DE2605162C2 - Enzyme-linked ligand - Google Patents

Description

MDH- (COCH ₂ (CH ₂ ), Z) _nl

corresponds, in which the symbols have the following meanings:

U is 0 or 1;

/? ' is on average in the range 1 to 10;

M DH is malate dehydrogenase; and Z is 3-iodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy-1') -phenyl-1 or 3,5-diiodo-4- (3'-iodo-or 3 ', 5 '-diiodo-4'-hydroxyphenoxy-1 ') -phenyl-1.

11. TIM-bound polyiodothyronine according to claim 1, characterized by the formula

TIM

XY CO ₂ D

C - AL - CNH - CHCH ₂ Z

where the symbols have the following meanings:

TI M is triosephosphate isomerase; a is 0 or 1;

η is at least 1 and not more than 10;

X and Y, which are the same or different from one another, are chalcogen or imono; D is hydrogen or an alkyl group having 1 to 6 carbon atoms; Z is 3-iodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy-1') -phenyl-1 or 3,5-diiodo-4- (3'-iodo- or 3 ', 5' -diiodo-4'-hydroxyphenoxy-1 ') -phenyl-1; and

A is a bond or an organic divalent group having 1 to 12 carbon atoms and 0 to 6 Heteroatoms, namely oxygen, sulfur or hydrogen, being the total number of heterofunctionalities ranges from 0-4.

The concentration of thyroxine in the blood is within relatively narrow limits for proper use Body function critical. The thyroxine concentration is extremely low and can only after very sensitive methods can be detected.

The triiodothyronines (T-3) are also an important factor for the proper functioning of the body. the Triiodothyronines differ from thyroxine (T-4) in that iodine is absent in the 5- or 5'-position. Man assumes that the (T-3) markedly influence the overall metabolic effects of thyroid hormones.

One method for determining thyroid hormones is radioimmunoanalysis (radioimmunoassay). Although this method has many variations, the principle behind it consists in making an antibody to the thyroid hormone and a radioactive or "hot" thyroid hormone combined with blood serum and that bound hormone is separated from unbound hormone. The amount of "hot" hormone delivered to the Antibody is bound or remains free is a function of the amount of the hormone in the serum. By determination the radioactivity of the solution freed from the antibody can be the amount of hormones, based on Calculate standards with known amounts of hormones.

In the case of radio immunoanalysis, a separation stage is required through which errors are introduced and which can be very time consuming. Furthermore, you have to work with radioactive substances that disintegrate and therefore only have a limited shelf life. Furthermore, working with radioactive substances is in the generally undesirable because of the associated health risks. There is a continuing need using a simple method in which the number of treatment stages is reduced to a minimum while ensuring high sensitivity.

In US-PS 38 17 837 an enzyme analysis (enzyme assay) is described, which is very general for a has shown a wide variety of ligands to be useful.

The invention is intended to provide an enzyme-linked ligand which, in a homogeneous enzyme immunoanalysis method for the detection or determination of thyroid hormones (thyroid hormones), in particular in the method according to the divested patent application P 26 60 911.0-52 is suitable, provided

The invention thus relates to an ENZ-bound ligand with the formula

ENZ

X \ Y CO ₂ D

Il Il!

CA L-CNH-CHCH ₂ Z

where the symbols have the following meanings:

ENZ is malate dehydrogenase or triose phosphate isomerase reversibly inhibited by the ligand;
a is 0 or 1;

π averages at least 1 and not more than 10;

X and Y, which are the same or different from one another, are chalcogen or imino;

D is hydrogen or an alkyl group having 1 to 6 carbon atoms;

Z is S-iodine ^ -fS'.S'-diiodine ^ '- hydroxyphenoxy-rj-phenyl-1 or 3,5-diiodo-4- (3'-iodo- or 3', 5'-diiodo-4 ' -hydroxyphenoxy-l ') - phenyl-dog

A is a bond or an organic divalent group with 1 to 12 carbon atoms and 0 to 6 heteroatoms, namely oxygen, sulfur or nitrogen, the total number of heterofunctionalities being between 0 and 4.

The compounds 3-iodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy-1') -phenyl-1 and 3,5-diiodo-4- (3'-iodo- or 3 \ 5'- diiodo-5'-hydroxyphenoxy-1 ') -phenyl-1 are in the following for the sake of simplicity with the collective term »Polyiodothyronine« designated. The enzymes used are too much after conjugation with the polyiodothyronine inhibited than 50% and become partially or completely after the binding of a receptor for dad polyiodothyronine reactivated. The conjugated enzyme used therefore has a low rate of conversion, which in The presence of a receptor, for example an antibody for polyiodothyronine, increases considerably.

The ligands of the invention can be used to determine molar Polyjodthyroninen in concentrations as low as 10 ⁷ or less, wherein they are reacted with the antibody for the Polyjodthyronine. In homogeneous enzyme immunoanalysis, the reagents used are the ligands according to the invention with antibodies, substrates for the enzyme and an aqueous, normally alkaline buffer medium which usually also contains additives to improve the enzyme stability. The order in which the reagents are added to the aqueous analysis medium is generally not critical. Furthermore, incubation after the addition of certain reagents may be desirable.

After all the reagents have been added, the rate of reaction of the enzyme is monitored, normally spectrophotometric. By comparing the result obtained with the unknown substance with the Results obtained using known amounts of polyiodothyronine can be used to determine the amount of Determine polyiodothyronine in the unknown sample.

In the ligands according to the invention, the phenolic hydroxyl group of the polyiodothyronines is used as the conjugation site not used.

If the enzyme used is malate dehydrogenase (MDH), the mitochondrial MDH is preferred, in particular from pork hearts; if triosephosphate isomerase (TIM) is used, use; man especially those made from rabbit muscles.

