DK152313B - REAGENT FOR DETERMINING AN IMMUNOLOGICALLY ACTIVE MATERIAL - Google Patents

Description

DK 152313 B

The present invention relates to a reagent for the determination of an immunologically active material, which reagent consists of a receptor that specifically binds the immunologically active material, an enzyme, an enzyme substrate and the immunologically active material labeled with a compound which is in capable of modifying the activity of the enzyme.

Many analytical systems have been developed in recent years, which are based on competing protein binding analysis (also called saturation analysis).

The terms "saturation assay" and "competing protein binding assay", which are synonymous, cover analytical systems used to determine immunologically active materials (ligands). The results of these assays, performed in biological fluids, are used for medical and veterinary diagnoses. The diagnosis depends on whether the level of

2 DK 152313B

the particular substance is normal or pathological. The analytical principle builds, for example. on a competition between a ligand and a labeled ligand for a common specific binder (receptor), as illustrated by the following equation: brand "" fke "" yke

I II

ligand + ligand + receptor - ^ ligand + ligand + ligand + ligand

I I

receptor receptor (1) (2) (3) (4) Equation 1

The primary reaction is a combination of 1 molecule ligand with 1 molecule receptor to form a bimolecular ligand / receptor complex (1). Another reaction is caused by the addition of labeled ligand, which is similarly combined with the receptor to form labeled ligand / receptor complex (2). In saturation analysis, the concentrations of the receptor and of the labeled ligand are constant. The concentration of receptor is limited so that the labeled ligand is in excess of the receptor. Under these conditions, the addition of ligand causes competition between ligand and labeled ligand for binding to the receptor. Therefore, an increase in ligand concentration will decrease the amount of labeled ligand / receptor complex. The test principle is based on a determination of the percentage of the total labeled ligand amount bound to the receptor. This percentage is inversely proportional to the amount of ligand added to the reaction mixture from the sample to be measured or from the standard used during the test. The reduction in the concentration of labeled ligand / receptor complex or the increase in the concentration of labeled ligand in the reaction mixture can be used to determine the ligand concentration.

The sensitivity of saturation analysis depends on the use of a receptor which has a very high affinity for the ligand and the labeled ligand. Moreover, the sensitivity also depends on the use of a mark which can be detected at very low concentration.

3 DK 152313 B

The specificity of the saturation assay depends on the receptor's ability to bind only ligand and labeled ligand in a complex mixture of different molecules.

Saturation analysis has been used in a variety of techniques, the difference of which is primarily due to the type of brand used. Usually, these techniques are classified by the broad terms "radio test" or "non-radio test" depending on whether a radioactive tracer is used as a label.

Radio experiments have been used to a greater extent than non-radio experiments. Radio experiments may further be classified as radioimmunoassays or radio-receptor assays depending on the type of receptor used in the assay. In radioimmunoassays, an antibody is used which specifically binds the ligand and the labeled ligand. In radio-receptor analysis, on the other hand, another type of biological receptor is used which similarly specifically binds the ligand and the labeled ligand.

In all radio test techniques, it is physically important to separate the bound fraction (1 and 2 in equation 1) from the unbound fraction (3 and 4 in equation 1) in the reaction mixture. An index of ligand concentration can then be obtained by counting these fractions in a radioactivity counter and comparing the counts of the unknown samples with counts of appropriate standard ligand samples tested in the same experiment. Many different and divergent methods have been described for separating the bound and free fractions in the radio test reaction mixture using techniques such as e.g. gel filtration, absorption and ion exchange chromatography, fractional precipitation, solid phase or electrophoresis.

Following the development of radio test techniques in saturation analysis, methods have been developed using non-radioactive labels. Methods have been demonstrated that use enzymes as a label. These methods may have the advantage that a physical separation of the bound (1 + 2) and free (3 + 4) fractions of labeled ligand is not required in the experimental procedure if enzyme activity changes when an antibody is changed.

4 DK 152313B

binds a ligand which is labeled with an enzyme. The degree of change in the activity of the enzyme is indicative of the concentration of the labeled ligand in the bound fraction and therefore gives an index of the ligand concentration in the reaction mixture.

