GB1570818A - Assay for catecholamines - Google Patents

Description

(54) ASSAY FOR CATECHOLAMINES (71) We, F. HOFFMANN-LA ROCHE & CO., AKTIENGESELL SCHAFT, a Swiss Company of 124-184 Grenzacherstrasse, Basle, Switzerland, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention relates to an assay for catecholamines. More particularly, the invention is concerned with an immunoassay for the determination of epinephrine- and norepinephrine-type catecholamines and with antigens and antibodies useful for carrying out such an immunoassay.

The immunoassay provided by the present invention is directed against the side-chain common to epinephrine-type or norepinephrine-type catecholamines and involves an oxidation procedure which selectively converts one or more of these compounds to another compound which is not bound by the antibody. By measuring the difference between the total epinephrine-type or norepinephrinetype catecholamine content and that after each of the oxidation steps, it is possible to determine the quantity of individual catecholamines of either type present in the original sample.

The use of a radioimmunoassay for the detection of catecholamines has been described in United States Patent No; 3 704 282. While this is an extremely sensitive assay procedure, the antibody used therein cannot readily distinguish between closely related catecholamines and thus there is obtained only an indication of total catecholaniine content in the sample. Thus it would not be possible using such procedure to determine the specific presence of the individual concentrations of epinephrine-type catecholamines such as epinephrine, metanephrine, synephrine or phenylephrine or norepinephrine-type catecholamines such as norepinephrine, normetanephrine, octopamine or norphenephrine in a sample. An improved fluorometric assay of catecholamines has been described by Laverty and Taylor, Analytical Biochemistry 22, 269 (1968).In such an assay the catecholamines in a sample are oxidised with iodine to produce a fluorescent indole derivative which is then detected in a fluorometer. To a limited extent it is possible to measure an individual catecholamine in the presence of other related compounds. This requires the use of different oxidation pH's, different treatments of the final solution and different wavelength maxima. However, cross-interference can still arise even with the use of such conditions and thus only a very few catecholamines can actually be detected in admixture without the use of chemical separation prior to detection.

The present invention relates to an improved assay method which allows for the individual quantitative detection of epinephrine-type catecholamines (i.e.

epinephrine, metaneprine, synephrine and phenylephrine) or norepinephrinetype catecholamines (i.e. norepinephrine, normetanephnne, octopamine- and norphenephrine) when present in admixture in a sample. In particular, the immunoassay provided by the present invention involves oxidation procedure whereby, under different conditions of pH and temperature, one or more selected epinephrine-type or norepinephrine-type catecholamines present in order of increasing lability to oxidation is converted into a form which is not immunoreactive with an antibody specific to that catecholamine type. The initial sample and each of the products from the oxidation procedure are assayed after treatment with the sulphite reducing agent by immunoassay with an antibody specific for the epinephrine or norepinephrine side-chain.Since the antibody used in the present assay does not have an affinity for the products produced in the selective oxidation step, the concentration of the specific epinephrine-type or norepinephrine-type catecholamine being converted can be determined by substracting the catechol amine content found in a sample oxidised under one condition from the catechol amine content determined in a duplicate sample without oxidation or in a duplicate sample oxidised under a less rigorous condition.

More particularly, the present invention is concerned with a method for the assay for individual epinephrine-type catecholamines selected from epinephrine, metanephrine, synephrine and phenylephrine or for individual norepinephrine type catecholamines selected from norepinephrine, normetanephrine, octopamine and norphenephrine in a test sample, which method comprises in combination the steps:: (A) determining the total epinephrine-type or norepinephrine-type catechol amine concentration by carrying out an immunoassay on said test sample using an antibody having specificity for epinephrine-type or norepinephrine-type catechol amines; (B) treating an aliquot of said test sample with iodine at a pH of 7.4 so as to selectively convert any epinephrine or norepinephrine present in said test sample into a form which is not bound by said antibody, treating said aliquot with excess aqueous sulphite solution so as to reduce any remaining iodine, adjusting the pH of the resulting solution to that required for said immunoassay and then determining the epinephrine-type or norepinephrine-type catecholamine concentration by carrying out said immunoassay on said resulting solution representing the sum total of metanephrine, synephrine and phenylphrine present in said test sample for epinephrine-type catecholamines or normetanephrine, octopamine and norphene phrine for norepinephrine-type catecholamines; (C) treating a second aliquot of said test sample with iodine at a pH of 8.6 so as to selectively convert any epinephrine and metanephrine or norepinephrine and normetanephrine present in said test sample into forms which are not bound by said antibody, treating said aliquot with excess aqueous sulphite solution, adjusting the pH to a level compatible for said immunoassay and then determining the concentration of epinephrine-type or norepinephrine-type catecholamines by carrying out said immunoassay on said test solution, said concentration representing the sum total of synephrine and phenylephrine or octopamine and norphenephrine present in said.test sample; and (D) treating a third aliquot of said test sample with iodine at a pH of 9.2 so as to selectively convert any epinephrine, metanephrine and synephrine or norepine phrine, normetanephrine and octopamine present in said test sample into forms which are not bound by said antibody, treating said aliquot with excess aqueous sulphite solution, adjusting the pH to a level compatible for said immunoassay and then determining the concentration of epinephrine-type or norepinephrine-type catecholamine by carrying out said immunoassay on said test sample, said concen tration representing the phenylephrine or norphenephrine present in said test sample; wherein the concentration of epinephrine or norepinephrine is determined by subtracting the concentration found in step (B) from that of step (A); the concen tration of metanephrine or normetanephrine is determined by subtracting the concentration found in step (C) from that of step (B); and the concentration of synephrine or octopamine is determined by subtracting the concentration found in step (D) from that of step (C).

