PatentDe  


Dokumentenidentifikation EP1835290 31.10.2007
EP-Veröffentlichungsnummer 0001835290
Titel Katecholamin-Assay
Anmelder LDN, Labor Diagnostika Nord GmbH & Co. KG, 48531 Nordhorn, DE
Erfinder Manz, Bernhard Dr., 48527 Nordhorn, DE
Vertragsstaaten AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HU, IE, IS, IT, LI, LT, LU, LV, MC, NL, PL, PT, RO, SE, SI, SK, TR
Sprache des Dokument EN
EP-Anmeldetag 17.03.2006
EP-Aktenzeichen 060055316
EP-Offenlegungsdatum 19.09.2007
Veröffentlichungstag im Patentblatt 31.10.2007
IPC-Hauptklasse G01N 33/94(2006.01)A, F, I, 20070821, B, H, EP
IPC-Nebenklasse G01N 33/74(2006.01)A, L, I, 20070821, B, H, EP   

Beschreibung[en]
TECHNICAL FIELD OF THE INVENTION

The present invention relates to the analysis of catecholamines. In particular, the invention relates to affinity extraction and detection and/or quantification of catecholamines.

BACKGROUND OF THE INVENTION

Catecholamines like norepinephrine, epinephrine and dopamine play an important role as neurotransmission substances or hormones. In order to study the functions and activities of sympathetic nerves, it is important to determine quantitative changes of catecholamines in a living body sample such as urine or plasma. Additionally, abnormal catecholamine concentrations are associated with a variety of diseases, for example hypertonia, pheochromocytoma, sympathetic neuroblastoma, degenerative cardiac diseases, schizophrenia, and alternating psychosis ( Manz B. et al. (1990) GIT Labor-Magazin 5/90, 245-254 ).

Catecholamines are very unstable and readily undergo oxidation, in particular under basic conditions. Furthermore, the concentration of catecholamines especially in plasma samples is quite low. Thus, there is a need for quick and sensitive determination methods for catecholamines.

To date, the following methods are available for the determination of catecholamines: fluorometric assays, radio enzymatic assays (REA), high-performance liquid chromatography (HPLC) in combination with different detection techniques, gas chromatography with mass spectrometric detection (GC-MS), radio immunoassays (RIA) and enzyme immunoassays (EIA) ( Manz B. et al. (1990) GIT Labor-Magazin 5/90, 245-254 ; Wolthers B.G. et al. (1997) Clinical Chemistry 43, 114-120 ). However, the fluorometric and radio enzymatic assays lack accuracy and reproducibility and the fluorometric assays do not differ between the different catecholamines. HPLC and GC-MS are time consuming, need complex technical equipment and, therefore, are cost-intensive. Thus, the most suitable methods for determining catecholamines in view of reliability, cost, accuracy, reproducibility and rapidness are the immunoassays.

In currently available EIA- or RIA-based assays for determining catecholamines, the catecholamines are first extracted out of the sample, then derivatized for antibody recognition and to enhance their stability, and thereafter detected using antibodies specific for catecholamines. Thereby, the most commonly used extraction method is binding of the catecholamines via their cis-diol structure to a boronate affinity gel, see e.g. DE 198 18 485 . The subsequent modification to stabilize the catecholamines is done using the enzyme catechol-O-methyl-transferase (COMT) which O-methylates the 3-hydroxyl group of the catechol moiety. Thus, for the COMT to access the 3-hydroxyl group of the catechol moiety, the catecholamines have to be released from the affinity gel which is achieved by adjusting the pH to acidic conditions. However, COMT is not active under acidic conditions. Therefore, in order to allow the reaction catalyzed by COMT to take place and prevent rebinding of the catecholamines released from the boronate affinity gel, the released catecholamines have to be separated from the boronate affinity gel by transferring them into another reaction vessel.

This transfer of the sample, however, creates an additional source of error and complicates the assay.

A known principle for reducing the transfer steps in methods for analyzing catecholamines by EIA is to perform the enzymatic O-methylation and the subsequent EIA in one reaction vessel. However, a drawback of this method is the fact that derivatization of the catecholamines and binding to the specific antibodies proceed in a parallel manner. Since only derivatized catecholamines are detected by the used antibodies this results in lowered precision and kinetics of the EIA and thus, in a prolonged assay time. Furthermore, this method cannot be performed in standard automated EIA analyzers due to the preceding enzymatic reaction taking place in the same reaction vessel.

Thus, there is a need for a method for analyzing catecholamines wherein no transfer step during the extraction and derivatization procedures is necessary. Such a method would eliminate a source of error and enable the separation of the extraction and derivatization procedures from the EIA, thereby allowing the use of standard automated EIA analyzers.