The chemical structure of the ligand-enzyme complex is extremely difficult to determine, and this is the major disadvantage of enzyme immunoassays. This is undoubtedly due to the large amount of amino acid side chains present on the enzyme surface for complexation with the ligand. This causes major problems in the reproduction of the ligand enzyme by various preparations of the complex. The general lack of control of the complex-forming reaction resulted in the binding of many ligand molecules to one enzyme molecule, although it is likely that the binding of only a few of these ligand molecules to the antibody is involved in the inhibition of enzyme activity. Therefore, not all antibody / tagged antigen "interactions result in a modification of the enzyme activity, thereby reducing the sensitivity of the technique.

Protein-protein interaction between different antibody molecules and antibody and enzyme molecules is another consequence of the amount of ligand molecules on the enzyme surface. The protein-protein interaction is further enhanced if the ligand is also a polypeptide. Therefore, the enzyme molecule induces a localized micro region of high protein concentration. In such situations, it has been shown that protein precipitation occurs, causing one of the main benefits of enzyme immunoassay to be lost as it becomes necessary to separate antibody bound and free fractions.

A modification of the enzyme immunoassay has been described, in which these problems are partially solved, labeling the ligand with a low molecular weight detector molecule. In this experiment, the antibody binding of labeled ligand sterically inhibits binding of the detector molecule to another antibody specific to the detector molecule. The degree of inhibition is determined by competition for binding of the free ligand detector molecule and detector molecule labeled enzyme with detector molecule antibody. The degree of change in enzyme activity,

5 DK 152313B

which is determined by normal enzyme immunoassay is indication of the concentration of the free ligand detector molecule which is also indication of the ligand concentration in the reaction mixture. The advantage of this technique is that a small molecule, rather than an enzyme, binds to the ligand, thereby allowing a determination of the chemical structure of the labeled ligand, thereby overcoming many of the disadvantages of the enzyme immunoassay described above. However, in this system, only the disadvantages of the primary binding reaction involving the ligand and the labeled ligand are overcome and they are transferred to the detector system where the degree of antibody bound and antibody-free fractions of the labeled ligand is determined.

The disadvantages of the known methods are overcome by the present invention, according to which an enzyme mode is used: i -F i k a r o r .9 nm label.

More specifically, the present invention relates to a reagent of the type described initially, which is characterized in that the labeled immunological material is an antigen-enzyme inhibitor or anti-gene enzyme activator complex.

The term "immunologically active material" or "ligand" refers to any immunologically active substance or part thereof which may be immunologically determined, e.g. using saturation analysis technique. The essential requirement is that there is a receptor that can specifically bind the ligand. When the receptor is an antibody, the ligand is haptic or antigenic so that specific antibody can be formed. A ligand is referred to as a hapten when it triggers antibody formation only when it binds to a compound having antigenic properties. Alternatively, a ligand is referred to as antigen when it triggers antibody formation without chemical modification. The molecular weight of the ligand can vary in a wide range from ca. 100 to approx. 1,000,000. This molecular weight range is not limiting the assay, provided that a receptor is found that can specifically bind the ligand.

The ligand may have polymer or non-polymer structure. When the ligand has polymer structure, it usually has biological origin and can be classified as a nucleic acid, a polysaccharide and / or polypeptide. On the other hand, when the ligand is non-polymeric, it usually has a molecular weight of less than 2,000 and can have a

e DK 152313B

Particularly important ligands which can be used in the present invention are amines, amino acids, peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acids, mono- and polysaccharides, alkaloids, vitamins, drugs, drugs, antibiotics, metabolites, pesticides, toxins, industrial pollutants, flavors, hormones, enzymes, co-enzymes, cellular or extracellular tissue components and antibodies isolated from humans or animals.

Immediately potential constituents for analysis with the present system are hepatitis B surface antigen, ferritin, tumor antigens such as CEA, α-fetoproteins, rheumatoid factor, C-reactive proteins, immunoglobulin classes IgG, IgM or IgA, myoglobin, thyroid hormones comprising T3 and, insulin, steroid hormones. testosterone or estradiol, addictive drugs, including narcotic analgesics such as morphine, barbiturates, stimulants such as amphetamine, drugs for the treatment of epilepsy including diphenylhydantoin and phenobarbital, cardiac glycocytes such as digoxin, vitamins such as vitamin and folic acid. Furthermore, antibodies which are immediately potential for analysis with the system are those associated with infections by syphilis, gonorrhea, brucellosis, rubella and rheumatism.