The sample may be first treated so as to eliminate non-catecholamine containing substances such as protein and other compounds which may interfere in the oxidation reaction.

The antibody used in the aforementioned immunoassay can be obtained in a manner known per se. Thus, for example, haptens of the general formula

wherein R, represents a hydrogen atom or a hydroxy group, R2 represents a hydrogen atom or a lower alkyl group, R'2 represents a hydrogen atom or a conventional amine-protecting group, and R3 represents a hydrogen atom or a hydroxy or lower alkoxy group and n stands for 1, 2 or 3, are used in the preparation of an antigen.

Preferred haptens used in the present invention are derived from synephrine for the epinephrine-type catecholamines and from octopamine for the norepinephrine-type catecholamines.

The tert.butoxycarbonyl group is used as a protecting group in haptens of formula I to prevent self-condensation reactions during further transformations in the preparation of the required antigens. The tert.butoxycarbonyl group can be readily cleaved to yield an antigen in which R'2 represents a hydrogen atom.

In order to prepare the antigens provided by the present invention, it is necessary to covalently bond a hapten of formula I through the carboxyl group to a conventional immunogenic carrier material. As used herein, the term "immunogenic carrier material" includes those materials which have the property of independently eliciting an immunogenic response in a host animal and which can be covalently coupled to the foregoing haptens. Suitable carrier materials include for example, proteins, natural or synthetic polymeric compounds such as polypeptides (e.g. polylysine or copolymers of amino acids), polysaccharides and the like.

Particularly preferred carrier materials are proteins and polypeptides, especially proteins.

The identity of the protein material used in the present invention is not critical.

Examples of suitable proteins which can be used include mammalian serum proteins such as, for example, human gamma globulin, human serum albumin, bovine serum albumin, methylated bovine serum albumin, rabbit serum albumin and bovine gamma globulin. Other suitable proteins will be readily apparent. It is generally preferred, but not necessary, to use proteins which are foreign to the animal hosts in which the resulting antigen will be used.

The covalent coupling of the hapten of formula I to the immunogenic carrier material can be carried out in a well-known manner for establishing amide bonds.

However, in order to insure an adequate degree of coupling under the mildest possible conditions so as to minimise any possible deleterious effect on the carrier material, it may be desirable to convert the hapten into an isolatable activated form prior to the coupling. One particularly preferred isolatable activated form is a Nhydrocysuccinimide ester of the general formula

wherein R, R2, R'2, R3 and n have the significance given earlier.

Other suitable isolatable activated derivatives include thep-nitrophenyl esters, acylimidazoles and the like. Other coupling methods in which the activated intermediates need not be isolated can be used. Such methods include the mixed anhydride method, the use of EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydro- quinoline) as the coupling agent and the like.

The coupling of the hapten of formula I or, more preferably, as an activated derivative thereof (e.g. a N-hydroxysuccinimide ester of formula II) to the immunogenic carrier material can be readily carried out according to well-known techniques for establishing amide bonds. Thus, for example, one such technique involves dissolving the carrier material and coupling agent in a suitable inert solvent followed by addition of the desired hapten of formula I. The reaction may be carried out at a temperature in the range of from about 0 C to about 50"C, although it may be carried out at higher or lower temperatures depending on the nature of the reactants. A most preferred temperature is about room temperature.

The coupling agent which may be used in the aforementioned reaction will be selected from those commonly used in organic chemistry for initiating amide bond formation. A particularly suitable class of coupling agents comprises the carbodiimides, most preferably dicyclohexylcarbodiimide or l-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide. The molar ratio of the hapten to the carrier material will, of course, depend on the identity of the hapten and the protein selected for the reaction.

The coupling reaction can be carried out under conventional conditions. Thus, when a carbodiimide is used as the coupling agents, it is desirable to carry out the reaction in a slightly acidic medium (e.g. a medium having a pH in the range of from about 3 to 6.5, most preferably in the range of from about 4 to 6.5). Upon completion of the reaction; the excess hapten may be removed by dialysis.