In the formula given above, η is preferably a number from 1 to 8, in particular from 2 to 6. The group A usually contains at least 2 and not more than 10 carbon atoms, preferably 3 to 7 carbon atoms and preferably 0 to 4, usually 0 or 1 up to 3, usually 1 to 2 heteroatoms.
A is / can be a hydrocarbylene group (an aliphatic, alicyclic, aromatic or a combination of these groups) or a non-hydrocarbylene group (a substituted aliphatic, alicyclic, aromatic, heterocyclic or a combination of these groups), with generally 0 to 1 cyclic Groups are present in the chain, usually 5 to 6 ring members having 0 to 2 hetero ring members, usually 0 to 1 hetero ring members, the substituents of the cyclic group being separated from one another usually by 2 to 4, usually by 3 to 3 ring members; usually the hydrocarbyl group is the only one

so aliphatic unsaturation 0 to 1 ethylenic groups; it is preferably saturated and, in particular, represents represents an alkylene group having 0 to 1 carboxamido, imino or oxy groups in the chain; when A is carbocyclic is an aromatic group, it usually contains 6 to 10, usually 6 to 8 carbon atoms.

The total number of functional groups in the chain (oxy, amino, amido groups or the sulfur and nitrogen analogs) is generally 0 to 4, usually 0 to 3, and usually 0 or 1 to 2.

The general group also includes ENZ-bound ligands, which are generally as follows have given formula

X ¹ Y ¹ CO ₂ D

MDH [C —d, —ff, —Λ— y, —C-NHCHCH ₂ Zj, '

where the symbols have the following meanings:
MDH is malate dehydrogenase;

/ j ¹ is in the range from 1 to 8, in particular between 2 and 6,
X 'and Y ¹ , which are the same or different from one another, are oxygen, sulfur or imino;

p. q and r are each 0 or 1, the sum of p, q and / - preferably at least = 1 \ si (p + q + r ^ 1);
s is 0 to 2, usually 0 to 1;

λ and γ are hydrocarbon groups with 1 to 8 carbon atoms, usually with 1 to 6 carbon atoms, usually with 1 to 3 carbon atoms, with a total of 1 to 12 carbon atoms, usually a total of 2 to 10 carbon atoms, preferably a total of 2 to 8 carbon atoms and especially 2 to 6 Carbon atoms are present;

for ρ or r = 0, ac or y preferably contain 1 to 8 carbon atoms, in particular 1 to 6 carbon atoms; / χ and γ can be aliphatic, alicyclic or heterocyclic; together with β and ό they can satisfy the definition of A;

oc and γ are preferably saturated aliphatic groups, in particular unbranched groups, e.g. B. methylene or polymethylene groups or aromatic, especially monocyclic groups, where only one of oc and / is an aromatic group; /? Is an oxy, thio, sulfonyl or alkylamino group, where the alkyl group is 1 to 6, preferably 1 contains up to 3 carbon atoms, with the proviso that /? is an amino or alkylamino group when ρ is 0;
rf is

- CHNHC—

YES

wherein T ^{1 is} hydrogen or a lower alkyl group having 1 to 3 carbon atoms;

Z is 3,5-diiodo-4- (3,5'-diiodo-4'-hydroxyphenoxy-1 ') -phenyl-1 and D is hydrogen or an alkyl group with 1

up to 6 carbon atoms.

According to a preferred subgroup, the polyiodothyronine can be mixed with a dibasic dicarboxylic acid be combined to form an amic acid (monoamide of a dibasic acid). The dibasic acid usually contains 2 to 8 carbon atoms, usually 2 to 6 carbon atoms and 0 to 1 ethylenic unsaturation in the chain; it also contains 0 to 1 atoms with atomic numbers 7 to 8 (nitrogen or Oxygen), where the nitrogen or oxygen atoms are only connected to carbon atoms. d. H. in shape of tertiary amino groups or ether groups. Preferred dibasic acids form cyclic anhydrides with 5 to 7, especially 5 to 6 ring members. To ensure that the carboxyl group of the dibasic Acid conjugated with the thyronine is the one that reacts with the ENZ becomes the original Thyronine carboxyl group usually with an alkyl group of 1 to 3 carbon atoms, preferably esterified with one carbon atom (methyl).

The modified polyiodothyronine or its analog, which is used for conjugation with the ENZ, has generally the formula given below

Z-CH ₂ -CH-CO ₂ D '

NH

(CY) - RC ₀ E

40

where the symbols have the following meanings:

X, Y, Z and a are as defined above;

D 'is hydrogen or an alkyl group having 1 to 3 carbon atoms, preferably 1 carbon atom; R. has the same definition as A, but is preferably a divalent aliphatic group of 0 to 1 Ethylenically unsaturated sites and 0 to 1 heteroatoms with an atomic number of 7 to 8, the heteroatoms are fully substituted by carbon atoms and have 1 to 6 carbon atoms: and

E is hydroxyl or can form a double bond together with a terminal nitrogen atom of R,

z. B. isocyanate or isothiocyanate

Another useful series of connections has a carbocyclic or heterocyclic ring of 5 to 6 Ring members in the chain, the heterocyclic ring containing 1 to 2, usually 1, heterocyclic member, wherein it is O, S or N. These compounds preferably have an isocyanate or isothiocyanate group, which is directly or indirectly connected to a ring link, especially if it is one In most cases, these compounds have the following formula

Y ² CO ₂ DX ² -C-NArCNHCHCH ₂ Z

where the symbols have the following meanings:

D and Z are as defined above;

X ² and Y 'are chalcogen (O or S), where X ^{2 is} preferably S;

Ar is an aryl or aralkyl group, including carbocyclic and heterocyclic groups with 5 to 6 ring members, 1 to 2, preferably 1 hetero ring members, namely O, N and S being present, and 4 to 10 carbon atoms. usually 4 to 8 carbon atoms, and if the compounds are heterocyclic, preferably 4 to 6 carbon atoms, and if the compounds are carbocyclic, preferably 6 to 8 carbon atoms are present.
The ENZ conjugal of the series of compounds given above has the formula

ENZ-

X ² Y ² CO ₂ D

I! ! I!

^ C-NHArCNHCHCH ₂ Zy _n

wherein all symbols are as defined above.

The polyiodothyronine analog is used in an active or activated form, making it capable is, with available reactive non-oxo-carbonyl groups from ENZ, e.g. B. amino and hydroxyl groups, too react.

ίο For the carboxy group, a mixed anhydride, an N-hydroxysuccinimide, a p-nitrophenyl, a phenylthio etc. ester derivative or a carbodiimide is used as the coupling agent, while the imidate esters and the isothiocyanates are used directly for conjugation with ENZ. The reaction is conducted at moderate temperatures, which are generally in the range of about -5 to 3O ⁰ C, carried out with a mixed aqueous buffer medium is used, which has usually a mild alkaline pH value in the range of about 7 to 10 degrees. As an auxiliary solvent, for. B. hexamethylphosphoramide can be used. This is usually used in amounts of from about 10 to 40%, preferably from about 20 to% by volume.

