DE2660911C2 - Method for the detection or determination of a ligand - Google Patents

Description

are brought together, characterized by

that ENZ means malate dehydrogenase or triosephosphate isomerase, which is reversible by the ligand are inhibited;

that the antiligen is present in such a concentration that a substantial increase in the Enzyme activity occurs in the absence of the ligand; and

that in the zone mentioned the effect of the medium on the enzymatic activity of the ENZ-bound Ligands studied

2. The method according to claim 1, characterized in that there is used as ligand thyroxine

3. The method according to claim 1 or 2, characterized in that the aqueous liquid zone at a pH in the range of about 7 to 10.5, and that the ENZ-bound ligand is less than about 50% of the enzyme activity of ENZ before conjugation to form the ENZ-bound ligand, however has no less than about 0.5% of the enzyme activity of the ENZ prior to conjugation.

4. The method according to claim 3, characterized in that when using malate dehydrogenase the enzymatic activity by adding NAD and malate to the aqueous liquid zone at one pH determined in the range from 8.5 to 10.5

5. The method according to claim 3, characterized in that when using triosephosphate isomerase the enzymatic activity by adding glyceraldehyde-3-phosphate, Λ-glycerophosphate dehydrogenase and NADH to the aqueous liquid zone at a pH in the range of 7 to 9 is determined.

6. The method according to claim 1, characterized in that the medium and the antiligand before the addition of the MDH-bound ligand combined.

7. The method according to claim 1, characterized in that the MDH-bound ligand is about 1 to 10 Ligands each MDH has, the MDH is mitochondrial MDH and up to about 65-99.5% of the enzyme aclivity of the MDH is deactivated prior to conjugation to form the MDH-bound ligand.

8. The method according to claim 1, characterized in that the MDH in the MDH-bound ligand from mitochondrial MDH is obtained.

The invention relates to a method for detecting or determining a ligand which is a Polyiodothyronine represents with 3 to 4 iodine atoms.

The concentration of thyroxine (tetraiodothyronine; T-4) in the blood is within relatively narrow limits critical for the proper functioning of the body. The thyroxine concentration is extremely low and can can only be detected by very sensitive methods.

The triiodothyronines (T-3) are also an important factor for the proper functioning of the body. the Triiodothyronines differ from thyroxine in that iodine is absent in the 5- or 5'-position. One takes suggest that the (T-3) noticeably 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 radioimmunoanalysis, 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 Pat. No. 38 17 837, an enzyme analysis (enzyme assay) is described which is generally used for has found a wide variety of ligands useful.

In this process, inter alia. Polyiodothyronines with 3 to 4 iodine atoms as ligands are determined, whereby to a sample containing the ligand, a receptor for the ligand and a conjugate of the ligand that is attached to a Enzyme is bound, is given. The enzyme-bound (ENZ-bound) ligand then joins the ligand of the sample for the ligand receptor (antiligand) in competition, so that the amount of antiligand that binds to the ENZ-bound ligand in the presence of the ligand to be determined, is less than in Absence of the ligand to be determined. Since this binding of the antiligand to the ENZ-bound

Ligand has a measurable influence on the enzymatic activity of the ENZ-bound ligand, the Ligand can be detected or determined in the sample.

However, this publication does not contain any suggestions as to which enzymes should be used for the ENZ-bound ligand are to be selected so that the new process principle underlying the invention, according to which the Inhibits enzyme activity by binding to the ligand and binding the antiligand to the ligand is recovered, was not apparent.

The invention thus relates to a method for the detection or determination of a ligand, which is a polyiodothyronine with 3 to 4 iodine atoms, being in an aqueous liquid zone

(1) a medium suspected of containing the ligand;

(2) a soluble ENZ-linked ligand; and

(3) an antiligen with sites that are common to the ligand and the ENZ-bound ligand and which can bind to them,

be brought together.

The procedure is characterized by

that ENZ means malate dehydrogenase or triosephosphate isomerase, which is reversible by the ligand inhibit. ' are;

that the antiligen is present in such a concentration that a substantial increase in the Enzyme activity occurs in the absence of the ligand; and that in said zone the effect of the medium on the enzymatic activity of the ENZ-bound Ligands studied.

The enzyme in the ENZ-bound ligand is after conjugation with the polyiodothyronine to more than 50% deactivated and becomes partially after the binding of the receptor or antiligand for the polyiodothyronine or fully reactivated. The conjugated enzyme used therefore has a low turnover rate, which increases considerably in the presence of a receptor such as an antibody to polyiodothyronine.

The process according to the invention is generally carried out in such a way that the aqueous liquid zone buffers at a pH of about 7 to 10.5, with the ENZ-bound ligand being less than about 50% of the Enzyme activity of ENZ prior to conjugation to form the ENZ-bound ligand, but not has less than about 0.5% of the enzyme activity of the ENZ prior to conjugation.

The serum sample to be determined, the antiligand, the ENZ-bound polyiodothyronine and the enzyme substrate are combined, and the rate of the reaction catalyzed by the enzyme is determined. Optionally, one or more incubation periods can be added between the addition of the individual reagents are interposed. By using standards with known amounts of T-3, reciprocal T-3 or T-4 produces a calibration curve with which the samples to be determined in Relationship can be set.

In the case of the ENZ-bound polyiodothyronines, there is on average at least one polyiodothyronine over one aliphatic carbon atom or a non-oxo-carbonyl group (including nitrogen and its thioiinalogues) bound to the enzyme, an amide (amidine or thioamide) and / or an ester being formed.

The method according to the invention can be used for the determination of polyiodothyronines in concentrations of only 10 ~ ⁷ molar or below, the ENZ-bound ligands being reacted with the antiligand (antibodies). In homogeneous enzyme immunoanalysis, the reagents used are the ENZ-bound ligands 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 spectropholometric. By comparing the result obtained with the unknown substance with that under Using known amounts of polyiodothyronine contained results one can determine the amount of the Determine polyiodothyronine in the unknown sample.