As mentioned earlier, one preferred technique for preparing the antigens of the present invention is to first prepare and isolate an activated derivative (e.g. a N hydroxysuccinimide ester of formula II) and then to react this ester with the carrier material to form the blocked antigen. Such activated derivatives are conveniently prepared by reacting a hapten of formula I with a desired activating compound such as N-hydroxy-succinimide and a coupling agent such as dicyclohexylcarbo diimide in an inert solvent. The reaction is usually allowed to proceed for 16-60 hours at a reduced temperature (OO--SOC). The activated derivative may then be isolated by filtering off the by-product, dicyclohexylurea, and distilling off the solvent.

The coupling to the carrier material may then be carried out by contacting the activated derivative with the chosen carrier material. When the activated derivative is the N-hydroxysuccinimide ester and the carrier material is bovine serum albumin, this may be carried out by adding the activated derivative in a water-miscible solvent to an aqueous solution of the carrier material containing a base such as sodium bicarbonate.

Another coupling method comprises activating the carboxyl group of the hapten and adding the activated hapten to the carrier protein without isolation of the activated derivative. An example of such a method is the mixed anhydride method involving the reaction of the hapten with isobutylchloroformate. The hapten is dissolved in an anhydrous, water-miscible organic solvent, usually dioxane, and - the solution is neutralised with an eqliimolar quantity of triethylamine. After stirring at room temperature, the temperature of the mixture is reduced to between 0 C and 8"C. an equimolar quantity plus a 10% excess of isobutyichloroformate is then added and stirring is continued.Meanwhile, the carrier protein (e.g. bovine serum albumin) is dissolved in water and the pH is adjusted to 9.0 with sodium hydroxide. The quantity of carrier used is equivalent to the molar quantity of hapten divided by the theoretical number of reactive groups on the carrier. Organic solvent is added to the carrier solution and the resulting soluton is cooled to between 0 C and 8"C. The solution is then added to the activated hapten and coupling is allowed to proceed for 30 minutes to overnight.

The final ratio of organic solvent to water is 1:1. The mixture is then adjusted to neutrality, the aqueous-organic solvent is removed and aqueous solution is carried out. After dialysis and lyophilisation, the amine-protecting group is removed.

After the coupling of a hapten of formula I or a N-hydroxysuccinimide ester of formula II to the carrier material, it is necessary to remove an amine-protecting group denoted by R'2 in order ta restore the free primary or secondary amino function. Where the amine-protecting group is the tert.butoxycarbonyl group, this may be conveniently removed by treatment with trifluoroacetic acid in dichloro methane at room temperature. The relative amounts of trifluoroacetic acid and dichloromethane and the time required to complete the treatment may be varied to suit particular cases. In general, from 1 to 3 volumes of dichloroniethane per volume of trifluoroacetic acid and treatment times of 30 to 60 minutes have heen found to give good results.

The aforementioned antigens may be used to induce formation of antibodies specific to epinephrine-type or norepinephrine-type catecholamines in host ,animals by injecting the antigen into such a host, preferably using an adjuvant.

Improved titres can be obtained by repeated injections over a period of time.

Suitable host animals include mammals such as rabbits, horses, goats, guinea pigs, rats, cows, sheep etc. The resulting antisera will contain antibodies which will selectively complex with the epinephrine-type or norepinephrine-type catechol amines depending on the hapten used to prepare the antigen used in the inoculation of the host.

The immunoassay used to detect the epinephrine-type or norepinephrine-type catecholamines in each step of the present method may be selected from wellknown immunoassays. A particularly preferred immunoassay for this purpose is a radioimmunoassay such as is described in United States Patent No. 3 704282 or 3 709 665 using the epinephrine-type or norepinephrine-type catecholamine specific antibody discussed earlier. Alternatively, it is also possible to use other immunoassays such as, for example, an enzyme amplification assay as is described in United States Patent No. 3 817837 or a free radical assay using spin labelled compounds as is described in United States Patent No. 3 690 834.

The specificity of an epinephrine-type catecholamine antibody suitable for use in the present method is set forth in Table 1: ANTIBODY SPECIFICITY