The molar ratio between the T-3 or T-4 analogs to the ENZ molecules is generally between about 1: 1 to 20: 1, usually between about 3: 1 to 10: 1.

The T-3 or T-4 analog can be added in multiple portions or all at once, and the reaction time is generally between about 1 minute and about 48 hours. The reaction mixture can then be converted into The usual way to be worked up. Preferably the mixture is chromatographed, but not everything to separate converted analogs from the ENZ-bound T-3 or T-4. A convenient chromatographic Material is Sephadex. One can isolate the fractions, determine their enzyme activity and the fractions collect with enzyme activity.

Instead of the analogue obtained by combining the polyiodothyronine with a di-non-oxo compound is formed, one can conjugate desamino-T-3 or -T-4 with ENZ. In the case of polyiodothyronine, the amino group must protected v / ground when ENZ is conjugated with the amino acid, while with desaminopolyiodothyronine no protecting group is required and the carboxy group in the same way as the others Carboxy groups can be activated.

The desaminopolyiodothyronine or its monocyclic analog are given by those given below Formula defined:

HO ₂ CCH ₂ (CH ₂ ), Z

wherein Z has the meaning given above and f is 0 or 1;

the ENZ conjugate of these compounds has the formula given below:

ENZ- (COCH ₂ (CH ₂ ), Z) _n .

wherein all symbols are as defined above.

The ENZ-bound T-3 or -T-4 is usually at least about 50% deactivated (compared to of the enzyme activity prior to conjugation) usually at least about 65% and no more than about 99.5%. The enzyme should expediently be almost completely deactivated, and its activity should be upon addition of excess Anti-T-3 or T-4 can be almost completely restored. However, it was found that the conjugated enzyme binds to the receptor up to between about 5 to 60% of the original Activity of the unconjugated enzyme, usually between about 10 to 50% of the original activity of the unconjugated enzyme is activated. First and foremost, it depends on the range of the units measured between ENZ-bound-T-3 or -T-4 in the absence of the antibody or in the presence of the excess Antibody. An excess is understood to mean that sufficient antibodies are present to to tie virtually any available T-3 or T-4.

The appropriate antibodies are obtained by injecting a T-3 or T-4 bound antigen into a Vertebrate, usually a domestic animal, e.g. B. a sheep, a rabbit or a goat generated. Because the antigen is usually a polypeptide or protein, usually having a molecular weight of about 5,000-10 Has millions. the conjugated antigen is obtained by combining a T-3 or T-4 analog with the antigenic Polypeptide or protein formed It can either be the same or a different analog as it is for the Conjugation with ENZ is needed. In addition to the analogs used to produce the Conjugates are used, other analogs can also be used. Furthermore, thyroglobulin can can be used to produce the antibodies.

Depending on the analog used, various methods known from the literature can be used for the preparation the antigens are used. The antigens have at least one analog molecule, preferably one Molecule to about 2,000 to about 50,000 molecular weight units of the antigen. The relationship between the T-3 or T-4 analogs and the molecular weight increases with decreasing molecular weight of the antigen to.

Injection of the antigen into the animal is done in the usual manner, although it may be advantageous to Use Freund's complete adjuvant with the main injection.

A wide variety of proteins can be used as antigens, e.g. albumins, globulins, Keyhole limpet hemocyanin and the like

ι | A wide variety of buffers can be used, e.g. B. phosphate, carbonate, glycinate, tris and

I have similar buffers. Phosphate buffers should not be used in high concentrations, preferably in concentrations less than about 0.1 molar. Certain buffers are preferable, for example Glycinate or triethanolamine buffer.

Depending on the course of the reaction, the substrates for MDH are malate and NAD or oxaloacetate and NADH. For triosephosphate isomerase (TIM) the enzymatic reaction of the enzyme with a second enzyme coupled so that a spectrophotometric determination is possible. So the analysis medium contains that Substrate for TIM, glyceraldehyde phosphate, NADH and «-glycerophosphate dehydrogenase. The latter Enzyme and NADH are used in considerable excess so that they are not rate-limiting Substances are.

A small amount of ethylenediaminetetraacetic acid is expediently added to encourage bacterial growth reduce and bind heavy metals. A protein and glycerine can also be added to the analysis medium can be added to improve enzyme stability. Other stabilizers such as dithioerythritol or others Antioxidants can also be added. Furthermore, small amounts of sodium azide can be used as a preservative can be added.

The analysis method is explained in detail in the patent application P 26 60 911.0-52, which has been dropped out

The following examples serve only to illustrate the invention. All temperatures are in ⁰ C, unless otherwise stated. All percentages relate to weight, unless stated otherwise.

20 Example 1

N-methyl, N-carboxymethylglycyl-thyroxine-methyl ester (T-4-MEMIDA)

In a 25 ml flask equipped with a stirrer and a membrane stopper, 1.054 g (1.27 10 ^-3 mol) of the methyl ester of thyroxine hydrochloride was placed. The thyroxine ester hydrochloride was dissolved in 8 ml of dimethylformamide (DMF) under an argon atmosphere, followed by the addition of 10 ml of dry tetrahydrofuran (THF); finally 253 μΐ dry triethylamine were added.

The mixture was stirred for 15 minutes, after which 0.279 g (2.16 × 10 ^-3 mol) of N-methyliminodiacetic anhydride in 2.5 ml of dry THF were added all at once. The reaction obviously started immediately. The volatiles were removed in vacuo on a rotary evaporator, leaving a foam-like solid which was dissolved in 25 ml of THF. The THF solution was extracted with a mixture of 30 ml of deionized water and 50 ml of ethyl acetate. After extraction and separation, the aqueous layer was extracted three times with 25 ml of ethyl acetate each time. The organic layers were then combined, extracted once with 50 ml of saturated NaCl solution and then dried with anhydrous magnesium sulfate. The organic layer was suction filtered and the solvent was removed on a rotary evaporator to give a solid white substance which was dissolved in 30 ml of THF became. 35 ml of chloroform was added to the THF, the solution was refluxed and n-heptane was slowly added. The volume of the solution was reduced until permanent turbidity occurred. The solution was brought to room temperature

& then allowed to cool in a freezer to give a white, fluffy product that

washed with 1-hexane and dried under vacuum over phosphorus pentoxide. 1.26 g (75%)

of a white, flaky product.