SUMMARY OF THE INVENTION

The present invention relates to a method for analysis of catecholamines which does not require the step of transferring the sample in a different reaction vessel to avoid rebinding of catecholamines to the cis-diol specific affinity medium used for extraction of the catecholamines from the sample. In particular, according to the present invention, the released catecholamines have not to be separated from the cis-diol specific affinity medium prior to adjusting the pH to non-acidic conditions which may be required, for example, for subsequent enzymatic reactions, in particular that catalyzed by COMT.

Accordingly, one aspect of the present invention relates to a method for analyzing at least one catecholamine comprising the steps of (a) binding the at least one catecholamine to a cis-diol specific affinity medium, and (b) adding a substance that blocks the binding of catecholamines to the cis-diol specific affinity medium.

Preferably, the at least one catecholamine comprises a naturally occurring and/or a synthetic catecholamine. More preferably, the naturally occurring catecholamine is selected from the group consisting of adrenalin, noradrenaline, dopamine, epinephrine, norepinephrine and mixtures thereof.

In one embodiment of the method of the present invention, the at least one catecholamine is contained in a biological sample or a fraction thereof. Preferably, the biological sample is a urine sample or a plasma sample.

In a preferred embodiment of the method of the present invention, the cis-diol specific affinity medium is a boronate affinity medium, preferably comprising amino boronic acid groups, more preferably m-aminophenyl boronic acid groups.

In one embodiment of the method of the present invention, the cis-diol specific affinity medium comprises cis-diol specific groups linked to a reaction vessel which may be a member selected from the group consisting of macro- and microtiter plates, pin plates, multi-well plates, cell and tissue culture plates and wells, test tubes and flasks.

Preferably, step (a) takes place at about neutral to basic pH, more preferably at a pH of about 6 or higher, even more preferably at a pH from about 7 to about 10.

In one embodiment of the method of the present invention, the substance added in step (b) blocks the binding of catecholamines to the cis-diol specific affinity medium by competitive binding to the cis-diol specific affinity medium. Preferably, the substance contains at least one cis-diol structure, and more preferably is selected from the group consisting of 1,2-dihydroxyalkanes, polyphenols, 2-hydroxy acids, ascorbic acid, carbohydrates, derivatives thereof and mixtures thereof. The 1,2-dihydroxyalkane may be ethanediol, 1,2-propanediol, or an alkanediol comprising vicinal hydroxyl groups. The polyphenol may be a catechol, anthocyane, gallic acid, or tannin. The 2-hydroxyacid may be lactic acid, tartaric acid, or citric acid. The carbohydrate may be a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide. The carbohydrate derivative may be a nucleoside, a nucleotide, an oligo- or polynucleotide such as RNA, or a glycoprotein. Preferably, the substance is a saccharide such as sorbitol or fructose, ascorbic acid or a nucleotide.

In one embodiment, the method of the present invention further comprises the step of releasing the at least one catecholamine bound to the cis-diol specific affinity medium from the cis-diol specific affinity medium. In this embodiment, step (b) and the step of releasing the at least one catecholamine may be performed simultaneously or step (b) may be performed prior to or following the step of releasing the at least one catecholamine. Preferably, the at least one catecholamine is released from the cis-diol specific affinity medium by addition of an acid including organic acids, inorganic acids and mixtures thereof. Organic acids include acetic acid, propionic acid, pyruvic acid, malonic acid, succinic acid, maleic acid, fumaric acid, benzoic acid, cinnamic acid, methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, salicylic acid, glycolic acid, lactic acid, oxalic acid, malic acid, citric acid, and mandelic acid.

Inorganic acids include sulfuric acid, nitric acid, phosphoric acid, perchloric acid, and hydrohalic acids such as hydrochloric acid, hydrobromic acid and hydroiodic acid. Preferably, the acid is hydrochloric acid or perchloric acid. Furthermore, in the step of releasing the at least one catecholamine from the cis-diol specific affinity medium the pH is preferably about 3 or lower, more preferably from about 1 to about 2.

In one embodiment, the method of the present invention further comprises a rinsing step following step (a) and prior to the step of releasing the at least one catecholamine from the cis-diol specific affinity medium.

In one embodiment of the method of the present invention, the substance added in step (b) is added to a final concentration that (i) is sufficient to block the binding of at least 90%, preferably at least 95%, more preferably at least 98% and most preferably at least 99% of the at least one catecholamine released from the cis-diol specific affinity medium to the cis-diol specific affinity medium, and/or (ii) equals or exceeds the binding capacity of the used cis-diol specific affinity medium. Preferably, the ratio between the final concentration of the substance added in step (b) and the binding capacity of the used cis-diol specific affinity medium is in the range of from about 1:1 to about 5000:1.