R, 1 R3 C- C -No 50% INHIBITION R1 H OR COMPOUND nH R5 H Ab-I125 dl-SHET* R2 COMPLEX, ng . R1 R1 R1 R2 R3 R4 R5 R6 l-Epinephrine H OH OH OH H CH3 / 0.250 dl-Xetanephrine H OH OCH3 OH H CH3 0.050 dl-Synephrine H OH H OH H CH3 i 0.100 Epinine H OH OH H H CH3 0.350 l-Phenylephrine OH H H OH H CH3 0.050 (Neosynephrine) l-Norepinephrine H OH OH OH H H > 500 Dopamine H OH OH H H H > 500 Amphetamine H H H H CH3 H > 500 3 Xethamphetamine H H H H CH3 CH3 > 500 Ephedrine H H H OH CH3 CH3 > 500 Isoproterenol H OH OH OH H C(CH3)2H > 500 DOPA H OH OH H COOH H > 500 OH |VMA CH3O C- C- OH acid) N' oll HO S CH2CH2NH2 Serotonin -CH2-CH2NH2 H *Ab-I125 dl-MET = antibody complex with I125 labelled dl-metanephrine A suitable procedure for carrying out the method provided by the present invention can be summarised as follows:: (A) test a sample aliquot with immunoassay to determine the total epine phrine-type catecholamine content (epinephrine + metanephrine + synephrine + phenylphrine); (B) adjust another sample aliquot to pH 7.4 with phosphate buffer and treat with 0.1-N iodine (in aqueous solution, complexed with sodium iodide) for 5 minutes at 40C so as to selectively convert epinephrine into the adrenochrome form; treat the resulting mixture with excess aqueous sodium sulphite (as a 10% solution) to reduce the unreacted iodine; if necessary, adjust the pH to a range required for immunoassay with buffer and run the immunoassay on the mixture to determine the remaining epinephrine-type catecholamine content (metanephrine + synephrine + phenylephrine); (C) adjust another sample akiquot to pH 8.6 with phosphate buffer and treat with iodine as described earlier for 5 minutes at 40C to selectively convert both epinephrine and metanephrine into the adrenochrone form, treat the resulting mixture with excess aqueous sulphite solution as described earlier, adjust the pH to the desired range for immunoassay (pH 7.4 with the antibody described earlier) and then carry out the immunoassay on the mixture to determine the remaining epinephrine-type catecholamine concentration (synephrine + phenylephrine);; (D) adjust an additonal sample aliquot to pH 9.2 with carbonate/bicarbonate buffer and treat with iodine as described earlier for 5 minutes at room temperature to selectively convert epinephrine, metanephrine and synephrine into the cyclised form, treat the resulting mixture with excess aqueous sulphite solution as described earlier, adjust the pH to the desired range for immunoassay and then carry out an immunoassay on the mixture to determine the remaining epinephrine-type catecholamine concentration (phenylephrine).

The phenylephrine content of the sample is thus determined directly by the immunoassay results in step D. The epinephrine content is determined by subtracting the epinephrine-type catecholamine content found in step B from the total epinephrine-type catecholamine content determined by direct immunoassay of the sample in step A. Similarly, the metanephrine content is determined by subtracting the concentration level found in step C from that of step B. Finally, the synephrine content is determined by subtracting the result of step D from that of step C.

The immunoassay described earlier is also useful for assaying for individual norepinephrine-type catecholamines when an antibody having specificity to the norepinephrine-type side-chain is used. Thus, in step A a sample aliquot is tested to determine the total norepinephrine-type catecholamine content (norepinephrine + normetanephrine + octopamine + norphenephrine). Step B is run under the same conditions and in the same manner and provides the remaining norepinephrine-type catecholamine content (normetanephrine + octopamine + norphenephrine). Similarly, step C is run in the same manner to provide the octopamine +' norphenephrine concentration. Finally, step D is carried out in an identical manner to give the norphenephrine concentration.Finally, the individual norepinephrine-type catecholamine concentrations are calculated in the manner set forth earlier for the respective cases in the epinephrine-type catecholamine series.

Thus, the norphenephrine content is determined from step D, the norepinephrine content is determined by subtracting the content of step B from that of step A, the normetanephrine content is determined by subtracting the level of step C from that of step B and, finally, the octopamine content is determined by subtracting the result of step D from that of step C.

The individual epinephrine-type or norepinephrine-type catecholamine contents of various test samples can be determined by the method of the present invention. Examples of such test samples include biological fluids such as urine, blood, tissue extracts and the like. In some instances, pre-treatment of the test sample may be required to meet the specific needs of the immunoassay used. Thus, for example, deproteinisation in a manner known per se may be required before the assay is initiated.

As used herein, the term "lower alkyl" denotes straight-chain or branchedchain hydrocarbon groups containing 1 to 7, preferably 1 to 4, carbon atoms such as methyl, ethyl, n-propyl, n-butyl etc. The term "lower alkoxy" denotes lower alkoxy groups in which the lower alkyl portion is as defined earlier. A most preferred lower alkyl group is the methyl group and a most preferred lower alkoxy group is the methoxy group.

The following Examples illustrate the present invention: Example 1.

41.8 g (0.28 moles) of racemic synephrine and 53.6 g (0.375 moles) of tert.butoxy-.

carbonyl azide were stirred together in l litre of 50% aqueous dioxane in the presence of 4 g of magnesium oxide for 22 hours at 400--450C. The dioxane was distilled from the pale amber solution and to the residue were added 500 ml of water. On chilling, 58.7 g of solid separated; melting point l4l.50-1430C (after drying at 100 C for 3 hours). Recrystallisation of a sample of this solid gave white crystals of the tert.butyl ester of racemic N-(4,alpha-dihydroxyphenethyl)-N- methylcarbamic acid of melting point 141"--141.5"C.