Example 2

45 conjugation of T-4-MEMIDA with bovine serum albumin (RSA)

In a reaction vessel equipped with a stirrer and glass stopper with membrane were 0.10 g (1.09 χ 10 ~ ⁴ mol) of T-4-MEMI-DA and 0.013 g (1.1 χ 10 ~ ⁴ mol) of N-hydroxysuccinimide charged to the reaction mixture 1 ml of dry THF and then 15 μΐ of dry triethylamine was added under an argon atmosphere

Cooling the mixture in an ice bath to ⁰ ° C was added 0.024 g (1.25 χ 10- ⁴ mol) of l-ethyl-3- (3'-dimethylaminopropyl) carbodiimide hydrochloride (ECDI) in the form of a powder was added The mixture was Stirred for 2 hours at ⁰ ° C. and then for 12 hours at 4 ° C. Then a solution of 0.100 g (1.55χ 10 ~ ⁶ mol) of bovine serum albumin (BSA) in 0.3 ml of sodium bicarbonate carbonate buffer (pH = 9, 4) and the pH was adjusted to 0.5 with 6N sodium hydroxide solution. The RSA solution was cooled to 0 ° C., whereupon the previously prepared T-4-MEMIDA ester solution at a rate of 50 μΐ each Minute was added dropwise with vigorous stirring. The mixture was then stirred ^{at 0 ° C. for 1.3 hours;} then it was slowly stirred at 4 ° for a further 2 days

The solution was then a Sm-hydroxylamine hydrochloride solution made with 6N sodium hydroxide solution was neutralized to a pH of 8.9, added dropwise. After the mixture was stirred at 4 ° for 10 hours, 60 l

it was placed in a dialysis bag and exchanged for two portions of 500 ml each for a day.

a Tris-HCl buffer (0.05 molar, pH = 7.8) dialyzed. The volume of the protein mixture was then with Aquacide II (manufacturer Calciochem) concentrated to 25 ml, whereupon the mixture was subjected to gel filtration chromatography twice was subjected to, in each case freshly packed Sephadex G-15 originally in Tris-HCl buffer (0.1m, pH = 9.0) was swollen was used. The column size was 2.6 x22.2 cm, the flow rate 48 drops per minute, with fractions of 40 drops being collected in each case became; the buffer used for elution was Tris-HCl (0.1 molar, pH = 9.0). The protein fractions were collected and dialyzed against deionized water (5 × 2000 ml) for 3 days. The conjugate solution was then

lyophilized to give 0.15 g of a white, fluffy solid substance which was deposited under vacuum over P2O5 Was dried for 3 days to obtain 0.140 g of conjugate. UV analysis showed that each RSA molecule 22 haptens were bound.

Example 1 3

Carboxymethoxyacetyl-thyroxine-methyl ester (DG MA)

To a solution of 1.65 g of the methyl ester of thyroxine hydrochloride in 80 ml of dry THF and 30 ml Chloroform in a flask protected from light, 300 μΐ triethylamine were injected while the mixture was stirred. Then 255 mg (0.0022 mol) of diglycolic anhydride was added and the mixture was stirred overnight. The solution was then washed three times with water, dried over sodium sulfate and the volatiles were removed in vacuo. The residue was deposited on a 30 g Sephadex LH-20 column purified using a solution of 20% methanol in dichloromethane as the eluant became. The clear fractions were collected, the solvent removed and the residue with water Methanol precipitated, 1.53 g of substance (84%) being obtained.

Example 4
DGMA conjugate with bovine serum albumin

To a solution of 100 mg BSA in 30 ml of aqueous, 8M urea solution of 0 ⁰ C 25 ml of DMF were added slowly, followed by 453 mg (5.9 χ 10 ~ ⁴ mol) were added dropwise DGMA in 5 ml DMF. After completion of the addition, the pH was adjusted the reaction mixture to 4.5, followed by 100 mg (0.0005 mole) of ECDI hydrochloride in proportions of 10 mg were added at half hour intervals held at 0 ⁰ C, stirred solution. After 5 hours, the reaction mixture was adjusted to a pH of 9 and dialyzed against 2 liters of 10% DMF in 4m urea solution (pH = 9). The precipitate was centrifuged off and the supernatant liquid was dialyzed in a Dow Beaker Dialyzer against about 95 liters of a 0.05 m carbonate solution (pH = 9) and then against about 19 liters of ammonia water (pH = 9). Lyophilization yielded 95 mg conjugate which, according to UV analysis, contained 23 DGMA groups per RSA molecule.

Example 5

Methyl methoxycarbimidomethoxyacetyl thyroxinate conjugate to RSA

A. To a suspension of 125 mg (1.1 mol) of cyanomethoxyacetic acid in 1 ml of dichloromethane were 98 μΙ Oxalyl chloride (146 mg, 1.15 mmol) was added. First a drop of dimethylformamide was added to to start the reaction and stir the reaction mixture for 30 minutes until foaming had stopped and the acidic starting material had dissolved. Then the solvent was on a

Rotary evaporator removed, and the remaining yellow oil became a suspension of 827 mg (1 mmol) Methyl thyroxinate hydrochloride in 10 ml of dry tetrahydrofuran containing 280 μΐ triethylamine (2 mmol), given. The reaction was terminated after 2 hours as determined by analytical thin layer chromatography (Silica gel, 25% ethyl ether in chloroform) no more residues of thyroxine ester starting material could be proven. The reaction mixture was poured into water covered with a layer of ethyl acetate, and the aqueous layer was extracted three times with 50 ml of ethyl acetate. The organic layers were combined, washed once with water and once with brine, dried over magnesium sulfate and evaporated in vacuo to give 888 mg (quantitative yield) of a pale yellow foam. Chromatography on Sephadex LH-20 (25 mg, 10% methanol in ethyl acetate) revealed a polar impurity removed. Yield 85%.

so B. A solution of 89 mg of methyl cyanomethoxyacetyl thyroxinate (0.1 mmol) in 1 ml of dry methanol was placed in a dried, nitrogen-purged flask fitted with a serum stopper, brought. Then a solution of sodium methoxide in methanol was added (1.1 base equivalents) and that The reaction mixture was stirred in the dark overnight. After 20 hours, thin layer chromatography showed (25% ethyl ether in dichloromethane, silica gel) a practically complete implementation, with only a few colored impurities were present.

