DE2605162A1 - IMMUNOANALYSIS OF POLYIODTHYRONINES - Google Patents

Description

The concentration of thyroxine in the blood is, within relatively narrow limits, critical to the proper functioning of the body. The thyroxine concentration is extremely low and can only be detected using very sensitive methods.

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 missing in the 5- or 5 'position. It is believed that the (T-3) markedly influence the overall metabolic effects of thyroid hormones.

One method for determining thyroid hormones is radioimmunoanalysis (radioimmunoassay). Although these

Method has many variants, it basically consists in combining an antibody for the thyroid hormone and a radioactive or "hot" thyroid hormone with blood serum and separating the bound hormone from the unbound serum. The amount of "hot" hormone bound or left free to the antibody is a function of the amount of the hormone in the serum. By determining the radioactivity of the solution freed from the antibody, the amount of hormones can be calculated based on standards with known amounts of hormones.

In radio immunoanalysis, a separation stage is required which introduces errors 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 generally undesirable because of the associated health risks. There is a continuing need for a simple method in which the number of treatment steps can be minimized while maintaining high sensitivity.

US Pat. No. 3,817,837 describes an enzyme analysis (enzyme assay) which has generally proven useful for a wide variety of ligands.

The invention now relates to a homogeneous enzyme immunoanalysis method for the detection or determination of thyroid hormones (thyroid hormones). The enzyme reagent used in immunoanalysis is an enzyme-linked polyiodothyronine (EBP), which uses an enzyme that is more than 50% deactivated after conjugation with a polyiodothyronine and that after the binding of a receptor for the polyiodothyronine is partially or completely reactivated. The conjugated enzyme used thus has a low turnover rate, which increases considerably in the presence of a receptor, for example an antibody for polyiodothyronine.

The analysis is carried out in such a way that a serum sample to be determined, the suitable receptor, EBP and the enzyme substrates are combined with one another in an aqueous buffered medium and the rate of the reaction catalyzed by the enzyme is determined. If necessary, one or more incubation periods can be interposed between the addition of the individual reagents. By making standards with known amounts of T-3, reciprocal T-3, or T-4, a calibration curve can be obtained against which the unknown samples can be related.

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

The invention specifically relates to a method and compositions of matter for the determination of polyiodothyronines (T-3 and T-4) at concentrations of only 10 [high] -7 molar or below, i.e. it is a homogeneous enzyme immunoanalysis. By analogy with the invention described in U.S. Patent 3,817,837, T-3 and T-4 are the ligands, the enzyme-linked ligand is enzyme-linked T-3 or T-4, and the antiligand is the antibody to T-3 or T -4. In particular, the enzymes are malate dehydrogenase or triose isomerase.

In the homogeneous enzyme immunoanalysis according to the invention, enzyme-bound polyiodothyronine (in particular T-3 and T-4), a receptor for polyiodothyronine (in particular an antibody, e.g. anti-T-3 and anti-T-4), substrates for the Enzyme and an aqueous, normally alkaline buffer medium, which usually contains one or more additives to improve enzyme stability, are used for determining the product of the enzyme reaction or the like. The order in which the reagents are added to the aqueous analysis medium is not critical, although certain procedures are preferred. 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, usually spectrophotometrically. By comparing the result obtained with the unknown substance with the results obtained using known amounts of polyiodothyronine, one can determine the amount of polyiodothyronine in the unknown sample.

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

The reagents

Enzyme-linked polyiodothyronine

In making the enzyme-linked polyiodothyronine, one can use the polyiodothyronine directly, activating the carboxyl group to be attached to available groups of the enzyme, e.g., lysines and tyrosines, or preferably from the available functionalities e.g. 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.

Enzymes which can be used are those which are deactivated practically reversibly during conjugation with a polyiodothyronine, so that the activity is largely regained upon binding to an antibody for the polyiodothyronine. It has been found that only a certain group of enzymes has this activity, in particular malate dehydrogenase, and of these again the mitochondral, preferably the mitochronic, malate dehydrogenase (MDH) from pig hearts; and triosephosphate isomerase, in particular triose isomerase (TIM) from rabbit muscles. Usually the polyiodothyronine is linked to the enzyme through a linking group linked to the amino group of the polyiodothyronine, the carboxy group being esterified, particularly with a lower alkyl group which usually contains 1 to 3 carbon atoms. The polyiodothyronine can expediently be bound by a functional group which contains two non-oxo-carbonyl functions. According to the invention, non-oxo-carbonyl is understood to mean the oxygen-containing carboxy group the nitrogen-containing imidine group the sulfur-containing thionocarboxy group

and their derivatives such as amides, esters and anhydrides.

The number of polyiodothyronines conjugated with the enzyme is on average at least 1 and no more than about 10 per enzyme, usually 1 to 8 and usually 2 to 6. The polyiodothyronines can be attached to available sites (e.g. amino or hydroxyl groups to form amides and Esters), namely by suitable linking groups starting from the amino group or the carboxy group (in particular from the amino group of polyiodothyronine)
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Since the amino group must be protected during conjugation if the carboxyl group is involved in conjugation, the linking groups will in most cases be attached to the amino site. Conveniently the ester of polyiodothyronine is used and a group with a non-oxo-carbonyl functionality (see definition above) is used for conjugation to the amino and hydroxyl groups of the enzyme.

A bond or a linking or linking group can connect the polyiodothyronine to the enzyme. As such, the linking group is not critical to the invention, although certain classes of linking groups are preferred. The number of atoms in the linking group (other than hydrogen) is generally not more than 20, usually not more than 16; the linking group can contain 0 to 7, usually 1 to 6, heteroatoms, especially chalcogen atoms (O to S), which are normally only bonded to carbon, and nitrogen which is only bonded to carbon and hydrogen and which is neutral when it is to Hydrogen is bound, for example in the form of amido nitrogen.

