and that of a blank sample is cell-free media. In this method, the ability of different types of white blood cells (neutrophils and eosinophils granulocytes, monocytes, immunocytes) of different experimental animals or humans to determine the biosynthesis of 5-HETE is determined. Expected concentrations may vary within wide limits depending on the cell type being tested, the experimental conditions and the treatments used, and the sample dilution required for measurement is adjusted accordingly in pre-experiments so that the measured values are within the calibration curve. In order to calculate the exact concentration, the RIA data should be corrected for the dilution factor and the blank value.

In our own studies, 5-HETE biosynthesis on human neutrophil granulocytes after 10 minutes treatment with calcium inophor (Ionophore-A _{23 Ig7} , Sigma, USA) is 10-30 ng / 10 ⁷ cells, accordingly the buffer solution obtained after extraction is preferably 1050 ng / 10 ⁷ cells. measured in dilution.

Example 3

Determination of lung tissue 5-HETE content G lung tissue is homogenized in 5 ml of methanol, followed by 5 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 5 000 Hb of radiolabelled 5-HETE (1 ml). ³ H) -5- (S) -HETE from Amersham International plc, England]. 0.5 ml of the solution is set aside to measure the extraction efficiency ("Sample A") and an additional 1 ml is purified by the solid phase extraction of Example 1. One ml of RIA buffer sample after extraction is discarded in 0.1 ml ("sample B"), and the remaining RIA is determined by dilution optimized based on preliminary experiments. The exact tissue concentrations are calculated taking into account the dilution factor and the processing loss. The latter is determined by measuring the radioactivity of samples A and B by liquid scintillation and forming the B / A quotient. This value for the exemplary processing method is generally greater than 0.8, i.e. a processing loss of less than 20%.

The above method is generally applicable to the determination of lung tissue 5-HETE in various experimental animals (eg rat, mouse, rabbit). Tissue concentrations vary widely across animals and depending on the treatment, so the appropriate weight of tissue to be processed and optimal dilutions should be determined in preliminary experiments. According to our own measurements, 6-12 ng of 5-HETE can be measured in rat, 1 g of wet lung tissue after treatment with endotoxin (Escherichia coli endotoxin, Sigma, USA), for which 1 g of tissue is sufficient and 10-fold dilution of the extraction samples. -definition.

Example 4

Production of specific 5-HETE antiserum in saliva

To the process was used 5 mg of synthetic 5- (±) -HETE (Cayman Company, USA) chemically bound to 20 mg of bovine serum albumin (BSA, lyophilized and crystallized powder, Sigma, USA), Eicosanoids 4, 1. , 1991. The resulting immunogenic protein was treated with 3 male New Zealand white rabbits as follows:

mg of protein in 0.5 ml of physiological saline (0.9% aqueous NaCl), vigorously mixed with 0.5 ml of complete Freundadjuvant (Sigma, USA), and the resulting emulsion is uniformly injected into the dorsal skin of the test animal at 40-60 injection sites. distributed. At 1 month intervals, the above treatment was repeated using 0.5 mg protein instead of 1 mg and replacing the complete Freund's adjuvant with incomplete Freund's adjuvant (Human Vaccine Production Company, Gödöllő). This treatment is repeated monthly and 10 days after the treatment, 1/2 ml of blood is drawn from the ear vein of the animals, which is centrifuged at 1000 rpm after collection and the serum is separated. The resulting sera are classified according to the general RIA procedures as follows:

- Measure the binding capacity ("titer") of the sample with high specific activity [ ³ H] -5- (S) -HETE (Amersham International plc., England) and the ¹²⁵ I-labeled tracer of the invention.

HU 209,849 Β

- Using the same tracers, prepare standard curves for antiserum dilution as defined above using 5- (S) HETE and 5- (±) -HETE (both manufactured by Cayman Chemical Co., USA) as standard.

Based on the measurement data, the inoculations were continued as described above until an antiserum capable of binding both tracers was obtained, however, the binding of the ¹²⁵ I-labeled tracer was inhibited by 5-HETE and the inhibitory capacity was the same as that of 5- (S). HETE and 5- (±) -HETE.

Claims (4)

PATENT CLAIMS CLAIMS 1. A method for reactively determining the content of 5- (S) -hydroxy-6 (trans), 8 (cis), 11 (cis), 14 (cis) -ecatetraenoic acid [5 (S) -HETE] in a biological sample at known dilutions. serially in the presence of different concentrations of standard 5-HETE and the biological samples to be assayed, measuring the binding of the tracemes to the specific antibody and the known amounts of standard 5- (S) -HETE and 5- (±) -HETE, respectively. The active substance content of the sample is calculated using the ¹²⁵ I-labeled high specific activity 5- (±) -HETE-monoiodo-tyrosine methyl ester derivative of formula (I). Process according to Claim 1, characterized in that the tracer of formula (I) has a specific activity of between 3000 and 100,000, preferably between 10,000 and 60,000 cpm, with a specific activity greater than 40 TBq / mmol, preferably 74 TBq / mmol. employed. 3. The method of claim 1 or 2, wherein the antibody is a specific anti-5-ΗΕΤΕ immune plasmid or antiserum generated in rabbit, preferably against bovine serum albumin-bound 5- (±) -HETE as an immunogenic substance. In a sample containing no 5-HETE, tracer is capable of binding at a concentration of 10-80%, preferably 30-60%. 4. A process according to any one of claims 1 to 5, wherein 5- (S) HETE or preferably 5- (±) -HETE is used as the standard material. HU 209 849 Β Int.Cl. ⁵ : GOI N 33/50 C-NH-CH-CH (1) COOCH,