C. A solution of the above imidate (0.1 mmol) in 2 ml of basic methanol (the untreated imidate formation mixture) was added to a solution of 112 mg of bovine serum albumin (-0.1 mmol of lysine) in 5 ml of 0.05 M Tris buffer pH = 8.5 added dropwise. The pH of the reaction mixture was adjusted to 9.5 with 0.05 M hydrochloric acid, and the reaction mixture was stirred in the cold for 24 hours. The mixture was dialyzed against 10 liters of dilute sodium bicarbonate solution and 3 liters of deionized water Analysis showed a conjugation number of about 20, based on an experimentally determined extinction coefficient for thyroxine of 6.2 × 10 ³ M- ¹ Cm- '. Gel filtration with Sephadex G-15 (0.05 m Tris, pH = 9 as eluent) did not result in any change in the conjugation number.

Example 6

(N-Carboxymethyl-S-Aza-S-methylglutaramic acid amide of
Methyl thyroxinate) conjugate to MDH

A. A stirred solution of 0.201 g (0.22 mmol) T-4-MEMIDA, 1 ml dry THF, 25 mg (0.22 mmol) N-hydroxysuccinimide and 30 μΐ dry triethylamine of 0 ° were added to 0.048 g (0.25 mmol) of ECDI. The mixture was stirred at 0 ° for 35 minutes, then at 4 ° for 14 hours

The above solution was added to 0.033 g (0.44 mmol) of glycine in 1.5 ml of deionized water and 0.5 ml of pyridine, which had been adjusted to a pH of 9 with In NaOH, at 0 ^° C. with vigorous stirring added dropwise. After stirring had been continued at 4 ° C. for 36 hours in the dark, the solvent was removed in vacuo and the residue was dissolved in 5 ml of abs. Ethanol dissolved and the ethanol evaporated, the treatment with ethanol being repeated three times to obtain a white foamy solid.

The residue was dissolved in 10 ml of methanol and the solution was dissolved in 15 ml of deionized water poured and extracted three times with 25 ml of ethyl acetate each time. The ethyl acetate layers were combined with 25 ml of saturated sodium chloride solution extracted and dried over MgSÜ4

The volatiles were removed in vacuo and the residue was dissolved in 2 ml of methanol and chromatographed on silica gel

(At ₃ N: CH _{3 OH: CH 2} Cl ₂ -Zl: 10:90).

The product was desorbed with CH3OH / CH2Cl2 in a ratio of 1: 1, the mixture was filtered, the volatile substances were evaporated and the residue was dissolved in 5 ml of THF and filtered again. After the volatile substances had been removed, the residue was taken up in THF and extracted from THF / HCCl ₃ / cyclohexane recrystallized, a yield of 0.046 g (21.6%) being obtained.

B. In a 1-mL vial was added 4.9 mg of radioactive ¹⁴ C labeled N-carboxymethyl-S-aza-S-methylgluta-

ϊ ramic acid amide of methyl thyroxinate, dissolved in 39 μΐ DMF and cooled to 4 ° C. The DM F solution

^ä 51 μΐ of a 0.11-molar ECDI solution in DMF of 4 ° and 10 μΐ of 0.5-molar N-hydrox-

ysuccihimide solution in DMF at 4 ° was added, and the mixture was stirred in the dark overnight.

C. Mitochondrial porcine heart-MDH was dialyzed against 0.05 M carbonate buffer (pH = 9.0) with 4 ml of a 1.31 χ 10 ~ ⁵ molar solution of MDH were obtained. 445 μΐ DMF were added to the solution at a rate of 15 μΐ / min. The ester prepared above was added in proportions of 2 μl, and the enzyme activity was determined after a waiting time of 5 minutes. In a first reaction 5 μΙ ester were used, while in a second reaction 10 μΐ were added, a ratio of thyroxine to MDH of 4.1 and 7.4 being set.

Both reaction mixtures were dialyzed three times against 300 ml of 1 molar K ₂ HPC> 4 solution (pH = 9.8), once against 300 ml of 0.05 molar carbonate buffer (pH = 9.0) and then twice against the phosphate buffer. The two reaction mixtures were then chromatographed at 4 ° on a Sephadex G 50M column (0.9 χ 54 cm) which had been equilibrated with the phosphate buffer, and with the same phosphate buffer at a flow rate of 4 drops / min eluted and collected in fractions of 20 drops each. The active fractions were pooled, the volume adjusted to 5 ml with the phosphate buffer, and a 0.5 ml portion was dialyzed against a 50 ml portion of deionized water at 4 °. The radioactivity was determined of an aliquot and assuming no protein was lost. the number of haptens per protein molecule was 2.0 and 3.0, respectively.

45 Example 7

T-4-MEMIDA-K conjugate to malate dehydrogenase

10 mg (11 μΜοΙ) T-4-MEMIDA and 1.3 mg N-hydroxysuccinimide were dissolved in 250 μΐ dry DMF. The reaction mixture was kept at 0 ° under a nitrogen atmosphere with stirring, whereupon 2.3 mg ECDI were added. The mixture was held at 0 ° until all of the ECD1 had dissolved. the Solution was left to stand at 4 ° overnight.

It becomes a general procedure for preparing the conjugate with greater or lesser hapten numbers indicated what was the amount of T-4-MEMIDA-hydroxysuccinimide ester in relation to MDH depends. To 4 ml of a stirred solution of MDH (from pig hearts, mitochondrial. Miles, 5.0 mg / ml) in Carbonate buffer (pH = -9.2) was added to 1 ml of DMF. The subsequent additions of the T-4 MEMIDA ester were done at intervals of about 60 to 90 minutes, with aliquots removed and checked for enzyme activity were examined. The table below shows the order of addition, the amount of additives, the Time of addition, the ratio between added T-4 and MDH and the observed percentage Deactivation. For the determination of the conjugate numbers, 2 to 20 μΐ of the conjugation mixture were added 0.5 ml in potassium monohydrogen phosphate given at 0 °. To determine the enzyme activities were 2 to 20 μΙ - aliquots of the diluted conjugate with 0.1% rabbit serum albumin in glycine buffer (0.1m, pH = 9.5) to which 100 μΐ 0.108m NAD and 100 μΐ 2m sodium malate solution (pH = 9.5) were added, to 0.8 ml diluted. The speeds were measured between 60 and 120 seconds after being inserted into a Gilford spectrophotometer, Model 300-N measured at 30 °.