Furthermore, in one embodiment of the method of the present invention, following step (b) and the step of releasing the at least one catecholamine from the cis-diol specific affinity medium the at least one catecholamine is to be subjected to at least one reaction which requires non-acidic conditions, preferably an enzymatic reaction. Preferably, the reaction is an O-alkylation, more preferred an O-methylation. Most preferably, the O-methylation is effected by means of the enzyme catechol-3-O-methyl-transferase in its soluble or membrane bound form. Furthermore, the non-acidic conditions may include a pH of about 6 or higher, preferably of from about 6 to about 9.

In one embodiment, the method of the present invention further comprises the step of acylating the at least one catecholamine. The acylation may be effected by Bolton-Hunter reagent. Preferably, the at least one catecholamine is acylated while bound to the cis-diol specific affinity medium. Furthermore, a rinsing step following acylation of the at least one catecholamine and prior to the step of releasing the at least one catecholamine from the cis-diol specific affinity medium may be included in the method of the present invention.

In one embodiment, the method of the present invention further comprises the step of detecting and/or quantifying the at least one catecholamine, preferably using an antibody specific for a catecholamine or modified catecholamine. Preferably, the detection and/or quantification comprises an enzyme or radio immunoassay.

In one embodiment of the method of the present invention, the substance added in step (b) does not interfere with the following reactions or with a subsequent immunoassay.

In one embodiment of the method of the present invention, step (a), step (b), the step of releasing the at least one catecholamine from the cis-diol specific affinity medium and the at least one reaction which requires non-acidic conditions are all done within the same reaction vessel. Preferably, the step of detection and/or quantification is also done within the same reaction vessel.

Another aspect of the present invention relates to a reaction vessel suitable for performing the method of the present invention, wherein the reaction vessel comprises a cis-diol specific affinity medium and a catecholamine or an antibody specific for a catecholamine specific antibody immobilized to the reaction vessel.

In one embodiment, cis-diol specific groups are linked to the reaction vessel. Furthermore, the cis-diol specific affinity medium is preferably a boronate affinity medium. The reaction vessel includes macro- and microtiter plates, pin plates, multi-well plates, cell or tissue culture plates or wells, test tubes and flasks. Preferably, the reaction vessel is in a pin plate format wherein the catecholamine or the antibody specific for a catecholamine specific antibody is immobilized on one or more pins of the pin plate. Preferred embodiments of the cis-diol specific affinity medium and the catecholamine for use in this aspect of the invention are as described herein. In particular, the catecholamine immobilized to the reaction vessel is preferably acylated and/or O-alkylated and the antibody immobilized to the reaction vessel is preferably specific for an antibody specific for an acylated and/or O-alkylated catecholamine.

A further aspect of the present invention relates to a kit suitable for performing the method of the present invention comprising (a) a cis-diol specific affinity medium, and (b) a substance that blocks the binding of catecholamines to the cis-diol specific affinity medium. Preferred embodiments of the cis-diol specific affinity medium and the substance are as described herein.

In one embodiment, the kit of the present invention further comprises at least one member selected from the group consisting of a reagent for releasing catecholamines bound to the cis-diol specific affinity medium, in particular an acid as described herein, a reaction vessel, an extraction buffer, an acylation reagent, an acylation buffer, an enzyme solution, reagents for detection of catecholamines and a washing buffer. Preferably, the enzyme solution contains an enzyme for O-methylating catecholamines, more preferably COMT. Furthermore, the reagents for detection of catecholamines are preferably reagents suitable for an enzyme or radio immunoassay.

In one embodiment of the kit of the present invention, the substance that blocks the binding of catecholamines to the cis-diol specific affinity medium and the reagent for releasing catecholamines bound to the cis-diol specific affinity medium are contained in one composition.

In a further embodiment of the kit of the present invention, the cis-diol specific affinity medium comprises cis-diol specific groups coupled to a reaction vessel. Preferably, the reaction vessel is a macro- and microtiter plate, pin plate, multi-well plate, cell or tissue culture plate or well, test tube or flask, in particular, a reaction vessel according to the present invention.

DEFINITIONS

According to the present invention, the term "catecholamine" refers to naturally occurring catecholamines such as adrenalin, noradrenaline, dopamine, epinephrine and norepinephrine as well as synthetic catecholamines, i.e. catecholamines not found in nature, such as benserazide, carbidopa, dobutamine, dopexamine, isoprenaline and alpha-methyldopa, mixtures thereof and derivatives thereof. The general formula of naturally occurring catecholamines is as follows: wherein R1 is OH for adrenalin and noradrenalin, and H for dopamine, and R2 is H for noradrenalin and dopamine, and CH3 for adrenalin. Epinephrine and norepinephrine are the physiologically active R-enantiomeres (according to the Cahn-Ingold-Prelog system) of adrenaline and noradrenaline, respectively.