Example 2.

26.7 g (0.1 mol) of the tert.butyl ester of raemic N-(4,alpha-dihydroxy phenethyl)-N-methylcarbamic acid were dissolved in 250 ml of hexamethy! phosphoric acid triamide and to the stirred solution under nitrogen were added 2.4 g (0.1 mol) of sodium hydride in small portions. The mixture was stirred until hydrogen evolution ceased (approximately 3 hours). To this stirred solution were added in a single portion 17.0 g (0.1 mol) of ethyl bromoacetate in 25 ml of benzene.

The external temperature rose from 7"C to 190C. The mixture was approximately neutral in 10 minutes. 500 ml of water and ice were added to the mixture, the resulting slightly turbid mixture was extracted five times with 150 ml portions of ether, the combined ether extracts were washed with a few small portions of water and dried over magnesium sulphate. After removal of the drying agent, the solvent was distilled in a rotary evaporator. The residue, 39 g of a viscous syrup, the ethyl ester of racemic 4-[2-(N-tert. butoxycarbonyl-N-methylamino)- I -hydroxvethvl] - phenoxyacetic acid resisted efforts to crystallise and was used as such in the hydrolysis to the free acid described hereinafter in Example 3.

Example 3.

The ethyl ester prepared as described in Example 2 was hydrolysed by suspending it in water at 800--850C and adding 10% sodium hydroxide solution until a permanent pH of 9-10 was obtained. After acidification with 20% citric acid solution to pH 3, the mixture was extracted with several portions of chloroform. The combined chloroform extracts were dried, the drying agent removed by filtration and the solvent distilled in a rotary evaporator. There was obtained a syrup of racemic 4-[2-(N-tert. butoxycarbonyl-N-methylamino)- 1 -hydroxyethyl] - phenoxyacetic acid which would not crystallise.It was therefore converted into the crystalline S-benzylthiuronium salt in the following manner: A weighed sample of the syrupy acid was treated with sufficient sodium hydroxide to form the sodium salt (pH 7.5-8.0). To this solution was added a concentrated solution of S-benzylthiuronium chloride, whereupon a precipitate formed. The solid was recovered, washed with cold water and then recrystallised from water to give white crystals of melting point l630-l650C. Analysis indicated that the crystals were the S-benzylthiuronium salt of the aforementioned acid.

Example 4.

3.25 g (0.01 mol) of the syrupy acid prepared as described in Example 3 were dissolved in 40ml of dimethoxyethane together with 1.15 g (0.01 mol) of N hydroxysuccinimide and to the resulting solution were added 2.06 g (0.01 mol) of dicyclohexyl carbodimide, whereupon the solution warmed perceptibly. The mixture was then stored at 50C for 21 hours. The dicyclohexylurea which had separated was removed by filtration, the filter cake washed with a small amount of dimethoxyethane and the combined filtrates were distilled in a rotary evaporator.

There remained 3.70 g of a turbid syrup which was dissolved in 75 ml of toluene and the undissolved dicyclohexylurea which had separated was removed by filtration.

Distillation of the solvent left a syrup which was dissolved in 50 ml of 2-propanol.

Petroleum ether (boiling range 60"--900C) was added up to turbidity and the solution stored at 5"C for 5 days. The crystals which separated were recovered, there being obtained 2.5 g of the N-hydroxysuccinimide ester of racemic 4-[2-(Ntert. butoxycarbonyl-N-methylamino)- 1 -hydroxyethyl]-phenoxyacetic acid of melting point l060-l08.50C.

Example 5.

47.5 g (0.25 mol) of racemic octopamine hydrochloride and 53.6 g (0.375 mol) of tert.butoxycarbonyl azide were stirred together in a mixture of 1 litre of 50% aqueous dioxane and 12 g (0.30 mol) of magnesium oxide at 37 H5 C for 21 hours under nitrogen. The dioxane was distilled from the solution in a rotary evaporator.

After adjusting the pH to 6 with acetic acid, the solution was extracted five times with 200 ml portions of chloroform. The combined chloroform extracts were washed with a small amount of water, the solution dried and the solvent distilled to skive 59.0 g of a crystalline solid of melting point 146 148 C. After recrystal libation from a mixture of ethyl acetate and petroleum ether (boiling range 60" 40"C), the product, namely the tert.butyl ester of racemic N-(4,alphadihydroxyphenethyl)-carbamic acid melted l470-l480C.

Example 6.