The term "labeled immunologically active material" or "labeled receptor" here refers to a ligand, a ligand analogue or a portion thereof, or a receptor labeled with an enzyme modifier. One, more than one, and usually less than 100 label molecules can be bound to a ligand or receptor molecule. Likewise, one, more than one, and usually less than 5 ligand or receptor molecules can be bound to a label molecule. The binding of extra labels to a ligand or receptor molecule usually increases the sensitivity of the experiment, provided that the extra labels do not affect binding.

Binding of a modifier atom to a ligand or receptor molecule results in the formation of intermolecular bonds, which in most cases, but not necessarily, are of a covalent nature. The binding may in some cases be carried out in the presence of a coupling agent by inserting a chain group between the label and the licorice or receptor.

7 DK 152313 B

in

The modifier molecule can be directly linked to the ligand or receptor molecule. However, it may be desirable to insert different length chemical bridges between the modifier molecule and the ligand or receptor molecules, depending on the intended specific experiment. In some cases, it may even be advantageous to bind the modifier molecule and the ligand or receptor molecules separately to the same carrier molecule, e.g. a macromolecule such as a polypeptide or a polysaccharide.

The term "receptor" as used herein refers to any substance that can specifically bind ligand and labeled ligand or part thereof. Usually, the receptor used in the assay is a specific antibody for the ligand that is formed in the blood of vertebrates after injection of a suitable hapten or an appropriate antigen. Naturally occurring receptors may also be used in the experiment. This latter group comprises proteins, nucleic acids and cellular membranes. Such receptors have been used in radio test techniques for thyroxine, insulin, angiotensin and various steroid hormones.

When the ligand is an antibody, the receptor may be the antigen used to induce this antibody in a host animal. In another embodiment, the receptor may be an antibody to the antibody to be determined.

It is not possible to determine for sure the receptor mode of action which, by binding of the labeled ligand, reduces the interaction between enzyme and modifier. The most likely explanation is that the affinity of the enzyme to the modifier is reduced as a result of a change in size and net charge of the receptor / ligand-modifier complex in comparison with the values of ligand-modifier alone.

The term "modifier" here means any substance capable of interacting with an enzyme so that the degree and manner of enzyme activity are modified. This modification may result in an inhibition, activation or change in specificity or any other of the enzyme's properties which can detect

8 DK 152313B

is either directly or indirectly caused by a change in enzyme activity or in the mode of activity, e.g. a change in reaction conditions, e.g. co-factor requirements or pH value optimum, in kinetic properties or in activation energy. The modifier can range in size from a small molecule to a macromolecule, and its interaction with an enzyme molecule can be either reversible or irreversible depending on whether the inter-molecular bond is ionic or covalent.

The sensitivity of the experiment is i.a. depending on the affinity of the receptor for the ligand and the ability of the modifier to cause a change in the degree or mode of enzyme activity. Preferably, the change in enzyme activity degree or mode is achieved with a minimal modifier concentration. The closer this concentration is to the enzyme concentration on a molecular year basis, the greater the sensitivity of the experiment.

The modifier is preferably an enzyme inhibitor which, upon interaction with the enzyme, inhibits its activity. The mode of action of the inhibitor may be competing, non-competing, aliosteroic, or a combination of two or more of these ways. Preferably, the inhibitor should have an inhibition constant (the inhibitor concentration required for 50% inhibition of the enzyme system) of less than 10 moles / liter of gout of the test performed. The inhibition constant is particularly preferred between 10 and 10

Any enzyme may be used in the assay, provided that a modifier exists which specifically modifies the enzyme activity in the manner described above. Preferred enzymes are stable, readily available at low cost and have a high turnover rate and a simple elaborate experimental system. The turnover rate (number of product molecules formed per enzyme molecule in 1 minute) is preferably above 100 depending on the specific test. More preferably, the turnover rate is as high as possible and at least 200.

Enzyme modifier systems which are particularly useful in the present invention are dihydrofolate reductase / methotrexate, dihydrofolate reductase.