It is noted that synthetic catecholamines may comprise further substituents in addition to those indicated in the above formula.

Catecholamines determined by the method of the present invention may be contained in a sample derived from a natural source, i.e. a biological sample, in particular bodily fluids.

The term "biological sample" according to the present invention comprises any material which may be used for testing in the methods according to the present invention. The sample may be derived from any source and may include components of different sources. As used herein, a "biological sample" is any sample obtained from or present in a biological system such as humans and animals.

The biological samples can be obtained by methods known in the art such as venous puncture (blood, serum, plasma), directly collecting urine or collecting urine by use of a catheter. Preferably, the biological sample is a blood, serum, plasma, or urine sample, preferably obtained from a healthy animal or human or from an animal or human afflicted with a disease or other condition.

"Fraction of a biological sample" means for example a fraction obtained from a biological sample by purifying, fractionating, separating etc. the biological sample with methods such as chromatography, ultrafiltration, precipitation techniques, etc.

Terms such as "analyzing catecholamines" or "analyzing at least one catecholamine" relate to methods of measuring, collecting and analyzing data about catecholamines such as their relative or absolute concentration, their presence or absence, etc. and include, in particular, the relative or absolute quantification of catecholamines.

The term "analyzing at least one catecholamine" according to the invention refers to an analysis of all or only a fraction of the catecholamines contained in a sample, in particular 1, 2, up to 4, up to 6, up to 8 or up to 10 different catecholamines. This can be achieved, for example, by using antibodies being either specific for a particular catecholamine or derivative thereof or a particular group of catecholamines or derivatives thereof or all catecholamines or derivatives thereof and/or using a mixture of antibodies having different specificities.

Terms such as "analyzing catecholamines" or "analyzing at least one catecholamine" according to the invention also includes situations wherein no catecholamines are detected or the amount of said catecholamines is below the detection limit.

According to the invention, a "reference sample" may be used to correlate and compare the results obtained from a test sample. The composition of a "reference sample" is usually similar to a test sample but differs from the test sample in certain variables, e.g. the test sample may be derived from an individual suspected of being afflicted with a disease while the reference sample is obtained from a healthy individual and/or an individual known to be afflicted with said disease.

According to the present invention, the term "derivative" in connection with a particular compound or groups of compounds refers to a modified form of said compound(s). In one embodiment, a derivative of a compound retains the function or activity of the parent compound such as the ability to bind to cis-diol specific groups. In another embodiment the derivatization of a compound creates a property which was not present in a parent compound or modifies, preferably enhances a property of a parent compound such as the stability of catecholamines or capability of catecholamines to interact with antibodies. Preferably, a derivative of a compound comprises one or more substituents at one or more positions of the parent compound. Derivative of a compound also includes substances wherein said compound is only a minor constituent of the substance. For example, according to the present invention a RNA molecule may be considered as a derivative of a ribonucleotide. Particular examples of catecholamine derivatives or modified catecholamines are catecholamines modified by alkylation, preferably O-alkylation, more preferably O-methylation, and/or modified by acylation.

According to the present invention a cis-diol specific affinity medium may be used to extract catecholamines from a sample. As used herein, a "cis-diol specific affinity medium" comprises a support (matrix) which exhibits chemical structures (cis-diol specific groups) that specifically bind to substances comprising a cis-diol structure. A cis-diol structure comprises two hydroxyl groups, each bound to one of two adjacent carbon atoms, respectively.

The matrix of the cis-diol specific affinity medium may be a polymer, e.g. polyacrylamide, which preferably forms a gel-like structure, or the matrix may be the wall of a reaction vessel such as macro- and microtiter plates, pin plates, multi-well plates, cell and tissue culture plates and wells, test tubes and flasks.

A preferred cis-diol specific affinity medium is a boronate affinity medium. The boronate affinity medium comprises boronic acid groups of the formula -RB(OH)2 and/or of the formula -RB(OH)3 -, depending on the pH, wherein the boronic acid groups are coupled to the matrix of the affinity medium via R by covalent or non-covalent interactions. Preferably, R comprises an optionally substituted aryl or optionally substituted alkyl group, wherein the substituents may be any substituents as long as they do not obstruct the binding of catecholamines to the boronic acid groups. More preferably, the boronate affinity medium comprises amino boronic acid groups wherein R comprises an optionally substituted aryl or alkyl group substituted with a -NH- group which links the boronic acid group to the matrix. A preferred boronate affinity medium comprises m-aminophenyl boronic acid groups of the following formulas:

Another example of a cis-diol specific affinity medium is alumina.