The reagents are first described below, followed by the actual analytical procedure.

Reagents
ENZ-bound ligand (polyiodothyronine)

In the production of ENZ-bound polyiodothyronine you can use the polyiodothyronine directly, wherein the carboxyl group attached to available groups of the enzyme, e.g. B. Lysine and Tyrosine bound is to be activated, or preferably from the available functionalities, e.g. B. the amino group or Carboxy group, especially the amino group, creates an extended chain. The phenolic hydroxyl group is not used as a conjugation site. The ENZ-bound ligand that is the subject of patent 26 05 162 has the formula

'X \ Y CO ₂ D

Il I I

> C-A / "- CNH-CHCH ₂ Z
where the symbols have the following meanings:

ΓΜ7 ι η il Γκιυ PUPU7

ENZ is malate dehydrogenase or triose phosphate isomerase reversibly inhibited by the ligand;
a is 0 or 3;

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 3-iodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy-r) -phenyl-1 or 3,5-diiodo-4- (3'-iodo- or 3', 5'-diiodo- 4'-hy-

droxyphenoxy-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 heterofunction-ο between 0 and 4

If the enzyme used is malaf dehydrogenase (MDH), the mitochondrial MDH is preferred, in particular from pig hearts, if the triosephosphate isomerase (TIM) is used, this is how one uses 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 can be a hydrocarbylene group (an aliphatic, alicyclic, aromatic or a combination of these Groups) or a non-hydrocarbyte group (a substituted aliphatic, alicyclic, aromatic heterocyclic or a combination of these groups), with generally 0 to 1 cyclic groups in the Chain are present, usually 5 to 6 ring links with 0 to 2 hetero ring links, usually 0 to 1 Hetero ring members, where the substituents of the cyclic group are usually through 2 to 4, usually are separated from one another by 3 to 4 ring members; usually the hydrocarbylene group is the only one 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-I C - ö _s - a _p - A, _{- y r} - C-NHCHCH ₂ Z

where the symbols have the following meanings:

MDH is malate dehydrogenase;

n ¹ is in the range from 1 to 8, in particular between 2 and 6;

X ¹ and Y ¹ , which are the same or different from one another, are oxygen, sulfur or imino;

ρ, q and r are each 0 or 1, the sum of p, q and r preferably being at least = 1 (p + q + r> 1);

5 is 0 to 2, usually 0 to 1;

χ 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 up to 6 carbon atoms are present;

for ρ or r = 0, χ or γ preferably contain 1 to 8 carbon atoms, in particular 1 to b carbon atoms;

χ and γ can be aliphatic, alicyclic or heterocyclic; together with β and ö they can satisfy the definition of A;

are preferably χ and / saturated aliphatic groups, especially unbranched groups, e.g. B. methylene or polyrethylene groups or aromatic, especially monocyclic groups, only one of α and γ being an aromatic group;

β is an oxy, thio, sulfonyl or alkylamino group, the alkyl group containing I to b, preferably 1 to 3 carbon atoms, with the proviso that / is an amino or alkylamino group when p is 0;
"is

—CHNHC -

I Il
τ ¹ ο

wherein T ^{1 is} hydrogen or a lower alkyl group having 1 to 3 carbon atoms;
b5 Z is 3,5-diiodo-4- (3.5'-diiodo-4'-hydroxyphenoxy-r) -phenyl-1 and

D is hydrogen or an alkyl group having 1 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 having 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 / X \

IV

(CY) -1 rc Ae

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 with

0 to 1 ethylenically unsaturated sites and 0 to 1 heteroatoms with the atomic number 7 to 8, wherein the heteroatoms are completely substituted by carbon atoms and 1 to 6 carbon atoms available; and

E is hydroxyl or can form a double bond together with a terminal nitrogen atom of R form, e.g. B. isocyanate or isothiocyanate.

Another useful series of compounds 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 aromatic ring. In most cases, these compounds are as shown below formula

Y ² CO ₂ D

Il I

X ² - C-NArCNHCHCH ₂ Z

where the symbols have the following meanings:

D and Z are as defined above;

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

Ar is an arylene or aralkylene group including carbocyclic and heterocyclic groups with 5 to 6 ring members, with 1 to 2, preferably 1 hetero-ring member, namely O, N or S are present and wherein 4 to 10 carbon atoms, usually 4 to 8 carbon atoms, and when it comes to heterocyclic compounds, preferably 4 to 6 carbon atoms, and when they are carbocyclic compounds, preferably 6 to 8 carbon atoms are present.

The ENZ conjugate of the series of compounds given above has the formula

55 ENZ-IC-NHArCNHCHCH ₂ ZA ₁

X ² Y ² CO ₂ D \

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.

For the carboxy group, a mixed anhydride, an N-hydroxysuccinimide, a p-nitrophenyl, is used Phenylthio, etc. ester derivative or a carbodiimide is used as a coupling agent during the imidate ester and the isothiocyanates can be used directly for conjugation with ENZ. The response will be moderate Temperatures generally in the range of about -5 to 30 ° C, performed, with a mixed aqueous buffer medium is used, which usually has a mild alkaline pH in the range of about 7 to 10 has. Hexamethylphosphoramide, for example, can be used as an auxiliary solvent. This is

usually used in amounts of about 10 to 40%, preferably of 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.

ίο Instead of the analogue that is created 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 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 J ₁ Z
wherein

Z has the meaning given above and
t 0 or list;

the ENZ conjugate of these compounds has the formula given below:
ENZ- (COCH ₂ (CH ₂ ), Z) "1

wherein all symbols are as defined above.