9 ..... DK 152313 B

tase / 4-aminopterin, dihydrofolate reductase / other specific inhibitors of this enzyme; p-glucoronidase / 4-deoxy-5-aminoglucaric acid and its derivatives; biotin containing enzymes / avidin-like carboxylase / avidin; chymotrypsin / TPCK

CH2-Ph \ I TPCK = CH, - // A-SOO-NH-C-C-CH, Cl |

V \ J I II J

γ-cystathionase / propargylglycine; alanine racemase / trifluoroalanine; tryptophanase / trifluoroalanine; tryptophansynthetase / trifluoroalanine; β-cystathionase / trifluoroalanine; pyruvate-glutamattransaminase / tri-fluoroalanine; mælkesyreoxidase / 2-hydroxy-but-3-yn-acid; monoamine-Oxidase / N, N-dimethylpropargylamine and diamine oxidase / H ^ N-CH2 -CSCC ^ -N ^.

The enzyme activity assay can be performed by directly or indirectly monitoring the substrate consumption or product formation at a suitable pH and temperature using a detector system, e.g. colorimetry, spectrophotometry, fluorospectrophotometry, gasometry, thermometry (heat production) or scintillation counting.

In order to increase the sensitivity of the system, it is possible to use bioluminescence and enzyme cycle techniques, e.g. the techniques described by J. Lee et al. in Liquid Scintillation Counting:

Recent Developments, Stanley P.E. and Scoggins, B.A., Academic Press, New York, page 403 and Lowry O.H. et al. in J. Biol. Chem.

236, pages 2746 - 2755.

In a particular embodiment of the present invention, a labeled ligand can be used to determine the presence of a ligand in an unknown sample by simultaneous or sequential addition at an appropriate pH of the labeled ligand and the unknown sample to an aqueous medium which contains a receptor specific to the ligand and the labeled ligand.

After a suitable incubation period, the distribution of receptor bound to ligand and labeled ligand is determined by the addition of enzyme and substrates. The receptor specific for the ligand

10 DK 152313B

also binds the labeled ligand, thereby reducing the interaction between the modifier and the enzyme so as to reduce the modification of the enzyme activity. Addition of ligand in the experiment results in a competition with the labeled ligand for binding to the receptor, thereby increasing the concentration of free labeled ligand in the experiment. The interaction between ligand modifier and enzyme is increased and enzyme activity is affected again. Therefore, the modification of enzyme activity is a function of the ligand concentration in the assay and is caused by unbound labeled ligand. Therefore, the difference between the resulting enzyme activity and the activity obtained in the absence of ligand is indicative of the ligand concentration in the unknown sample.

One of the main advantages of this process is that separation of bound and free fractions is unnecessary in the process. However, this does not preclude the use of such a separation step in the experiment after incubating the ligand and labeled ligand with the receptor prior to the enzyme activity assay. Separation of fractions may be desirable in some cases for the removal of substances in the sample which may interfere with the enzyme assay. The separation can be carried out using any of the techniques described in the radioimmunoassay, including gel filtration, adsorption and ion exchange chromatograph, fractional precipitation, solid phase and electrophoresis.

The present invention is further illustrated by the following examples. Example 1.

Preparation of "amino digoxin".

To a suspension of 156 mg (0.2 millimoles) of digoxin in 5 ml of absolute ethanol is added 10 ml of 0.2M sodium metaperiodate with stirring.

The mixture becomes homogeneous after 10 minutes, and then a precipitate is slowly formed. After 2 hours, add 5 ml of water + 5 ml of ethanol. After another 30 minutes, 122 µl (2.2 millimoles) of ethylene glycol is added and a dense white precipitate immediately begins to precipitate. After stirring for 80 minutes, add

11 DK 152313 B

133 µl (2.0 millimoles) of ethylenediamine, and the resulting pH of 11.0 is adjusted to 9.5 with 0.1M hydrochloric acid and the reaction mixture is allowed to stand at room temperature for 18 hours. The pH value does not change during this period.

151.4 mg (4.0 millimoles) of sodium borohydride is then added and the mixture is stirred for 3 1/2 hours. The pH was then adjusted from 10.5 to 6.5 with 1M formic acid (about 3 ml) and thin layer chromatography on this mixture showed a single large spot with an R R value of 0.15 (silica on aluminum developed in butanol : acetic acid: water (4: 1: 1)) · Digoxin has an R 2 value of 0.7 when developed in the same system.

The solvents are evaporated to near dryness on a rotary evaporator under vacuum with a water bath temperature of 60 ° C. The last few ml of water are removed by adding 95% ethanol (3 x 20 ml) and repeating the evaporation as above.