25.3 g (0.1 mol) of the tert.butyl ester of racemic N-(4,alpha-dihydroxy- phenethyl)-carbamic acid were dissolved in 350 ml of hexamethylphosphoric acid triamide and to the stirred solution under nitrogen were added 2.4g (0.1 mol) of sodium hydride in small portions. The mixture was stirred until hydrogen evolution ceased. To this stirred solution were then added in a single portion l7.0g (0.1 mol) of ethyl bromoacetate in 25 ml of benzene.The external temperature rose from 7"C to 190C. The mixture was approximately neutral in 10 minutes. 500 ml of water and ice were added to the mixture, the resulting slightly turbid mixture was extracted five times with 150 ml portions of ether, the combined ether extracts were washed with a few small portions of water and dried over magnesium sulphate. After removal of the drying agent, the solvent was distilled in a rotary evaporator. The residue, 31.7g of an amber syrup, was dissolved in hot carbon tetrachloride and, on cooling, crystals of the ethyl ester of racemic 4-[2-(N tert. butoxycarbonylamido)- 1 -hydroxyethyl]-phenoxyacetic acid were deposited.

These crystals melted at 560--590C. After drying in a high vacuum at 450C, the crystals were transformed into a colourless glass.

Example 7.

The ethyl ester prepared as described in Example 6 was hydrolysed by suspending it in water at 800--850C and adding 10% sodium hydroxide solution until a permanent pH of 9-10 was obtained. After acidification with 20% citric acid solution to pH 3, the solution was extracted twice with 100 ml portions of chloroform. The combined chloroform extracts were dried, the drying agent removed by filtration and the solvent distilled in a rotary evaporator. A pale amber syrup resulted. The syrup was dissolved in hot petroleum ether (boiling range 60"--90"C) and on cooling, crystals separated. These off-colour crystals weighing 22.55 g, were dissolved in 100 ml of acetonitrile, treated with charcoal and filtered.

From the cooled filtrate there were obtained 8.9 g of a solid of melting point 980-l0l0C. After a further recrystallisation from acetonitrile using decolorising carbon, there were obtained 6.65 g of slightly greyish crystals of racemic 4-(2 tert.butoxycarbonamido- 1 -hydroxyethyl)-phenoxyacetic acid of melting point 109 111 C.

Example 8.

The N-hydroxysuccinimide ester of racemic 4-[2- (N - tert. - butyoxy carbonyl - N- methylamino- 1 - hydroxyethyl]phenoxyacetic acid was coupled to bovine serum albumin (BSA) as follows: 300 mg (0.00447 mmol) of bovine serum albumin in 12 ml of water were treated with 6 ml of a 0.5-M solution of sodium bicarbonate and then with 8 ml of dimethoxyethane containing 60 mg (0.075 mmol) of the N-hydroxysuccinimide ester of racemic 4 [2- (N - terf.butoxycarbonyl- N- methylamino)- 1 - hydroxy ethyl] - phenoxyacetic acid. The mixture was stirred for 3 hours at room temper ature, 96 ml of ethanol were added and the solution was evaporated to a small volume.The residue was then dialysed against 200 volumes of water for 3 days with two changes per day and then the protected antigen was lyophilised. A second run was carried out under the same conditions with the exception that 162 mg of the activated ester were used.

Removal of the tert.butoxycarbonyl group was carried out by stirring 50 mg of the protected antigen with 50 Ó trifluoroacetic acid in 50 ml of methylene chloride for I hour at room temperature. The trifluoroacetic acid was then removed by flash evaporation the residue was washed with water and evaporated. The residue was then taken up in water and lyophilised to yield the desired antigen.

Examination of the antigen produced from both runs using protein analysis and differential ultraviolet spectral analysis indicated that the antigen from the first run contained 14 moles of hapten per mol of BSA (170/ substitution based on 85 theoretically- available amino groups, while the antigen from the second runv contained 25 moles of hapten per mol of BSA (29% substitution). This antigen is used to elicit antibody specific for epinephrine, metanephrine, synephrine and phenylephrine.

Example 9.

Racemic 4-(2-tert.butoxycarbonamido- 1 -hydroxyethyl)-phenoxyacetic acid was coupled to BSA by means of the mixed anhydride method as follows: 39.46 mg (0.1269 mmol) of the protected hapten were added to 1 ml of dry dioxane and then 0.1269 mmol of triethylamine in 0.5 ml of dioxane were added.

The mixture was stirred at room temperature for 10 minutes and then cooled to 8"C. 0.1395 mmol of isobutylchloroformate in 0.5 ml of dioxane was added and the solution was stirred for 20 minutes.

In a separate flask 100 mg of BSA were dissolved in 10 ml of water, the pH was adjusted to 9 with sodium hydroxide and 8 ml of dioxane were added slowly with stirring. The solution was cooled to 80C, the protected hapten solution from the preceding paragraph added and the mixture stirred for 30 minutes at 80C and then overnight at 40C at pH 9.