The ENZ-bound T-3 or T-4 is normally at least about 50% deactivated (compared to Enzyme activity prior to conjugation) usually at least about 65% and no more than about 99.5%. Appropriate the enzyme should be almost completely deactivated, and its activity should be with the addition of excess Anti-T-3 or T-4 can be almost completely restored. However, it has been found that the conjugated enzyme upon binding to the receptor 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 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 there are enough antibodies to practically tie 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. A sheep, rabbit or goat, Da produces the antigen is usually a polypeptide or protein, usually having a molecular weight of about 5000 to 10 million, the conjugated antigen is made by combining a T-3 or T-4 analog with the antigenic polypeptide or protein. 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 decreases 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. B. albumins, globulins, Keyhole limpet hemocyanin and the like

A wide variety of buffers can be used, e.g. B. phosphate, carbonate, glycinate and Tris 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 M DH are malate and NAD or oxaloacetate and NADH. Tnose phosphate isomerase (TIM) is the enzymatic reaction of the enzyme with a second enzyme coupled so that a spectrophotometric determination is possible. The analysis medium therefore contains the Substrate for TIM, glyceraldehyde phosphate NADH and «-glycerophosphate dehydrogenase. The last

called enzyme and NADH are used in a considerable excess, so that they are not the rate-limiting Substances are.

Appropriately, a small amount of ethylenediaminetetraacetic acid is added to allow the bacteria to grow 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, the dithioerythritol or others Antioxidants can also be added. Furthermore, small amounts of sodium azide can be used as a preservative can be added.

Analytical method

Reagent solutions

The buffer solution used normally has such a concentration that one in the analysis medium Concentration from about 0.001 to 0.5 molar, usually from about 0.01 to 0.2 molar, and preferably from about 0.05 to 0.15 molar is obtained. The added protein, which is suitably an albumin such as rabbit serum albumin and / or gelatin is in the finished analysis mixture generally in amounts of about 0.005 to 0.5% by weight, usually in amounts of about 0.01 to 0.2% by weight. Glycerine can be used in amounts from 0.1 to 5, usually present in amounts from 0.4 to 4 percent by weight. The pH of the solution is generally between about 6.0 and 10.5, usually between about 7 and 10.5, suitably between about 8 and 10, and preferably between 8.5 and 10.5 when using NAD and malate or NADH and oxaloacetate; he preferably lies between 7 and 9 when glyceraldehyde phosphate is used.

The concentration of ENZ in the analysis medium can fluctuate within wide ranges; However, it is generally in the range of about 10 ^-5 to IO ¹² molar, usually from about 10 ^-7 to 10 "molar. The antibody concentration depends on the ratio between the antibody-binding sites, and the concentration of the bound ENZ Polyjodthyronine from In general, the ratio of binding sites to T-3 or T-4 (as ENZ-bound T-3 or T-4) is at least 0.5 and not more than 1000, usually about 1 to 100 and suitably about 1 to 25 A sufficient amount of antibody is usually added to recover 25 to 75, preferably 25 to 50 percent of the recoverable enzyme activity.

With decreasing binding constants, larger amounts of antibodies are required. The specific amount the antibody used in a particular analysis is usually determined empirically.

Other additives which may optionally be added in minor amounts are Äthylendiamintetracssigsäure which .- in amounts of about 0.001 to 0.1 wt% may be present, which have to IO ³ molar sodium azide present in amounts of about 10 ^-5 and a surfactant such as Triton X-100 which can be present in amounts from about 0.0001 to 0.03 weight percent.

If MDH is used, either malate or NAD or, depending on the direction of the reaction, are used Oxaloacelate and NADH are used, the first-mentioned substances being preferred. The Concentration These substances directly affect the sensitivity of the analysis and must be determined empirically be the difference in reaction rates in the presence and in the absence of the added To optimize the antibody. The concentration of malate and oxaloacetate is generally about 0.01 to 0.5 molar, in particular about 0.05 to 0.3 molar. The concentration of NAD or NADH is generally about 0.01 to 0.05 molar, usually about 0.005 to about 0.2 molar. Usually the concentrations are on Enzyme, antibodies and substrates are chosen so that the sensitivity of the analysis is optimized.

TlM is used, the glyceraldehyde is generally molar in a concentration ranging from about 10 ~ to 10 ~ ^4; "-Glycerophosphatdehydrogenase is generally used in amounts of about 10 - used ⁵ to IO ⁹ molar, while the NADH concentrations generally between about 10 ^-2 and IO is ⁴ molar.

The individual reagents can expediently be added in the form of aqueous solutions. Normally a large proportion of the total analysis sample is the buffer solution.

The stages of analysis

The order in which the individual reagents are combined is not critical, although there are some orders are preferred and in some cases one order is preferred over another. An incubation can be carried out after adding a certain reagent or after adding all reagents will. Usually the analysis mixture is no longer available before the start of the speed measurement incubated for 10 minutes in the presence of the enzyme substrates.

All reagents can be added at the same time. But it can also be the sample, e.g. B. serum, buffer solution and antibodies are combined, optionally followed by incubation. In most cases the antibody and the conjugated ENZ will not be conjugated prior to the addition of the sample. But if a monovalent antibody is used to avoid precipitin formation, so it can be for certain Applications may be desirable to store a mixture of antibody and enzyme.

Depending on the order of addition, the events proceed differently. Will the sample and the Antibodies are combined, so the polyiodothyronine is bound to the antibody, using the available sites to bind to the ENZ-bound T-3 or T-4. The concentration of available Binding sites is therefore a function of the amount of T-3 or T-4 in the sample. If the ENZ-bound T-3 or T-4 is added, so is the amount of antibody that will be with the conjugated T-3 or T-4 connects, a function of T-3 or T-4 in the sample. This way of working is less sensitive to it Differences in the binding constants between T-3 or T-4 and ENZ-bound T-3 or T-4.

But you can also combine the sample, the appropriate antibody and the conjugated ENZ and that free T-3 or T-4 with the T-3 or T-4 conjugated to ENZ with respect to the available binding sites in Let competition arise.

If necessary, the analysis mixture can be incubated after each addition of a reagent, either before or after the addition of the enzyme substrates or other reagents. Incubation can take about 2 minutes

; ' to 1 hour and is generally at temperatures in the range from about 15 to 40 ⁰ C, usually at

; -, carried out around 25 ° C (room temperature) to 37 ° C.