The resulting pale yellow solid is extracted three times with absolute ethanol and these combined extracts are evaporated to ca. 4 ml and centrifuged to separate a small amount of salt, which is discarded. The pale yellow supernatant solution is evaporated to dryness under a stream of dry nitrogen to give a yellow oil having the same R R value by thin layer chromatography as the reaction mixture described above. It is shown that the product contains a free amino group by reacting it with Fluram® (4-phenylspiro [fluorane-2 (3H), 11-phthalane] -3,31-dione) to form an intensely fluorescent compound. This concentrated sulfuric acid product has a spectrum similar to digoxins with absorption peaks at 385 and 495 nm. The product also exhibits strong affinity for antisera (rabbit) specific for digoxin. The most likely structure of the isolated product is the following:, ro ™ r ° '' CH3 —p 1 li & & jTiCyit h2n-ch2ch? -N -

12 DK 152313B

Example 2.

Preparation of methotrexate-aminodigoxin conjuate.

Dissolve 11 mg of methotrexate in 5 ml of water and adjust the pH to 6.5. 10 mg of "aminodigoxin" is dissolved in this solution and the volume is adjusted to 20 ml with water. The pH is adjusted to 6.5 again and 484 mg of N-ethyl-Nn- (3-dimethylamino) propylcarbodiimide hydrochloride dissolved in 5 ml of water are added to the reaction mixture. The pH value is kept at 6.2 for 24 hours at room temperature. The conjugate is purified on a silica gel column using 3% ammonium citrate as solvent. Fractions containing the desired product are combined and shown to have the dual capacity to strongly bind antisera (rabbits) specific for digoxin and also to strongly inhibit the enzyme dihydrofolate reductase (chicken liver). The most likely structure of the methotrexate-aminodigoxin conjugate is the following:

ΠΟ 0N

ljH3 f -—? °

Q x ~ f

æ7 ch3 ch3 0H

HN-CH2CH2-n ~

H

O = C CH2 CH f 'NH2 λ i 2 I il 1 HO __ I _ T CH3 | __j CHNHC? CH2 NH2 / I I ch3.'? ®3 rtjtr I 1 hn-ch2-ch2-n ti

DK 152313B

13

Example 3

Enzyme inhibitor immunoassay for digoxin.

100 μΙ serum is incubated at 30 ° C for 15 minutes together with 100 μΙ anti-digoxin antibody solution, 100 μΙ NADPH solution, 100 μΙ 2-mercaptoeth anole solution and 500 μΙ sodium phosphate buffer with pH 7.5. Add 100 μΙ of methotrexate digoxin conjugate from Example 2 (70 μg / ml) and incubate for 15 minutes, then add 100 μΙ dihydrofolate reductase solution. The dihydrofolate reductase used is isolated from chicken livers by the method of Kaufman, B. T., & Gardiner, R. C., Journal of Biological Chemistry, Vol. 211, page 1319 (1966). The mixture is incubated for an additional 3 minutes and the enzyme activity is determined by adding 100 μΙ of dihydrofolate solution and measuring at 340 nm with a Varian® spectrophotometer with printer. The results are listed in Table I.

Table I.

Reactants Enzyme activity

Digoxin Antidigoxin- Methotrexate- Enzyme- DD / min Inhibition (sample con- antibody-digoxin con- -for- in% centration) jugate (in trial) Components _____ 0 absent 0 present 0.150 0 0 absent 7 present 0,100 33 0 present 7 ng present 0.145 3 5 ng / ml present 7 ng present 0.125 16 10 ng / ml present 7 ng present 0.115 23

The reactants are added in the order listed above.

OD = optical density.

X4 DK 152313 B

From the above results, it appears that a few nanograms of digoxin in serum can be determined by the system described.

Example 4

Preparation of methotrexate-human serum albumin conjugate.

Dissolve 45 mg of methotrexate in 1.0 ml of Ν, Ν-dimethylformamide and 25 mg of N-hydroxysuccinimide is added. Dissolve 41 mg of Ν, Ν-dicyclohexylcarbo-diimide and keep the mixture at room temperature for 13 hours. The by-product insoluble urea is filtered off and 100 µl of the filtrate is added to a solution containing 5 mg of human serum albumin in 800 µl 0.1 M sodium phosphate buffer with pH 7.5 plus 100 µl dioxane. The reaction mixture is kept at room temperature for 30 minutes and then poured onto a Sephadex G-25 column equilibrated with 0.1M sodium phosphate buffer of pH 7.5. The column is eluted with this buffer and two elution peaks containing methotrexate are obtained and the first one contains the methotrexate human serum albumin conjugate.