Time of addition total volume ¹ ) T-4 / MDK T-4-Konj. ² )%

νοηΤ-4-ester μ! Deactivation

μΐ

10:34 13 4996 0/1 0

11:24 5007 2/1

5003 2/1 30.1

1 ml taken as conjugate c 1.3

11:30 10.5 40Π 4/1

4009 4/1 85.7

1 ml taken as conjugate d 2.8

13:10 8 3001 6/1

13:37 2991 6/1 94.0

1 ml removed as conjugate e 3 7

13:58 5.5 1977 8.1 / 1

14:49 4 1941 9.6 / 1 98.8

1 ml taken as conjugate f 5.6

') Samples were taken periodically to determine enzyme activity, which is not specified, which is the specified total volume affected.

² ) Number of T-4 units bound to MDH

Example 8
T-4-MEMIDA conjugate to triose phosphate isomerase (TIM)

In 500 μΐ dry DMF in a glass tube, 6.0 mg (6.8 μΜοΙ) T-4-MEMIDA and 0.8 mg (7.3 μΜοΙ) N-hydroxysuccinimide given; the tube was purged and covered with dry argon, and that Mixture was cooled in an ice bath. 1.5 mg ECDI was then added to the stirred mixture, and that The tube was flushed with dry argon and stirred until everything dissolved. The tube was wiped dry, placed in a covered plastic container with a desiccant (Drierite®), with Wrapped in foil and left to stand overnight at 4 ° with stirring.

To 1 ml of triosephosphate isomerase (2 mg) in aqueous carbonate buffer (0.1 molar, pH = 9.2) at 4 ° were 0.3 ml of DMF given slowly with a syringe. The ester solution was slowly using in portions was added to a hypodermic syringe and enzyme activity was monitored. As the enzyme to about 42% was deactivated, DMF was added to make a 50 volume percent DMF solution.

The cold reaction mixture was passed through a Sephadex G-25 column (middle column) filled with 0.1M carbunal buffer (pH = 9.2) had been equilibrated, passed through. The column was a 50 cc burette with a diameter of 1.1 cm and a bed volume of about 19 ml. Elution was carried out with a Solution of 60 parts by volume of an aqueous solution of 0.1 M carbonate buffer (pH = 9.2) and 0.3 M ammonium sulfate as well as 40 parts DMF. The fractions, the volume of which varied from about 1 to 4 ml, were collected. the Fractions 5 and 6 were combined (2.4 ml) and first against about 100 ml of aqueous, 20% by volume DMF 0.02m triethanolamine (TEA) (pH = 7.9) and three times against 250 ml of aqueous 0.02 molar TEA (pH = 7.9) each time dialyzed. The ratio of conjugated T-4 to enzyme was approximately 6: 1.

Example 9
Desaminothyroxine conjugai to MDH

9,1xlO- Desaminothyroxin ³ g (1.2 χ 10 ~ ⁵ mol), l, 4xlO- ³ g (1.2 χ 10 ~ ⁵ mol) of NHS and 0.25 ml of dry

DMF were successively filled into a 1 ml reaction vessel. The reaction mixture was on a

Fisbad cooled, whereupon 2.5 χ 10 ~ ³ g (1.3 χ 10- ⁵ mol) ECDI was added under a nitrogen blanket. That

The reaction mixture was stirred at 4 ° overnight. I.

With cooling on an ice bath and with stirring, 0.23 ml of DMF were slowly added to 1.9 10 ^J g (2.8 10- " ^i!

MoI) MDH in 0.83 ml of 0.05m NaHCO ₃ -Na ₂ CO ₃ (pH = 9.0) was added. Then the above ester solution (5.8 μΐ) was added with stirring, and stirring was continued for a further 1 hour while the temperature indicated was maintained. The conjugation mixture was subjected to gel filtration on three |

Sephadex-G-SOM columns with the dimensions 0.9 χ 13 cm subjected, with a desaminothyroxine / MDH conjugate was obtained which was 91% deactivated and which had a hapten number of 2.5 (according to the Had iodine analysis). The enzyme activity of the conjugate was activated by 30% when used with anti-T-4 serum was treated.

Example 10
T-4 MEMIDA glycine

A 3 ml Pierce Reacti-Vial® was charged with 0.201 g (2.18 10- "mol) T-4-MEMIDA and 0.025 g (2.17 χ 10-" mol) N-hydroxysuccinimide (NHS). Then, 2 ml of dry THF and 30 μΐ was (2.15 χ 10- ⁴ mol) of dry

Triethylamine was added and the reaction mixture was cooled to 0 ° with the aid of an ice bath. Then, 0.048 g ECDI added in powder form (2,5Ox 10- ⁴ mol) and the reaction mixture was stirred for 35 minutes at 0 ° The Reaktiorisgemisch was then placed in a cold room (2 °) and stirred for 15 hours. I-iii Diinnschichlchroiiiatogrunini the reaction mixture * after 15 hours showed two hedges with Ri values of 0.0b (T-4-MEMIDA) and 0.60 (T-4-MEMIDA-NHS-Ester) on an analytical silica gel, where 10 % Methanol in dichloromethane was used as a rinsing agent. A 25-ml flask with stir bar and membrane plug was charged with 0.033 g (4.39 χ 10 ~ ⁴ mol) of glycine, then with 1.50 ml of distilled water, 0.50 ml of pyridine and 100 μΙ (l, 00x 10- ⁴ Mol) 1.0-n NaOH charged. The reaction mixture was cooled to 0 ° with the aid of an ice bath (2 °) vessel stirred for 36 hours. The solvents were then removed on a rotary evaporator to give an oily solid that smelled of pyridine. This solid substance was taken up in 10 ml of methanol and _{poured into 15 ml of H 2} O, and the solution was extracted twice with 25 ml of ethyl acetate each time. The combined organic layers were extracted once with 25 ml of saturated saline solution and then dried _{over anhydrous MgSO 4.} After filtering, the ethyl acetate was removed on a rotary evaporator leaving a white crystalline solid. The solid material was taken up in 2 ml of methanol and spread out on four preparative silica gel plates for thin-layer chromatography, which were developed in triethylamine / methanol / dichloromethane (2.1: 10: 90). The plates were treated twice, then scraped off and the product desorbed with methanol / dichloromethane (1: 1). The silica gel was filtered off and the filtrate was concentrated to 2 ml in vacuo. A thin-layer chromatogram of the product in THF showed only one spot with an Rr value of 0.50 on a silica gel plate for analytical thin-layer chromatography in triethyamine / methanol / dichloromethane (2.1: 10: 90). The product was recrystallized from THF / chloroform / cyclohexane