, & After adding the mixture to the substrates for the enzyme, the mixture can be used up to about 10 minutes before the

; γ initial reading to be incubated; this reading can be done with either a flow cell or with a

. ■ 5 cuvette can be made. The speed is at a temperature in the range of about 20 to

·; 40 ⁰ C, usually at about 25 to 37 ° C, determined. Generally, the readings are from about 0.1 to 45 times

[ ^l:. Minutes, usually about 0.5 to 2 minutes if a flow cell is used, and about 5 to 45 minutes

41 minutes when using a cuvette. Measurement periods of about 5 to 20 minutes are for certain

T ₁ ; automated instruments suitable.

: 'ίο If one uses standards with known amounts of T-3 or T-4 in the serum, one can create a calibration curve

?; that can be used to determine T-3 or T-4 in an unknown sample.

|! i Although spectrophotometric methods are most convenient for determining the course of the enzyme reaction

W; (i.e. by determining the absorption spectrum), other methods can also be used,

p. z. B. fluorimetry, titrimetry, etc.

I 15 The following examples serve only to illustrate the invention. All temperatures are in ⁰ C if

II nothing else is indicated. All percentages are by weight unless otherwise $ angege- is Ben.

1 B e i s ρ i e 1 1

|| 20 N-methyl, N-carboxymethylglycyl-thyroxine-methyl ester (T-4-MEMIDA)

If a 25 ml flask equipped with a stirrer and membrane stopper was added 1.054 g (1.27 10 ^-3 mol) of the methyl ester

H introduced by thyroxine hydrochloride. The thyroxine ester hydrochloride was under an argon atmosphere

Ί dissolved in 8 ml of dimethylformamide (DMF), after which 10 ml of dry tetrahydrofuran (THF) were added;

f25 Finally, 253 μΐ dry triethylamine were added.

_s , The mixture was stirred for 15 minutes, whereupon 0.279 g (2.16 χ 10 ^-3 mol) of N-Methyliminodiacigsäureanh-

|) hydride in 2.5 ml of dry THF was added all at once. The reaction obviously started immediately. the

I volatiles were removed under vacuum in a rotary evaporator, leaving a foam-like solid

I substance remained, which was dissolved in 25 ml of THF. The THF solution was with a mixture of 30 ml

It extracted 30 deionized water and 50 ml of ethyl acetate. After extraction and separation, the aqueous

f: Layer extracted three times with 25 ml of ethylene acetate each time. The organic layers were then combined, once

| » extracted with 50 ml of saturated NaCl solution and then dried with anhydrous magnesium sulfate. To

ίί The organic layer was filtered off with suction, the solvent was removed in a rotary evaporator, whereby

¹ 'f: a solid white substance was obtained, which was dissolved in 30 ml of THF. 35 ml of chloroform were added to the THF

; _r 35 was added, the solution was refluxed and n-heptane was slowly added. The volume of the

• The solution was reduced until permanent turbidity occurred. The solution was at room temperature

: {and then allowed to cool in a freezer to give a white fluffy product which

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

? of a white, flaky product.

¹ B example 2

; Conjugation of T-4-MEMIDA with Bovine Serum Albumin (RSA)

; 0.10 g (1.09 χ 10- ⁴ mol) of T-4-

; 45 MEMIDA and 0.013 g (1.1 χ 10 ~ ⁴ mol) of N-hydroxysuccinimide were charged. The reaction mixture was under

;. 1 ml of dry THF and then 15 μΐ of dry triethylamine were added to an argon atmosphere

'■' after cooling the mixture in an ice bath to 0 ° C, 0.024 g (1.25 10 ~ ⁴ mol) of l-ethyl-3- (3'-dimethy-

£ laminopropylj-carbodiimide hydrochloride (ECDI) was added in the form of a powder. The mixture was 2 hours

S the at 0 ⁰ C and then stirred for 12 hours at 4 ° C Then, a solution of 0.100 g

ί 50 (1.55 χ 10- ⁶ mol) bovine serum albumin (RSA) in 0.3 ml sodium bicarbonate carbonate buffer (pH = 9.4)

I 'and the pH was adjusted to 0.5 with 6N sodium hydroxide solution. The BSA solution was set to 0 ^° C

j cooled, whereupon the previously prepared T-4-MEMIDA ester solution was added dropwise at a rate of 50 μl per minute while stirring vigorously. The mixture was then stirred ^{at 0 ° C. for 1.3 hours;} then it was slowly stirred at 4 ° for a further 2 days

55 The solution was then a Sm-hydroxylamine hydrochloride solution made up with 6N sodium hydroxide solution a pH of 8.9 was neutralized, was added dropwise. After the mixture had been stirred at 4 ° for 10 hours was, it was placed in a dialysis bag and for a day in exchange for two portions of 500 ml each Tris-HCl buffer (0.05 molar, pH = 7.8) dialyzed. The volume of the protein mixture was then dialyzed with Aquacide II (manufacturer Calciochem) concentrated to 25 ml, whereupon the mixture was subjected to a gel filtration chromatography twice

60 graph, in each case freshly packed Sephadex G-15 originally in Tris-HCl buffer (0.1 m, pH = 9.0) was used. The Koionnen size was 2.6 22.2 cm, the Flow rate 48 drops per minute, with fractions of 40 drops being collected in each case; 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

65 siert, whereby 0.15 g of a white, fluffy solid substance were obtained, which under vacuum over P2O5 3 days

I was dried to give 0.140 g of conjugate. The UV analysis showed that each RSA Molckül

; 22 haptens were bound.