Example 5

Enzyme inhibitor immunoassay for human serum albumin.

100 µl of diluted serum is incubated at 30 ° C for 15 minutes together with 100 µl of anti-human serum albumin antibody (rabbit), 100 µl of NADPH solution, 100 µl of 2-mercaptoethanol solution, and 550 µl of sodium phosphate buffer with pH 7.5. Then 100 µl methotrexate-human serum albumin conjugate (8 µg / ml) is added and the mixture is incubated for 15 minutes, then 100 µl dihydrofolate reductase solution is added. The mixture is incubated for a further 3 minutes and the enzyme activity is determined by adding 100 µl of dihydrofolate solution and measuring at 340 nm with a Varian® spectrophotometer with printer. The results are listed in Table II.

15

Table II DK 152313 B

Reactants Enzyme activity

Human Serum Antihuman Methotrexate Enzyme-for-OD / min Inhibition Albumin (Trial Serum Album-Humane Sequence Component in% Concentrate Anti-Rumum Albumin) Substance Conjugate (in Experiment) 0 absent 0 to present 0.123 0 0 absent 0.8yug present 0.068 45 0 present 0.8 ^ ug present 0.105 16 S ^ ug / ml present 0.8 ^ ug present 0.092 25 10 ^ ug / ml present 0.8 ^, ug present 0.085 31

It is apparent from the above results that some few human serum albumin can be determined in the system described.

Example 6

Synthesis of methotrexate-aminoethylmorphine conjugate.

3 a) Synthesis of O -aminoethylmorphine.

400 mg of lithium aluminum hydride is suspended under a nitrogen atmosphere in 10 ml of tetrahydrofuran freshly distilled from lithium aluminum hydride. Over 5 minutes, a solution of 400 mg of morphine and 400 mg of chloroacetonitrile in 4 ml of freshly distilled tetrahydrofuran is added and then refluxed for 1 hour. The mixture is allowed to cool and 0.6 ml of water is added followed by 0.6 ml of 10% by weight sodium hydroxide solution and 2 ml of water. The mixture is filtered and the salts are washed with tetrahydrofuran, the tetrahydrofuran fractions combined, dried with magnesium sulfate under nitrogen and filtered and the filtrate is evaporated to give 380 mg of O-aminoethylmorphine.

16 DK 152313B

3 b) Synthesis of N-tert-butoxycarbonyl-γ- (O-aminoethylmorphine) glutamic acid α-benzyl ester.

3

A mixture of 50 mg of O-aminoethylmorphine (prepared as above), 34 mg of N-tert-butoxycarbonyl-glutamic acid α-benzyl ester and 25 mg of dicyclohexylcarbodiimide in 5 ml of dichloromethane is stirred for 4 hours at room temperature. The mixture is diluted with ethyl acetate and washed with dilute sodium carbonate solution. The ethyl acetate solution is then extracted twice with 0.1M hydrochloric acid and the combined acidic extracts are treated with a sufficient 10% by weight sodium hydroxide solution to adjust the pH to 8.0. The solution is extracted twice with ethyl acetate and the combined ethyl acetate extracts are washed with sodium chloride solution, dried over anhydrous sodium sulfate and evaporated to give 36 mg of a colorless oil.

c) Synthesis of glutamic acid γ- (0-amidoethylmorphine) -α-benzyl ester trifluoroacetic acid salt.

A solution of 36 mg of N-tert-butoxycarbonyl-glutamic acid morphine derivative (prepared as above) in 3 ml of dichloromethane is stirred at room temperature and 1 ml of trifluoroacetic acid is added. The mixture is stirred for 15 minutes and then evaporated to dryness. As the residue, 40 mg of a colorless glassy material is obtained.

d) Synthesis of methotrexate-γ- (O-amidoethylmorphine) -α-benzyl ester.