The solution was then treated with acid up to neutrality, the solvent removed and the residue taken up in 5 ml of water, sodium hydroxide being added to bring about solution. 5 ml of this solution were dialysed successively against 6000 ml each of 0.5%N sodium hydroxide, 0.1-N and water (twice). The amine-blocked antigen solution was removed and lyophilised. The tert.butoxycarbonyl group was removed using trifluoroacetic acid as described in Example 8.

Analysis of the resulting antigen by protein determination and ultraviolet analysis indicated that there were 64 moles of hapten per mole of BSA. This antigen is useful in eliciting antibodies which recognise norepinephrine, normetanephrine, octopamine and norphenephrine when injected into suitable animals.

Example 10.

Immunisation and Bleeding For the immunisation of rabbits, 10 mg of each of the products of Example 8 or Example 9 were dissolved in 1 ml of phosphate buffered saline and emulsified with I ml of complete Freund's adjuvant. A second inoculation was given 3 weeks later and cdnsisted of 1.5 mg of - imrnunogen in 1 ml of phosphate buffered saline emulsified with 1 ml of complete Freund's adjuvant. A third injection, 5 weeks after the first, comprised the same concentration of antigen, but was in incomplete Freund's adjuvant. Immunisations were carried out by the subcutaneous route.

Test bleedings were taken after 5 weeks and at monthly intervals thereafter.

30 ml of blood were withdrawn and the serum was separated by standard techniques.

Assay Procedure A competitive binding radioimmunoassay procedure similar to that described in United States Patent No. 3 704282 was used. An incubation volume of either 0.5 or 1.0 ml was used and antibody-label complex was separated from free label by precipitation with saturated ammonium sulphate.

Conditions of assay (and order of addition): 1) 100 ,ul sample containing 1 ng of catecholamine; 2) 100--200 cull of 0.2-M phosphate buffer or carbonate-bicarbonate buffer at an appropriate pIt 3) 0.1-N iodine (I22NaI), aqueous, followed by 10% Na2SO3, aqueous; 4) total incubation volume of I ml, brought to volume with 0.1--M phosphate buffer, pH 7.4 (this buffer being used to return the pH after oxidation to 7.4 for assay); 5) 100 lug label in phosphate buffer, pH 7.4, 0.1--M (I'25 dl-metanephrine, specific activity approximately 300 Ci/mmole, prepared by the chloramine T method); 6) 100 ,ul of a 1::400 dilution of antibody in 10% (v/v) normal rabbit serum solution in phosphate buffered saline (pH 7.4); incubation: 2 hours to overnight at 40C; precipitation of antibody-label complex: 1 ml of saturated ammonium sulphate; centrifugation: 3000 revolutions per minute, 30 minutes, 4"C; aspiration: supernatant; counting: pellet directly in tube in gamma counter;

. . I Quantity of catecholamine as determined by RIA with epinephrine antibody OH I CH3 R = -C- CH -No NH 1 2 sH H Treatment of sample neosyn epinephrine metanephrine synephrine ephrine HO R CH O R < R < R HC A I HO HO HO 'N OH none, pH 7.4 1 ng 1 ng 1 ng 1 ng 50 p1 0.1-N I2, 4aC, 5' reduce w/ O 1 ng 1 ng 1 ng 50 1 Na2SO3 10% 200 1 phosphate buffer, pH 8.8 (adj. soln. sample 0 O 1 ng 1 ng to 8.6), 50 iii 12, 5', 40C, reduce w/ 50 p1 Na2SO3 100 p1 carbonate/ bicarbonate buffer.

O.1-M pH 9.2, 50 1 0 0 0 1-N 12, 5'1 240C, 1 ng reduce w/50 cll Na2S 3 Preparation of Labelled Compounds Radiolabelled epinephrine-type catecholamines useful in a radioimmunoassay for epinephrine-type catecholamines are either commercially available (epinephrine 3H) or can be prepared using well-known methods such as the chloramine T method for introducing iodine labels (synephrine '25I, metanephrine '25I and phenylephrine '25I).

Similarly, norepinephrine-type catecholamines radiolabelled with tritium are commercially availabe (norepinephrine 3H and normetanephrine H) or can be prepared by the chloramine T method (normetanephrine l25I, octopamine l25I or norphenephrine 125I).