Example 3
Carboxymethoxyacetyl-throxine- M ethyl ester (DG MA)

W 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 mole) diglyic 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 were slowly added 25 ml of DMF was added, followed by 453 mg (5.9 χ 10 ~ ⁴ mol) were added dropwise DGMA in 5 ml DMF. After the addition was complete, the pH of the reaction mixture was adjusted to 4.5, whereupon 100 mg (0.0005 mol) of ECDI- ^

Hydrochloride in portions of 10 mg every half hour of the stirred solution kept at 0 ° C ΐ

were added. After 5 hours, the reaction mixture was adjusted to a pH of 9 and against Liters of 10% DMF in 4m urea solution (pH = 9) dialyzed. The precipitate was centrifuged off and the supernatant liquid in a Dow-Beaker dialyzer against about 95 liters of a 0.05 m carbonate solution (pH = 9) and then dialyzed against about 19 liters of ammonia water (pH = 9) The lyophilization resulted in 95 mg conjugate which, according to the UV analysis, contained 23 DGM Α groups per RSA molecule.

Example 5
Methyl methoxycarbimidorethoxyacetyl thyroxinate conjugate to RSA

A) 98 μΐ were added to a suspension of 125 mg (1.1 mol) of cyanomethoxyacetic acid in 1 ml of dichloromethane Oxalyl chloride (146 mg, 1.15 mmol) was added. First a drop of dimethylformamide was added, to start the reaction and the reaction mixture was stirred for 30 minutes until the Foaming had stopped and the acidic starting material had dissolved. Then the solvent became removed on a rotary evaporator, and the remaining yellow oil became a suspension of 827 mg (1 mmol) of methyl thyroxinate hydrochloride in 10 ml of dry tetrahydrofuran, the 280 μΐ triethylamine (2 mmol) was given. The reaction was stopped after 2 hours after going through analytical thin layer chromatography (silica gel, 25% ethyl ether in chloroform) no residues of thyroxine ester starting material more could be proven. The reaction mixture was poured into water covered with ethyl acetate, and the aqueous layer was washed three times with 50 ml of ethyl acetate extracted. The organic layers were combined, once with water and once with brine washed, dried over magnesium sulfate and evaporated in vacuo, yielding 888 mg (quantitative Yield) of a pale yellow foam. By chromatography on Sephadex LH-20 (25 mg, 10% methanol in ethyl acetate) a polar impurity was removed. Yield 85%.

H) 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 the reaction mixture was stirred overnight in the dark. After 20 hours, thin layer chromatography showed (25% ethyl ether in dichloromethane, silica gel) a practically complete conversion, only some 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 at pH = 8.5 was added dropwise. The pH of the reaction mixture was adjusted to 9.5 with 0.05M 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. The UV analysis showed a conjugation number of about 20, based on an experimentally determined extinction coefficient for thyroxine of 6.2 χ 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-methyiglutaramic acid amide of methylthyroxinate) 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 vigorously added ^{at 0} ° C. 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 Stir 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

Obtain solids.

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

The volatiles were removed in vacuo and the residue was dissolved in 2 ml of methanol

and Süikagel chromatographies (Ät3N: CH ₃ OH: CH ₂ Cb-2.1: 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 recrystallized from THF / HCCb / cyclohexane, 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-methylglutaramsäureamid of Methylthyroxinat dissolved in 39 μΐ DMF were charged and cooled to 4 ° C, the DMF solution was while stirring 51 μl of a 0.11 molar ECDI solution in DMF of 4 ° and 10 μl of 0.5 molar N-hydroxysuccinimide solution in DMF of 4 ° were added and the mixture was stirred overnight in the dark

C) Mitochondrial porcine heart-MDH was dialyzed against 0.05 M carbonate buffer (pH = 9.0) with 4 ml of a 1.31 molar solution of 10- ⁵ χ 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 μΐ and the enzyme activity was determined after a waiting time of 5 minutes.In a first reaction, 5 μΐ ester were used while in a second reaction 10 μΐ were added, with a ratio of thyroxine to MDH of 4 , 1 and 7.4 respectively.

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 combined, the volume adjusted to 5 ml with the phosphate buffer, and a 0.5 ml portion was dialysed against a 50 ml portion of deionized water at 4 °. The radioactivity of an aliquot was determined and, assuming that no protein was lost, the number of haptens per protein molecule was 2.0 and 3.0, respectively.

Example 7
T-4 MEMIDA conjugate to malate dehydrogenase

10 mg (11 μΜοΙ) T-4-MEM1DA and 1.3 mg N-hydroxysuccinimide were dissolved in 250 μΙ 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 ECDI 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 ^ 1 aliquots of the diluted conjugate with 0.1% rabbit serum albumin in glycine buffer (0.1m, pH = 9.5) to which 100 μΙ 0.108m NAD and 100 μΐ 2m sodium malate solution (pH = 9.5) are 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 °.

Uhrxeil

adding T-4 ester

Total volume ¹ ) μ "

T-4 / MDH

T-4 conj. ² )

Deactivation

10:34 13 4996 0/1 1.3 0 11i24 5007 2/1 5003 2/1 30.1 1 ml removed as conjugate c 2.8 ! 1JO 10.5 4011 4/1 4009 4/1 85.7 1 ml taken as conjugate d 3.7 13:10 8 3001 6/1 13:37 2991 6/1 94.0 1 ml removed as conjugate e 5.6 1358 5.5 1977 8.1 / 1 14:49 4 1941 9.6 / 1 98.8 1 ml removed as conjugate f

10

') 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 (TlM)

In 500 μΙ dry DMF in a glass tube, 6.0 mg (6.8 μΜο1) T-4-MEM1DA and 0.8 mg (7.3 μΜοΙ) N-hydroxysuccinimide given; the tube was purged and covered with dry argon and the mixture was cooled in an ice bath. To the stirred mixture was then added 1.5 mg ECDI, and the tube was purged with dry argon and stirred until everything dissolved. The tube was Wiped dry and placed in a covered plastic container with a desiccant (Drierite®), with Wrapped in foil and left to stand overnight at 4 ° with stirring.

0.3 ml of DMF was slowly added with an injection syringe to 1 ml of triosephosphate isomerase (2 mg) in aqueous carbonate buffer (0.1 molar, pH = 9.2) at 4 °. The ester solution was slowly added in increments using a hypodermic syringe and enzyme activity was monitored. As the enzyme was to about 42% deactivated, DMF was added to a solution containing 50 vol to generate ⁰ as DMF.