According to the present invention, the term "binding" refers to the covalent or non-covalent attachment of one entity to another entity such as catecholamines to the cis-diol specific groups of a cis-diol specific affinity medium. The term "binding" includes specific and non-specific binding. Binding may be achieved, for example, by covalent bonds or ionic, polar or nonpolar interactions. Binding may require special conditions to be met, including pH value, ionic strength, temperature, solvents and additives. Preferably, the binding is strong enough to withstand one or more rinsing steps.

If the cis-diol specific groups are boronic acid groups, binding to a catecholamine is achieved by a covalent bond between the cis-diol structure of the catechol moiety of the catecholamine and the boronic acid group, as shown in the following formula:

The term "releasing of catecholamines bound to a cis-diol specific affinity medium" refers to disrupting the binding of the catecholamines to the cis-diol specific affinity medium. This may be achieved by changing one or more of the conditions required for binding of the catecholamines to the cis-diol specific affinity medium and/or by addition of a releasing agent. Most preferably, catecholamines are released from a cis-diol specific affinity medium by adjusting the pH to acidic conditions.

According to the present invention the term "blocking the binding of catecholamines to a cis-diol specific affinity medium" refers to the inhibition of the binding of catecholamines, not bound to said cis-diol specific affinity medium, to said cis-diol specific affinity medium. This may be achieved by the addition of a substance (blocking substance) which blocks the binding of catecholamines to the cis-diol specific affinity medium under conditions which otherwise would be appropriate for binding. The blocking substance may be a single compound or a composition, i.e. a mixture, of compounds, including one or more blocking compounds. Furthermore, the blocking substance may block the binding of one particular catecholamine, a particular group of catecholamines or all catecholamines. The mechanism underlying the blocking may be any suitable mechanism, such as e.g. competitive binding. Preferably, the blocking does not alter the chemical structure of the catecholamines. "Competitive binding" in the context of the present invention refers to binding of the blocking substance to the cis-diol specific affinity medium, thus inhibiting its interaction with catecholamines. Preferably, the blocking substance is added in excess amounts.

According to the present invention, the term "enzymatic reaction" refers to any reaction that is catalyzed by an enzyme. An enzyme is any protein, polypeptide or derivative thereof, such as those containing co-enzymes, capable of catalyzing a chemical reaction.

According to the present invention, the term "modification of catecholamines" relates to any modification of catecholamines as found in a sample and includes, in particular, modifications by acylation and/or O-alkylation.

As used herein, the term "O-alkylation" of a substance refers to a reaction, wherein an alkyl group is covalently bound to an oxygen atom of said substance. The alkyl group may be any suitable alkyl group, including optionally substituted, branched and unbranched, saturated and unsaturated alkyl groups, optionally comprising heteroatoms. Preferably, the alkyl group is a methyl group. "O-methylation" of a substance refers to a reaction, wherein a methyl group is covalently bound to an oxygen atom of said substance.

According to the present invention, the term "acylation" of a substance refers to a reaction, wherein an acyl group is covalently bound to said substance. An acyl group is any group of the formula -C(O)R, wherein R is an alkyl or aryl group, including optionally substituted, branched and unbranched, saturated and unsaturated alkyl or aryl groups, optionally comprising heteroatoms. Acyl groups for covalent binding to catecholamines may contain a label such as a radioisotope. An example of an acylation reagent suitable for the use in the present invention is Bolton-Hunter reagent:

As used herein, the term "immunoassay" refers to a test system for detection and/or quantification of a compound which includes binding said compound to an antibody specific thereto and detecting the compound-antibody complex. An immunoassay may provide information on the presence or absence of the compound and/or on the relative or absolute concentration or amount of the compound. The detection and/or quantification may be accomplished using a detectable label such as enzymes, fluorescent labels, luminescent labels and radioactive labels. Examples of suitable enzymes are peroxidases such as horseradish peroxidase, &bgr;-galactosidase and phosphatases such as alkaline phosphatase.

In one example of an immunoassay, a defined amount of a competitor for the compound of interest is added together with an antibody specific for the competitor/compound of interest. The competitor may be the same compound as the compound of interest, but may differ from the compound of interest in that it bears a detectable label such as described above, e.g. a radioisotope such as 125I, or in that it is immobilized on a support. The antibody complexes formed may then be bound by a secondary antibody immobilized on a support, if the competitor is detectably labeled, or by a secondary antibody coupled to a detectable label, e.g. an enzyme such as horseradish peroxidase, if the competitor is immobilized on a support. A suitable support includes precipitation agents, e.g. beads such as sepharose beads, and the walls of a reaction vessel.