A solution of 37 mg of 4-amino-4-deoxy-N 2 -methylpteroic acid in 3 ml of dimethyl sulfoxide is treated with stirring at room temperature with 28 µl of triethylamine and 25 µl of isobutyl chloroformate and the mixture is stirred for 30 minutes. It is then added to a mixture of 75 mg of morphine derivative prepared as described under c) and 28 µl of triethylamine in 2 ml of dimethyl sulfoxide, and the mixture is stirred at 60 ° C for 1 hour. The mixture is cooled, diluted with water and extracted twice with ethyl acetate. The combined ethyl acetate extracts are washed with water and then extracted twice with 0.1M hydrochloric acid. The combined acidic extracts are treated with a sufficient 10% by weight sodium hydroxide solution to raise the pH

DK 152313B

to 8.0. The mixture is extracted three times with ethyl acetate and the combined extracts are washed with sodium chloride solution, dried over aqueous sodium sulfate and evaporated to give 15 mg of a yellow oil. This is purified by preparative thin layer chromatography on silica gel (developing with chloroform / methanol (4: 1)). The desired compound is obtained in the form of 4 mg of yellow solid.

E) Synthesis of methotrexate-γ- (0-amidoethylmorphine).

The product prepared as described under d), 4 mg, is mixed with 5 ml of 0.1M sodium hydroxide solution and the mixture is stirred at room temperature for 8 hours, resulting in a clear yellow solution.

To this is added 5 ml of 0.1M hydrochloric acid and the mixture is diluted to 25 ml with 0.05M sodium orthophosphate buffer pH 7.4 and a solution of the desired compound suitable for the enzyme experiment is obtained.

Example 7

Enzyme inhibitor immunoassay for morphine.

A mixture of 10 μΙ morphine solution of appropriate concentration, 50 μΙ methotrexate-γ (0-amidoethylmorphine) solution (4 ng / 25 ml), 50 μl anti-morphine antibody solution, 10 μΙ NADPH solution, 10 μΙ 2-mercaptoethane -ol solution, 50 μΙ potassium chloride solution, 150 μΙ tris-hydrochloric acid buffer solution (pH 7.5) containing EDTA and 10 μΙ dihydrofolate reductase solution (E. casei) are incubated for 20 minutes at 37 ° C. The enzyme activity of the mixture is determined after addition of 10 μΙ dihydrofolate solution by measuring the change in extinction of the solution at 340 nm using a Centrifichem® centrifugal analyzer. The results are given in Table III below.

18 DK 152313B

Table XII

Reactants Enzyme activity

Morphine Antimorphine- Morphine-Enzyme-for-OD / min Inhibition, µg / test antibody -methotreatment compo- in% 7 xate conjugates hole 0 absent absent present 0.54 0 -8 0 absent 3 x 10 M present 0.14 76 0 present 3 x 10 ^ M present 0.41 29 0.04 present 3 x 10 ® M present 0.37 36 0.40 present 3 x 10 ^ M present 0, 35 40 4.0 present 3 x 10 ^ M present 0.31 46 20 present 3 x 10 ® M present 0.27 54 40 present 3 x 10 ® M present 0.23 60

From the above table it can be seen that with increasing morphine concentration there is a reduction in dihydrofolate reductase activity. In this way, morphine solutions containing from 4 µg per ml to 4 mg per ml. ml of morphine is examined, provided there is 10 µl of the solution. However, this should not indicate that this is the required area, but rather that it can be used in practice.

Example 8.

Synthesis of ferritin-methotrexate conjugate.

A solution of 1.3 mg of human liver ferritin in 5 ml of 0.05 M sodium phosphate buffer pH 8.0 and 1 ml of dimethylformamide is stirred at room temperature and 100 µl of a solution prepared by treating 23 mg of methotrexate in 2 ml of dimethylformamide with 21 µl of triethylamine and 15 µl of isobutyl chloroformate at room temperature for 30 minutes. The mixture is stirred

Claims (1)

At room temperature for 2 hours, then dialyzed against 2x2 liters of 0.05M sodium orthophosphate buffer (pH 7.4 containing 0.1M sodium chloride and 0.05% by weight sodium azide) for 24 hours. The solution is then passed through a column of Sephadex G-25 which is equilibrated with the same buffer and the ferritin-containing fractions are combined and filled up to 10 ml with the buffer. The resulting solution is suitable for use in an enzyme immunoassay to determine ferritin. Reagent for determination of an immunologically active material, which reagent consists of a receptor that can specifically bind the immunologically active material, an enzyme, enzyme substrate and the immunologically active material labeled with a compound capable of modifying the activity of the enzyme, characterized in that the labeled immunological material is an antigen-enzyme inhibitor or antigen-enzyme activator complex.