The cold reaction mixture was passed through a Sephadex G-25 column (middle column) filled with 0.1 M carbonate 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 0.1m carbonate buffer (pH = 9.2) and 03m ammonium sulfal as well as 40 parts DMF. The fractions, the volume of which varied from about 1 to 4 ml, were collected. 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 Conjugate to MDH

9.1 χ 10- ³ gDesaminothyroxin (l, 2 χ 10- ⁵ mol), 1,4 χ ΙΟ- ³ g (1.2 χ 10 ~ ⁵ mol) of NHS and 0.25 ml of dry DMF were added sequentially to a reaction vessel of 1 ml filled. The reaction mixture was cooled on an ice bath and 2.5 χ 10- ³ g (1.3 MoI χ 10- ⁵⁾ ECDI was added under a nitrogen blanket. The reaction mixture was stirred at 4 ° overnight.

Under cooling on an ice bath and with stirring, 0.23 ml of DMF was slowly added to 1.9 χ 10- ³ g (2.8 χ 10- "mol) of MDH in 0.83 ml 0.05 M NaHCO ₃ -Na ₂ CO ₃ (pH = 9.0) 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-50M columns with the dimensions 0.9 χ 13 cm, the desaminothyroxine / MDH conjugate being obtained which was 91% deactivated and which had a hapten number of 2, 5 (after iodine analysis). The enzyme activity of the conjugate was activated by 30% when treated with anti-T-4 serum.

Example 10 T-4 MEMIDA glycine

A 3 ml Pierce Reacti-Vial® was charged with 0,201g (2.18 mol χ 10- *) T-4 MEMIDA and 0.025 g (2.17 χ 10- ⁴ mol) of N-hydroxysuccinimide (NHS) were charged . Then, 2 ml of dry THF and 30μ1 (2.15 χ 10- ⁴ mol) of dry triethylamine were added and the reaction mixture was cooled using an ice bath to 0 °. Then, 0.048 g ECDI (2.50 χ 10- ⁴ mol) in powder form was added and the reaction rates

20th

30th

40 45 50 55 60 65

The mixture was stirred for 35 minutes at 0 ° The reaction mixture was then placed in a cold room (2 °) and stirred for 15 hours A thin-layer chromatogram of the reaction mixture after 15 hours showed two spots with R _r values of 0.06 (T-4-MEMIDA) and 0.60 (T-4-MEMIDA-NHS ester) on an analytical silica gel plate, 10% methanol-dichloromethane being used as a rinsing agent. A 25 ml K.olben with stir bar and membrane plug was charged with 0.033 g (χ 439 10 ~ ⁴ mol) of glycine, then with 1.50 ml of distilled water, 0.50 ml of pyridine and 100 μ! (1.00 χ ΙΟ- ⁴ moles) of 1,0-n-NaOH The reaction mixture was charged with the aid of an ice bath to 0 "cooled with vigorous stirring, the T-4-MEMI-DA-NHS Esterlösung prepared above was added dropwise and after At the end of the addition, the reaction table was stirred in a cold (2 °) vessel for 36 hours. The solvents were then removed using a rotary evaporator,

ίο an oily solid substance smelling of pyridine was obtained. This solid was taken up in 10 ml of methanol and poured into 15 ml of H2O, 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} the ethyl acetate was stripped off on a rotary evaporator, leaving a white crystalline solid. The solid was taken up in 2 ml of methanol and spread 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 was desorbed with methanol / dichloromethane (1: 1) The silica gel was filtered off and the filtrate was concentrated in vacuo to 2 ml. A thin layer chromatogram of the product in THF showed only one spot with an R _f - Value voi, 0.50 on a silica gel plate for analytical thin-layer chromatography in triethylamine / methanol / dichloromethane (2.1: 10: 90). The product was recrystallized from THF / chloroform / cyclohexane

Example 11
T-4 MEMID A-Glycylglycine

^{0.202 g (2.2OxIO-4} MoI) T-4-MEMIDA and 0.025 g (2.17 χ ΙΟ- ⁴ MoI) NHS were poured into an S-ml Pierce Reacti vessel. 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 added and the reaction mixture was poured into a cold room (2 °) 8.25 Stirred for hours. A thin-layer chromatogram indicated the formation of the T-4-MEMIDA-NHS ester (Rf 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 stir bar and membrane plug was charged with 0.058 g (4.39 χ 10 ~ ⁴ mol) of glycylglycine and then with 1.50 ml H2O and 0.50 ml of pyridine and 100 μΙ (1.0 χ 10- ⁴ Mol) 1.0N NaOH 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, worked up in the same manner as T-4-MEMIDA-glycine, 0.018 g (yield 9%) of a brown-golden solid substance being obtained. The product was homogeneous after thin layer chromatography on silica gel with triethylamine / methanol / dichloromethane (2.1: 10: 80); the Rf value was 0.81 when the plate was developed twice.