DESCRIPTION OF A PREFERRED EMBODIMENT

In the following, a preferred embodiment of the method of the present invention is described, wherein catecholamines contained in a biological sample are analyzed.

In the first step, the sample is added into a reaction vessel comprising a boronate affinity medium attached to it. At neutral to basic conditions, e.g. a pH of about 8, the catecholamines contained in the sample bind to the boronic acid groups of the boronate affinity medium. The remainder of the sample is removed, e.g. by decanting or suction, and the reaction vessel rinsed.

In the second step, an acylation reagent is added, e.g. a solution containing Bolton-Hunter reagent, acylating the bound catecholamines. After completion of the acylation reaction the solution is removed and the reaction vessel rinsed.

In the third step, the bound catecholamines are released from the boronate affinity medium by adjusting the pH to acidic conditions, e.g. about 2. This is achieved by addition of an acid, e.g. hydrochloric acid. Simultaneously, a blocking substance, e.g. fructose or sorbitol, is added in a final concentration depending on the binding capacity of the boronate affinity gel and the affinity of the blocking substance to the boronate affinity gel.

In the fourth step, an enzyme solution for O-methylation of the released catecholamines is added, e.g. comprising S-adenosylmethionine and the enzyme COMT, which transfers a methyl group to the 3-hydroxyl group of the catechol moiety of the catecholamines. Simultaneously, the pH of the solution containing the catecholamines is adjusted to neutral to basic conditions, e.g. a pH of about 7.5. Rebinding of the catecholamines to the boronate affinity medium under these pH conditions is blocked by the blocking substance added in the previous step.

In the fifth step, the presence and/or concentration of the modified catecholamines contained in the reaction vessel is analyzed using an immunoassay, e.g. an enzyme or radio immunoassay, as described above.

As obvious from the disclosure of the present invention, a method is provided that allows to perform the extraction and modification of catecholamines all in one reaction vessel, resulting in the final catecholamine derivative useful for enzyme or radio immunoassays. Thus, the catecholamines prepared according to the method of the invention are suitable for analysis via an EIA using a standard automated analyzer. Thereby, multiple EIAs analyzing different catecholamines may be performed using the modified catecholamines prepared by only one extraction procedure.

Alternatively, by using the appropriate reaction vessel as described herein it is possible to perform the EIA in the same reaction vessel which was used for the extraction and modification of the catecholamines.

A suitable reaction vessel according to the invention comprises a cis-diol specific affinity medium and a catecholamine or an antibody specific for a catecholamine specific antibody immobilized to the reaction vessel. One example of such a reaction vessel is a pin plate which consists of a plate with one or more wells and a top cover comprising one or more pins which project into the recesses of the wells. For the use of a pin plate in the method of the present invention, a cis-diol specific affinity medium may be attached to the bottom and/or wall of one or more wells of the pin plate and catecholamines or antibodies specific for a catecholamine specific antibody may be attached to one or more pins of the pin plate. Pin plates are know in the art and a pin plate according to the invention may be produced by a conventional process.

In the following, an example of performing the method of the present invention according to the preferred embodiments is described.

EXAMPLE 1 Extraction of epinephrine from urine and its quantitative determination by EIA in one reaction vessel

10 µl urine and 25 µl assay buffer (1 M sodium hydrogencarbonate) are added into a reaction vessel coated with (i) a boronate affinity medium comprising m-aminophenyl boronic acid groups and (ii) 3-O-methyl-norepinephrine attached to the reaction vessel via an acyl group bound to the nitrogen atom. After 30 min incubation at room temperature (RT) on a shaker, the reaction vessel is washed 3 times with 300 µl washing buffer (10 mM Tris/HCl pH 7.4, 150 mM sodium chloride, 0.1% (w/v) Tween 20). 100 µl assay buffer and 25 µl acylation reagent (0.8 M succinic acid anhydride in dimethyl sulfoxide/dimethylformamide, v/v, 1/1) are added and the reaction is incubated for 15 min at RT on a shaker. After removal of the fluid the reaction vessel is again washed 3 times with 300 µl washing buffer. 150 µl release buffer (25 mM hydrochloric acid, 20 g/l sorbitol, 4 mg/l phenol red) are added and after 5 min incubation at RT on a shaker 50 µl enzyme solution (COMT, 0,125 g/l S-adenosyl-L-methionine, 350 mM Tris/HCl pH 9.1, 5 mM MgCl2, 0,125 g/l dithiotreitol) are added. The reaction is incubated for 15 min at RT on a shaker and 50 µl antiserum from rabbit immunized against N-acyl metanephrine (N-acylated and 3-O-methylated epinephrine) are added. After incubation for 60 min at RT on a shaker the fluid is removed and the reaction vessel is washed 3 times with 300 µl washing buffer. 100 µl enzyme conjugate (goat anti-rabbit IgG monoclonal antibody conjugated to a peroxidase) are added and incubated for 30 min at RT on a shaker. After removal of the fluid the reaction vessel is washed 3 times with 300 µl washing buffer and 100 µl substrate (3,3',5,5'-tetramethylbenzidine (Blue Star TMB)) are added. The reaction is incubated for 30 min at RT on a shaker and thereafter stopped by adding of 100 µl stop solution (0.25 M H2SO4). The product of the enzymatic reaction is detected at 450 nm. The concentration of epinephrine in the sample can be determined by comparing the detected OD with an established calibration curve, whereby the OD increases with decreasing epinephrine concentration in the sample.