lized.

Example 11

T-4-M EM ID A-Glycy Iglycine

In a 3 ml Pierce Reacti-vial was T-4 MEM1DA and 0.025 g (2.17 χ 10- ⁴ mol) of NHS charged 0.202 g (2.20 mol χ 10- ^4). Then 2 ml of THF and 31 μΐ dry triethylamine were added and the reaction mixture was cooled to 0 °. Then 0.051 g (2.66 χ 10 ~ ⁴ mol) ECDI was added and the reaction mixture was stirred (2 °) 8.25 hours in a cold room. A thin-layer chromatogram indicated the formation of the T-4-MEMIDA-NHS ester (R / value 0.63 on an analytical silica gel plate with triethylamine / methanol / dichloromethane in a ratio of 2.1: 10: 90). A 25 ml flask with a stir bar and membrane stopper was filled with 0.058 g (4.39 χ ΙΟ- ⁴ mol) of glycylglycine and then with 1.50 ml of H ₂ O and 0.50 ml of pyridine and 100 μΐ (1.0 χ ΙΟ- ^{4 mol) of glycylglycine} Mol) 1.0 nNaOH charged. The reaction mixture was cooled to 0 ° and the T-4-MEMIDA-NHS ester solution was added dropwise with stirring. After the activated ester was added, 1.0 ml of deionized water and 0.50 ml of pyridine were added. The reaction mixture was stirred in a cold room (2 °) for 93 hours in the same way as T-4-M EMIDA-glycine, whereby 0.018 g (yield 9%) of a brown-golden solid substance was obtained. The product was homogeneous after thin layer chromatography on silica gel with triethylamine / methanol / dichloromethane (2.1: 10: 80); the R / value was 0.81 when the plate was developed twice.

Example 12

45 T-4-MEMIDA-glycine / MDH-K conjugate

I A 1 ml Pierce Reacti vessel with a stir bar was filled with 0.049 g (5, OxIO- ⁶ mol) T-4-MEMIDA and 39 ul §

charged with dry DMF. The reaction mixture was cooled to 0 °, whereupon 10 µ (5.2 10- ^b MoI) of a 5, Ox 10-'m NHS solution in DMFvonO "and 51 µΐ (6.1 χ ΙΟ- ⁶ mol) of a l, 2x 10 -'m ECDI solution in DMF of 0 ° was added; the reaction mixture was stirred in a cold room (2 °) for 49 hours. A thin layer chromatogram of the reaction mixture after 49 hours showed two spots with R 1 values of 0.49 (T-4 -MEMIDA-Glycine) and 0.68 (T-4-MEMIDA-Glycine-NHS-Ester) on analytical silica gel plates developed with triethylamine / methanol / dichloromethane in a ratio of 2.1: 10: 90. MDH (4.0 ml. 1.3 χ 10 ~ ⁵ mol, 0.05m NaHCOa-Na ₂ CCh, pH was 9.2) in a 10 ml round bottom flask with stir bar and membrane plug 55 e

at 0 ° and the enzyme activity was determined in the manner described above. 445 μl dry DMF were added to the reaction mixture at a rate of 15 μl / min until a reaction mixture with 10% DMF was obtained. The enzyme activity was then redetermined. The 4.3 χ 10- ² -molareT-4-MEMIDA-glycine-NHS Esterlösung was added to the reaction mixture in aliquots 1-2 μΙ, and the enzyme activity was determined after each addition. 9 μl of the ester solution activated above resulted in an enzyme conjugate which was 82% deactivated. After the last addition of activated ester, the enzyme reaction mixture was dialyzed exhaustively against 0.1m K ₂ HPO ₄ (IrUt I ₁ Ox 10 " ³ m NaN ₃ ) at 2 °. After the dialysis, the enzyme conjugate was carefully removed from the dialysis bag and passed through two Sephadcx-G-50M columns (in 1.0m K ₂ HPO ₄ , pre-swollen with I ₁ QxIO- ³ M NaN ₃ ) The sizes of the two columns were 0.9x54.0 cm and 0.9 χ 51.0 cm, the flow rates were 4-5 drops per minute, and 20 drop fractions were collected, the protein fractions were concentrated using a collodion bag device in a cold room, and the hapten number was found to be 3.0.

Example 13
T-4 MEMIDA glycylglycine / MDH conjugate

0.015 g (7.8 χ 10 ~ ⁵ mol) ECDI was dissolved in 0.50 ml of dry DMF in a 1.6 χ lO-'moIaren solution dissolved. 0.75 g (6.5 χ 10- ⁴ mol) of NHS were dissolved in 1.0 ml of dry DMF in a 6.5-χ 10'molaren solution, both solutions were cooled before use in an ice bath to 0 °. A 1 ml Pierce Reacti vessel with a stirring rod was charged with 2.4 × 10 ~ ³ g of T-4-MEMIDA glycylglycine and then with 78 μΐ ice-cold dry DMF (cooled in an ice bath), whereupon 4 μ1 of a 6.5 × 10 'm NHS solution in DMF of 0 ° and 18 μΐ of a 1.6 χ lO-'m ECDI solution in DMF of 0 ° were added.