Example 12
T-4-M EM ID A-glycine / M DH conjugate

A 1 ml Pierce Reacti-vial with stir bar was charged with 0.049 g of dry (5.0 χ 10- ⁶ mol) 4-T-MEMIDA and 39 μΙ DMF. The reaction mixture was cooled to 0 °, whereupon 10 μΐ (5.2 10 ^-6 mol) of a 5.0 χ ΙΟ- ' _m NHS solution in DMF of 0 ° and 51 μΐ (6.1 χ 10- ⁶ mol) of a 1.2 χ 10- 'm ECDl solution in DMF of 0 ° were 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 Rf values of 0.49 (T-4-MEMIDA-glycine) and 0.68 (T-4-MEMIDA-glycine-NHS-ester) on analytical silica gel plates that were marked with Triethylamine / methanol / dichloromethane in a ratio of 2.1: 10: 90 were developed. MDH (4.0 mL, 1.3 χ 10- ⁵ MoI, 0.05 m NaHCO ₃ -Na ₂ CO _3, pH = 9.2) was filled in a 10 ml round bottom flask with stir bar and membrane plug at 0 ° , and the enzyme activity was determined in the manner described above. 445 μΐ dry DMF were added to the reaction mixture at a rate of 15 μΙ / ιτ) ϊη until a reaction mixture with 10% DMF was obtained. The enzyme activity was then redetermined. The 4.3-molar ² χ 10 T-4 MEMlDA-glycine-NHS Esterlösung μΐ was added to the reaction mixture in aliquots of 1 to 2, 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 esters, the enzyme reaction mixture was exhaustively against 0.1 m K.2HPO4 (1.0 χ 10 ~ ^J m NaN ₃₎ dialyzed at 2 ° After dialysis, the enzyme conjugate was carefully removed from the dialysis bag and passed through two Sephadex G 50M columns (in 1.0 m K ₂ HPO ₄ , pre-swollen with 1.0 χ 10 ^-3 M NaN _3). The sizes of the two columns were 0.9 54.0 cm and 0.9 51.0 cm, the flow rates were 4-5 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 3.0.

Example 13
T-4-MEMIDA-Glycylglycine / MDH Conjugate

0.015 g (7.8 10- ⁵ χ Mo!) ECDI was dissolved in 0.50 ml of dry DMF was added to a 1.6 molar solution χ lO -'- 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 molar solution χ lO -'-. Both solutions were cooled to 0 ° in an ice bath before use. A 1 ml Pierce Reacti-vial with stir bar was GT-4-MEMIDA-glycylglycine and then (cooled in an ice bath) with ice-cold 78 μΐ of dry DMF was charged with 2.4 χ 10- ³ whereupon μΐ 4 a 6, 5 10- 'm NHS solution in DMF of 0 ° and 18 μΐ of a, 6 χ 10-' 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 MoI in 0.05m NaHCO ₃ -Na ₂ CO ₃ -LoSMIg (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 1.9-molar ² χ 10 T-4 MEMIDA-GIycylgIycin-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 dialyzed against 1.0m K2HPO4 (with 1.0 10 ^-3 M NaN ₃ ) at 2 °. After dialysis, the conjugate was passed through three Sephadex G-50M columns (pre-swollen in 1.0m K2HPO4 with 1.0 χ 10 ^-3 M NaN ₃ ) and eluted with the same buffer. The column dimensions were 0.9 55 cm, 0.9 χ 56 cm and 0.9 χ 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.

Antibodies were produced using the antigen conjugates. Initially, 2 mg of conjugate injected. Then 0.25 mg conjugate was injected every two weeks. In sheep it was single injection total of 2 ml, of which 0.5 ml is based on the conjugate dissolved in saline, and 1.5 ml is based Freund's complete adjuvant was omitted. There were aliquots of 0.25 ml subcutaneously at 4 locations and 0.5 ml aliquots injected intramuscularly into each hind leg.

In rabbits, the total injection volume was 0.75 ml, with 25 mg of the conjugate in 0.25 ml Saline were dissolved and 0.5 ml Freund's complete adjuvant was added. There were Injections of 0.09 ml were made subcutaneously in 4 sites and injections of 0.19 ml were made in each hind leg.

The animals are usually bled 5 to 7 days after each injection and the antibodies are raised after isolated methods known per se.

To demonstrate the utility of the thyroxine-MDH conjugate for T-4 analysis, a number of Analyzes carried out In the first series of analyzes, the analyzes were carried out with varying amounts of antibodies performed in order to increase the activity of the enzyme conjugate with increasing amounts of antibody demonstrate. In a second series of analyzes, varying amounts of T-4 were added to the antibody by effective concentration of the antibody available for binding to the MDH-bound thyroxine stands to change. These results show that by drawing up a standard curve from samples with Known Levels of Thyroxine The amount of free thyroxine in serum can be determined by looking at the relates observed values of enzyme activity to the standard curve

The analytical method is as follows: 0.8 ml of a solution of 0.1% by weight of rabbit serum albumin (KSA) in a 0.1 molar glycinate buffer (pH = 9.5). the ΙΟ- ³ mol EDTA contains is made from the antibody solution and the MDH-bound T-4. The solution is incubated for 45 minutes, after which substrates (100 μΐ 2m Malal and 100 μΙ 0.108m NAD) are added; the solution is placed in a spectrophotometer and the values are read at 30 ° C as the change in optical density between 120 and 60 seconds after insertion into the spectrophotometer. For the conjugates according to Example 7 with sheep antibodies, the results are given in the table below.

Table II

AbT-4 ¹ ) Conjugate of Example 7

μΙ c ² ) d ² ) e ² ) f ² )

Change change change change

1 +17 113 246 34
2 +17 124 358 111

3 +17 130 389 190

4 +17 126 399 290

5 +17 129 427 376
10 +17 130 467 665

15 +17 136 471 725

20 +17 139 503 829

25 +17 144 485 825

') Etwa9,0 χ 10- ⁶ mAt> T.4, based on the binding sites of K = 1.12 χ 10 ⁹ ~
² ) Molar concentration of enzyme
c = 5 χ ΙΟ- ' ⁰

d = 2.56 χ 10- ⁹
e = 1.4 χ 10- ⁸
f = 3.7 χ 10- ⁸

A series of analyzes was then carried out in which a thyroxine solution of 9.7 mg in 25 ml of 0.05N sodium hydroxide solution was successively diluted with 0.05N sodium hydroxide solution. The analyzes were μΐ by combining 600 0, l% sodium rabbit serum albumin 100 μΐ antibody in 0.1 M glycine buffer (pH = 9.5), 10- ³ M EDTA, and 100 μΐ of T-4 solution carried out and the mixture was Incubated for 15 minutes at room temperature. A volume of the MDH-bound T-4 solution was then added to the solution and the mixture was incubated for 10 minutes. The substrates were then added and readings were taken in a spectrophotometer at 30 ° C in the manner previously described. The results are given in the table below:

Table IIP)

-

Τ-4-sample! Conjugate ¹ ). ² )

molar conc. ede

Λ OD Λ OD ziOD

- 91 81 248

5 χ 10- ¹⁰ 89 83 254

5 χ ΙΟ- ⁹ 88 82 255

5 χ ΙΟ- ⁸ 90 81 249

5 χ 10- ⁷ 88 77 212

5 χ 10- ⁶ 95 54 93

5 χ ΙΟ- ⁵ 99 56 85

') Volume and concentration of the enzyme conjugate used
c = 15 μ.1 1.11 χ 10- ⁷ molar

d = 5.12 χ 5μ1 10- ⁷ -molar

e = 2.81 χ 5μ1 10- ⁶ -molar

² ) Anti-thyroxine 9 χ 10- ⁶ -moIar at binding sites, diluted to 1:33 before adding to 100 μΐ.

³ ) 4ODx 10 ³ .

Following the procedure of Example 7, another conjugate was prepared which was run on a Sephadex G-50 column, which had been brought into equilibrium with 1 molar potassium monohydrogen phosphate solution, Chromatographies was. The flow rate was slow and fractions of 20 drops were obtained collected to tube 39, whereupon fractions of 99 drops were collected. The procedure was repeated with a second G-50 column, each time using the collected fractions with Sephadex-G-200 were concentrated by ultrafiltration. The number of T-4 groups per enzyme was determined by UV analysis determined to be 43 to 53 (average 4.8)

A number of analyzes were carried out using different and alternating sources of antiserum Thyroxine concentrations carried out. As described in the above procedure, the antibody and the thyroxine were combined in 0.1% KSA solution buffered with glycine and 15 Incubated minutes after which the specified amount of enzyme solution was added; the analysis mixture was incubated again for 10 minutes, the substrates added, and the mixture was then placed in a spectrophotometer introduced with the change in the optical density units being read off in the second minute. The volume of the added antibody is 1 μΐ, and the volume of the added enzyme is also 1 μΙ. The results are given in the table below.

Table IV ¹ )

T-4 analysc
molar conc.

Antisera

// OD

B Λ OD

C / IOD

5 χ 10-5
5 χ 10- ⁶
5 χ ΙΟ- ⁷
5 χ ΙΟ " ⁸
5 χ 10- "
5 χ 10- '°
5 χ 10- "

') // OD χ ΙΟ ³

76 20 28 30 38 73 81 78

61 24 29 39 50 59 61 63

87 26 29 32 62 84 94 92

When performing the T-4 analysis using TIM-T-4 conjugate, the following reagents were used used:

Reagents for TI M-T-4 analysis

Buffer:

0.02m triethanolamine (TEA) -HCl, pH = 7.9

0.0054m ethylenediaminetetraacetic acid (EDTA)

Rabbit serum albumin solution:

0.2% by weight KSA, in buffer solution T'M-T-4 solution:

About 7 χ 10 ~ ³ μΜο1 T-4 as T1M-T-4, in KSA solution (results in analysis speed of about 200 OD units / min).

Anti-T-4 solution:

About 4 χ 10 ⁵ m An.ti T-4, relative to binding sites in KSA solution.
NADH solution:

2.5 mg NADH / ml in buffer solution (stock solution), prepared according to the TEKIT instructions and diluted in a weight ratio of 1: 7 with buffer solution).

DL-glyceraldehyde-3-phosphate solution (GAP):

Made from DL-glyceraldehyde-3-phosphate diethylacetal, Ba-SaIz by Sigma Chemical Co., St. Louis,

Mon .: 1.5 g of salt were treated so that a final volume of 10 ml was obtained.
rt-glycerophosphate dehydrogenase («- GPDH) - solution:

0.2 mg / ml in buffer solution (Boehringer-Mannheim).

Thyroxine (T-4 solution):

2.57 χ 10- ⁴ m in KSA solution.

The analysis was carried out as follows:

A number of dilutions of the T-4 solution were made. First, 0.4 ml of the T-4 solution, 0.4 ml of the TIM T-4 solution and 0.4 ml of the antibody solution are mixed together and placed in a water bath at 12 minutes 3O ⁰ C. The solution was then added 200 μl of the NADH solution, 25 μl of the ar-GPDH solution and 100 μl of the GAP solution. The analysis tube was covered with parafilm and an aliquot was then drawn into a thermal cuvette in a 300N Gilford spectrophotometer. The first reading was taken after 30 seconds at a temperature of 30 ° C in the device. The optical density was read at 366 nm and 30 ° C. The remainder of the analysis solution was kept in a bath at 30.degree. C. and read off after 13 minutes. The results are given in the table below.

Table V

Sample no.

T-4 solution concentration

Decrease in optical density in 13 min

1 2 3 4 5 6 7

The results in the tables above show that extremely low concentrations as well as extremely small amounts of thyroxine can be detected or determined by the method according to the invention

0 X ΙΟ- ⁶ 0.543 3.84 X ίο- ⁶ 0.550 7.68 X 10-5 0350 1.54 X 10-5 0.490 6.14 X ίο- ⁴ 0.448 1.29 X 10- " 0.422 5.14 0.409

15th

can. The process is very simple and requires only a few handling steps. By getting the reagents combined in a buffered medium and the mixture optionally incubated and then the Enzyme substrates can be added to the T-4 by a spectrophotometric reading over a short Determine the period. The system can be automated so that the samples and reagents are processed automatically mixed and the readings can be taken automatically.

16

Claims (1)

Patent claims: 1. Method for the detection or determination of a ligand which is a polyiodothyronine with 3 to 4 Represents iodine atoms, being in an aqueous liquid zone (1) a medium suspected of containing the ligand; (2) a soluble ENZ-linked ligand; and (3) an antiligen with sites that are common to the ligand and the ENZ-bound ligand and who can bind to them,