EXAMPLE 2

Extraction of epinephrine from urine and its quantitative determination by EIA using a standard automated EIA analyzer

The extraction and derivatization of the catecholamines is performed as in example 1, except that a reaction vessel comprising only the boronate affinity gel is used. After the incubation of the enzymatic reaction using COMT, the solution containing the modified catecholamine is transferred into a reaction vessel suitable for a standard automated EIA analyzer and the EIA is performed in an automated manner using such an analyzer.

EXAMPLE 3 Extraction of epinephrine from urine and its quantitative determination by RIA

15µl urine and 25 µl assay buffer (1 M sodium hydrogencarbonate) are added into a reaction vessel coated with a boronate affinity medium comprising m-aminophenyl boronic acid groups. After 30 min incubation at room temperature (RT) on a shaker, the reaction vessel is washed 3 times with 300 µl washing buffer (150 mM sodium chloride). 100 µl assay buffer and 25 µl acylation reagent (0.8 M succinic acid anhydride in dimethyl sulfoxide/ dimethylformamide, v/v, 1/1) are added and the reaction is incubated for 15 min at RT on a shaker. After removal of the fluid the reaction vessel is again washed 3 times with 300 µl washing buffer. 150 µl release buffer (25 mM hydrochloric acid, 20 g/l sorbitol, 4 mg/l phenol red) are added and after 5 min incubation at RT on a shaker 50 µl enzyme solution (COMT, 0,125 g/l S-adenosyl-L-methionine, 350 mM Tris/HCl pH 9.1, 5 mM MgC12, 0,125 g/l dithiotreitol) are added. The reaction is incubated for 30 min at RT on a shaker and 50 µl antiserum form rabbit immunized against N-acyl metanephrine (N-acylated and 3-O-methylated epinephrine) and radioactively labeled N-acyl metanephrine (<100 kBq) are added. After further incubation for 1 h, 1 ml precipitating reagent (goat anti rabbit antibodies in 5% PEG 6000 solution) is added and incubated for 15 min at 2 - 8 °C. The reaction solution is then centrifuged for 15 min. at 3000 x g and the supernatant is carefully decanted or aspirated (except totals). The radioactivity retained in the reaction vessel is then counted for 1 min in a gamma counter.