WHAT WE CLAIM IS: 1. A method for the assay for individual epinephrine-type catecholamines selected from epinephrine, metanephrine, synephrine and phenylephrine and for individual norepinephrine-type catecholamines selected from norepinephrine, normetanephrine, octopamine and norphenephrine in a test sample, which method comprises in combination the steps:

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (6)

**WARNING** start of CLMS field may overlap end of DESC **. centrifugation: 3000 revolutions per minute, 30 minutes, 4"C; aspiration: supernatant; counting: pellet directly in tube in gamma counter; . . I Quantity of catecholamine as determined by RIA with epinephrine antibody OH I CH3 R = -C- CH -No NH 1 2 sH H Treatment of sample neosyn epinephrine metanephrine synephrine ephrine HO R CH O R < R < R HC A I HO HO HO 'N OH none, pH 7.4 1 ng 1 ng 1 ng 1 ng 50 p1 0.1-N I2, 4aC, 5' reduce w/ O 1 ng 1 ng 1 ng 50 1 Na2SO3 10% 200 1 phosphate buffer, pH 8.8 (adj. soln. sample 0 O 1 ng 1 ng to 8.6), 50 iii 12, 5', 40C, reduce w/ 50 p1 Na2SO3 100 p1 carbonate/ bicarbonate buffer. O.1-M pH 9.2, 50 1 0 0 0 1-N 12, 5'1 240C, 1 ng reduce w/50 cll Na2S 3 Preparation of Labelled Compounds Radiolabelled epinephrine-type catecholamines useful in a radioimmunoassay for epinephrine-type catecholamines are either commercially available (epinephrine 3H) or can be prepared using well-known methods such as the chloramine T method for introducing iodine labels (synephrine '25I, metanephrine '25I and phenylephrine '25I). Similarly, norepinephrine-type catecholamines radiolabelled with tritium are commercially availabe (norepinephrine 3H and normetanephrine H) or can be prepared by the chloramine T method (normetanephrine l25I, octopamine l25I or norphenephrine 125I). WHAT WE CLAIM IS:

1. A method for the assay for individual epinephrine-type catecholamines selected from epinephrine, metanephrine, synephrine and phenylephrine and for individual norepinephrine-type catecholamines selected from norepinephrine, normetanephrine, octopamine and norphenephrine in a test sample, which method comprises in combination the steps:

(A) determining the total epinephrine-type or norepinephrine-type catechoFamine concentration by carrying out an immunoassay on said test sample using an antibody having specificity for epinephrine-type or norepinephrine-type catechol gamines; (B) treating an aliquot of said test sample with iodine at a pH of 7.4 so as to selectively convert any epinephrine or norepinephrine present in said test sample in a form which is not bound by said antibody, treating said aliquot with excess aqueous sulphite solution so as to reduce any remaining iodine, adjusting the pH of the resulting solution tb that required for said immunoassay and then determining the epinephrine-type or norepinephrine-type catecholamine concentration by carrying out said immunoassay on said resulting solution representing the sum total of metanephrine, synephrine and phenylephrine present in said test sample for epinephrine-type catecholamines or normetanephrine, octopamine and norphenephrine for norepinephrine-type catecholamines; (C) treating a second aliquot of said test sample with iodine at a pH of 8.6 so as to selectively convert any epinephrine and metanephrine or norepinephrine and normetanephrine present in said test sample into forms which are not bound by said antibody, treating said aliquot with excess aqueous sulphite solution, adjusting the pH to a level compatible for said immunoassay and then determining the concentration of epinephrine-type or norepinephrine-type catecholamines by carrying out said immunoassay on said test solution, said concentration repre senting the sum total of synephrine and phenylephrine or octopamine and norphenephrine present in said test sample; and (D) treating a third aliquot of said test sample with iodine at a pH of 9.2 so as to selectively convert any epinephrine, metanephrine and synephrine or norepine phrine, normetanephrine and octopamine present in said test sample into forms which are not bound by said antibody, treating said aliquot with excess aqueous sulphite solution, adjusting the pH to a level compatible for said immunoassay and then determining the concentration of epinephrine-type or norepinephrine-type catecholamine by carrying out said immunoassay on said test sample, said concen tration representing the phenylephrine or norphenephrine present in said test sample; wherein the concentration of epinephrine or norepinephrine is determined by subtracting the concentration found in step (B) from that of step (A); the concen tration of metanephrine or normetanephrine is determined by subtracting the con centration found in step (C) from that of step (B); and the concentration of syne phrine or octopamine is determined by subtracting the concentration found in step (D) from that of step (C).

2. A method according to claim 1, wherein the pH at which the treatment with iodine in steps (B) and (C) is performed is maintained with phosphate buffer while the pH at which the treatment with iodine in step (D) is performed is maintained with carbonate/bicarbonate buffer.

3) A method according to claim I or claim 2, wherein said immunoassay is a radioimmunoassay.

4. A method according to any one of claims I to 3 inclusive which is for the assay of epinephrine-type catecholamines selected from epinephrine, metanephrine, synephrine and phenylephrine.

5. A method according to any one of claims I to 3 inclusive which is for the assay of norepinephrine-type catecholamines selected from norepinephrine, normetanephrine, octopamine and norphenephrine.

6. A method according to any one of claims 1 to 5 inclusive, wherein the sample is first treated so as to eliminate non-catecholamine containing substances such as protein and other compounds which may interfere in the oxidation reaction.