The reaction mixture was stirred in a cold room (2 °) for 20 hours. Thereafter, a thin-layer chromatogram of the reaction mixture showed two spots with Rr values of 0.25 (T-4-MEMIDA-glycylglycine) and 0.59 (T-4-MEMIDA-glycylglycine-NHS-ester) on analytical silica gel plates which were in triethylamine / methanol / Dichloromethane in a ratio of 2.1: 10: 90 were developed. 4.0 ml of MDH in a concentration of 1.5 ΙΟ- ⁵ mol in 0.05m NaHCO ₃ -Na ₂ CO ₃ solution (pH = 9.2) were placed in a 10 ml round bottom flask with a stir bar and membrane stopper. The reaction mixture was cooled to 0 ° with an ice bath, whereupon 440 μΐ dry DMF were added at a rate of 50 μΐ. The enzyme activity was determined before and after the addition of the DMF. The χ 1.9 10- ² molar T-4 MEMIDA-Glycylgrycin-NHS was 3-10 Esterlösung μΐ added to the reaction mixture in aliquots was determined after which the enzyme activity after each addition. When 29 μl of activated ester solution was added, an enzyme conjugate which was 82% deactivated was obtained. The reaction mixture was then exhaustively against 1.0 M K ₂ HPO ₄ (1.0 χ 10- ³ M NaN ₃₎ at 2 ° dialyzed After dialysis was the conjugate by three Sephadex G-50M column (pre-swollen in 1, 0m K ₂ HPO ₄ with 1.0x 10 ~ ³ M NaN ₃ ) and eluted with the same buffer. The column dimensions were 0.9 × 55 cm, 0.9 × 56 cm and 0.9 × 56 cm, the flow rates 4 to 5 drops per minute, and 20 drop fractions were collected. The protein fractions were concentrated in a cold room using a collodion bag device. The hapten number was determined to be 3.9 by the method described above.

Using the antigen conjugates, antibodies were produced with which the analytical procedure was carried out. Details are given in the dropped patent application P 26 60 911.0-52.

Claims (10)

Claims: 1. ENZ-bound ligand with the formula ENZ is malate dehydrogenase or triosephosphate isomerase reversibly inhibited by the ligand; a is 0 or 1; π averages at least 1 and not more than 10; X and Y, which are the same or different from one another, are 0 or S or imino; D is hydrogen or an alkyl group having 1 to 6 carbon atoms; Z is 3-iodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy-1') -phenyI-1 or 3,5-diiodo-4- (3'-iodo- or 3 ', 5' -diiodine-4'-hydro- xyphenoxy-1 ') - phenyl-1; and A is a bond or an organic divalent group having 1 to 12 carbon atoms and 0 to 6 Heteroatoms, namely oxygen, sulfur or nitrogen, the total number of heterofunctionalities ten is between 0 and 4. 2. ENZ-bound ligand according to claim 1, characterized in that ENZ is malate dehydrogenase reversibly inhibited by the ligand, η 1 to 8, D is an alkyl group with 1 to 3 carbon atoms, Z 3,5-diiodo-4- (3 ' , 5'-diiodo-4'-hydroxyphenoxy-l ') - phenyI-l and A denotes an aliphatic organic divalent radical having 1 to 10 carbon atoms and 0 to 2 heteroatoms in the chain between the non-oxo-carbonyl groups, the heteroatoms Oxygen and nitrogen are present as oxy, tert-amino or amido groups and where a total of 0 to 4 heteroatoms, namely oxygen or nitrogen, are present. 3. ENZ-bound ligand according to claim 1, characterized in that π an average of 1 to 8, X and Y 0 or S, D is an alkyl group with 1 to 6 carbon atoms, Z 3,5-diiodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy-1 ') -phenyl-1 and A is an aromatic group with 6 to 8 carbon atoms and 0 to 4 heteroatoms, namely oxygen, nitrogen and sulfur. 4. MDH-bound ligand, characterized by the formula MDH- Y ¹ CO ₂ D Il I _? —V _r —C-NHCHCH ₂ ZJ _n where the symbols have the following meanings: MDH is malate dehydrogenase; / J ¹ ranges from 1 to 8; X ¹ and Y ', which are the same or different from one another, are oxygen, sulfur or imino; ρ, q and r are each 0 or 1; sist0bis2; oc and / are hydrocarbon groups having 1 to 8 carbon atoms, /? is an oxy, thio, sulfonyl or alkylamino group, the alkyl group containing 1 to 3 carbon atoms, with the proviso that /? a Is amino or alkylamino when ρ is 0; Jist Il - CHNHC - T ¹ wherein T 'is hydrogen or a lower alkyl group having 1 to 3 carbon atoms; Z is 3,5-diiodo-4- (3,5'-diiodo-4'-hydroxyphenoxy-1 ') -phenyl-1 and D is hydrogen or an alkyl group with 1 to 6 carbon atoms. 5. MDH-bound ligand according to claim 4, characterized in that 5 = 0, /? An alkylamino group, ac and γ 1 to 3 carbon atoms and p, q and r each mean 1. 6. MDH-bound ligand according to claim 4, characterized in that β is an oxy group, oc and γ 1 to 3 carbon atoms, s = 0 and pq and r are each 1. 7. Miiliitdchydrogcnasc-gcbundcnes-Polyiodothyronine with 3 to 4 | od groups per thyronine and 1 to 10 Thyronine groups linked to malate dehydrogenase. 8. malate dehydrogenase-bound polyiodothyronine according to claim 7, characterized in that the Polyiodothyronine is thyroxine and has 2 to 6 thyroxine groups attached to malate dehydrogenase. 9. malate dehydrogenase-bound polyiodothyronine according to claim 7, characterized in that it is reversibly inhibited by the polyiodothyronine and 1 to 10 polyiodothyronines of the formula DO ₂ CCHCH ₂ Z
NH ₂ contains attached to the amino group by a non-oxo-carbonyl group, including its nitrogen and Sulfur analogs, bound to malate dehydrogenase, in which the symbols have the following meanings: D is hydrogen or an alkyl group having 1 to 6 carbon atoms; and Z is 3-iodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy-1') -phenyl-1 or 3,5-diiodo-4- (3'-iodo-or 3 ', 5 '-diiodo-4'-hydroxyphenoxy-1') - phenyl-1. 10. malate dehydrogenase-bound polyiodothyronine according to claim 7, characterized in that it is reversibly inhibited by the polyiodothyronine and the formula