Anspruch[en]
A method for analyzing at least one catecholamine comprising the steps of: (a) binding the at least one catecholamine to a cis-diol specific affinity medium; and (b) adding a substance that blocks the binding of catecholamines to the cis-diol specific affinity medium. The method of claim 1 wherein the at least one catecholamine comprises a naturally occurring and/or a synthetic catecholamine. The method of claim 2 wherein the naturally occurring catecholamine is selected from the group consisting of adrenalin, noradrenaline, dopamine, epinephrine, norepinephrine and mixtures thereof. The method of any one of claims 1 to 3 wherein the at least one catecholamine is contained in a biological sample or a fraction thereof. The method of claim 4 wherein the biological sample is a urine sample or a plasma sample. The method of any one of claims 1 to 5 wherein the cis-diol specific affinity medium is a boronate affinity medium. The method of claim 6 wherein the boronate affinity medium comprises amino boronic acid groups. The method of claim 6 or 7 wherein the boronate affinity medium comprises m-aminophenyl boronic acid groups. The method of any one of claims 1 to 8 wherein step (a) takes place at a neutral to basic pH. The method of any one of claims 1 to 9 wherein the substance added in step (b) contains at least one cis-diol structure. The method of claim 10 wherein the substance is selected from the group consisting of 1,2-dihydroxyalkanes, polyphenols, 2-hydroxy acids, ascorbic acid, carbohydrates, derivatives thereof and mixtures thereof. The method of claim 11 wherein the 1,2-dihydroxyalkane is ethanediol, 1,2-propanediol, or an alkanediol comprising vicinal hydroxyl groups. The method of claim 11 wherein the polyphenol is a catechol, anthocyane, gallic acid, or tannin. The method of claim 11 wherein the 2-hydroxyacid is lactic acid, tartaric acid, or citric acid. The method of claim 11 wherein the carbohydrate is a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide. The method of claim 11 wherein the carbohydrate derivative is selected from the group consisting of nucleosides, nucleotides, oligo- and polynucleotides, and glycoproteins. The method of claim 16 wherein the oligo- or polynucleotide is RNA. The method of claim 11 wherein the substance is sorbitol or fructose. The method of any one of claims 1 to 18 further comprising the step of releasing the at least one catecholamine bound to the cis-diol specific affinity medium from the cis-diol specific affinity medium. The method of claim 19 wherein step (b) and the step of releasing the at least one catecholamine from the cis-diol specific affinity medium are performed simultaneously, or step (b) is performed prior to or following the step of releasing the at least one catecholamine from the cis-diol specific affinity medium. The method of claim 19 or 20 wherein the at least one catecholamine is released from the cis-diol specific affinity medium by addition of an acid. The method of claim 21 wherein the acid is selected from the group consisting of hydrochloric acid, perchloric acid, and mixtures thereof. The method of any one of claims 19 to 22 wherein in the step of releasing the at least one catecholamine from the cis-diol specific affinity medium the pH is 3 or lower. The method of any one of claims 19 to 23 wherein the concentration of the substance added in step (b) is sufficient to block the binding of at least 95%, preferably at least 99%, of the at least one catecholamine released from the cis-diol specific affinity medium to the cis-diol specific affinity medium. The method of any one of claims 1 to 24 wherein the substance added in step (b) is added to a final concentration that equals or exceeds the binding capacity of the used cis-diol specific affinity medium. The method of any one of claims 19 to 25 wherein following step (b) and the step of releasing the at least one catecholamine from the cis-diol specific affinity medium the at least one catecholamine is to be subjected to at least one reaction which requires non-acidic conditions. The method of claim 26 wherein the at least one reaction is an enzymatic reaction. The method of claim 26 or 27 wherein the at least one reaction is an O-alkylation, preferably an O-methylation. The method of claim 28 wherein the O-methylation is effected by means of the enzyme catechol-3-O-methyl-transferase. The method of any one of claims 26 to 29 wherein the non-acidic conditions include a pH of 6 or higher. The method of any one of claims 1 to 30 further comprising the step of acylating the at least one catecholamine. The method of claim 31 wherein the at least one catecholamine is acylated while bound to the cis-diol specific affinity medium. The method of any one of claims 1 to 32 further comprising the step of detecting and/or quantifying the at least one catecholamine, preferably using an antibody specific for a catecholamine or modified catecholamine. The method of claim 33 wherein the detection and/or quantification comprises an enzyme or radio immunoassay. The method of any one of claims 26 to 34 wherein step (a), step (b), the step of releasing the at least one catecholamine from the cis-diol specific affinity medium and the at least one reaction which requires non-acidic conditions are all done within the same reaction vessel. The method of claim 35 wherein the step of detection and/or quantification of the at least one catecholamine is also done within the same reaction vessel. A reaction vessel comprising a cis-diol specific affinity medium and an immobilized catecholamine or immobilized antibody specific for a catecholamine specific antibody. The reaction vessel of claim 37 wherein the cis-diol specific affinity medium is a boronate affinity medium. The reaction vessel of claim 37 or 38 which is a member selected from the group consisting of macro- or microtiter plates, pin plates, multi-well plates, cell or tissue culture plates or wells, test tubes or flasks. A kit adapted for detection and/or quantification of at least one catecholamine comprising: (a) a cis-diol specific affinity medium; and (b) a substance that blocks the binding of catecholamines to the cis-diol specific affinity medium. The kit of claim 40 which further comprises at least one member selected from the group consisting of a reagent for releasing catecholamines bound to the cis-diol specific affinity medium, a reaction vessel, an extraction buffer, an acylation reagent, an acylation buffer, an enzyme solution, reagents for detection of catecholamines and a washing buffer. The kit of claim 40 or 41 wherein the substance that blocks the binding of catecholamines to the cis-diol specific affinity medium and the reagent for releasing catecholamines bound to the cis-diol specific affinity medium are contained in one composition. The kit of claim 41 wherein the reaction vessel is a reaction vessel according to any one of claims 37 to 39.






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