The particular enzyme used can be reversibly deactivated by a polyiodothyronine so that in the absence of the receptor for polyiodothyronine the enzyme has less than about 50%, usually less than about 35% and usually less than 25% of its original enzyme activity before conjugation, but with at least 0.5, usually at least 1% of the original enzyme activity is present prior to conjugation. When excess receptor for polyiodothyronine is added to the enzyme conjugate, the enzyme activity increases by at least 50%, usually by at least 100% and frequently by 200% or more. The value of the increase in activity depends in large part on the initial decrease in activity upon conjugation. Other enzymes, in particular dehydrogenases, which are inhibited by thyroxine, are in Wolff and Wolff, Biochimica et. Biophysica Acta, 26, 387 (1957). These enzymes are, for example, glutamine dehydrogenase, lactate dehydrogenase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase.

In general, the enzyme-linked polyiodothyronine (EBP) has the formula below where the symbols have the following meanings:

ENZ is an enzyme which is reversibly inhibited by a polyiodothyronine, in particular T-3 and / or T-4, so that the enzyme is activated when the polyiodothyronine is bound by a receptor, for example an antibody; the enzyme is generally a dehydrogenase or a triose phosphate isomerase;

X is 0 or 1

Z is wherein

m [high] 1 0 or 1;

G and G [high] 1 are hydrogen or iodine, provided that G [high] 1 is iodine when m [high] 1 is 0 and that there are at least 3 iodine atoms when m [high] 1 1;

one of W and W [high] 1 is a linking group, in particular an aliphatic group with 0 to 1 ethylenically unsaturated sites and 1 to 20 atoms other than hydrogen, which are carbon, chalcogen atoms or nitrogen, the chalcogen atoms as Oxy or oxo atoms (including the thio analogs) are only bonded to carbon, in particular non-oxo-carbonyl; and wherein the nitrogen is only bonded to carbon and hydrogen and is neutral when bonded to hydrogen, e.g., as amido nitrogen; the number of heteroatoms is usually in the range from 1 to 8, usually in the range from 2 to 7 and preferably between 2 and 4; there are usually no more than 5, usually no more than 4 and preferably 2 to 3 functionalities in the chain of the linking group, e.g. tertiary amino, oxy, amido, etc., provided that W [high] 1 is a Can be bond, in particular for x = O; if it is not a linking group, W is hydrogen and W [high] 1 is a hydroxyl group or an alkoxy group having 1 to 3 carbon atoms, especially the methyl group; and

m averages at least 1 and not more than 10, usually 1 to 8, usually 2 to 6.

In particular, the EBP used in the present invention has the formula given below where the symbols have the following meanings:

ENZ is an enzyme which is reversibly inhibited by a polyiodothyronine, in particular T-3 and T-4, so that the enzyme is activated when the polyiodothyronine is bound by a receptor; the enzyme is usually a dehydrogenase, particularly malate dehydrogenase, or triose phosphate isomerase;

n averages at least 1 and not more than 10, usually 1 to 8, usually 2 to 6;

a is 0 or 1, usually 1;

X and Y, which are the same or different from one another, are chalcogen (O or S) or imino (-NH), where Y is normally not imino;

D is hydrogen or an alkyl group having 1 to 6 carbon atoms, usually 1 to 3 carbon atoms, and preferably one carbon atom;

Z is 3,3 ', 5-, 3,5,3' -triiodo- or 3,3 ', 5,5'-tetraiodo-4- (hydroxyphenoxy-1') -phenyl-1 or 3,5-diiodo -4-hydroxyphenoxy; and

A is a bond or a divalent organic group having at least 1 carbon atom, usually at least 2 carbon atoms and usually no more than 12 carbon atoms, usually no more than 10 carbon atoms, with 0 to 12, usually 1 to 10, usually 2 to 8 and preferably 3 up to 7 atoms in the chain (for cyclic groups the larger number of ring atoms is included) between the non-oxo-carbonyl groups and there are 0 to 4, usually 0 or 1 to 3, usually 1 to 2 heteroatoms in the chain , which can be chalcogen (O or S) (as oxy-thio or sulfonyl groups) or nitrogen (tertiary amino or amido nitrogen); the total number of heteroatoms is 0 to 6, generally 0 to 5, usually 0 or 1 to 4, especially 1 to 5, being chalcogen (O or S) (as non-oxo-carbonyl, non-oxo-thiocarbonyl , Oxy, thio or sulfonyl) or nitrogen (imino, tert-amino or amido); there are usually 2 carbon atoms between the heteroatoms in the chain; A 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 in of the chain are usually 5 to 6 ring members with 0 to 2 hetero ring members, usually with 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 4 ring members; normally the only aliphatic unsaturation in the hydrocarbylene group has 0 to 1 ethylenic groups; it is preferably saturated and in particular represents an alkylene group with 0 to 1 carboxamido, imino or oxy groups in the chain; when A is a carbocyclic 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 analogues) is generally from 0 to 4, usually from 0 to 3 and usually from 0 or 1 to 2.

The general group also includes ENZ-bonded T-3 or T-4 compositions which have a di-non-oxo-carbonyl link with an aliphatic chain which is not interrupted by heteroatoms or which is interrupted by about 1 to 2 heteroatoms, namely chalcogen and nitrogen, is interrupted. These compounds generally have the formula given below where the symbols have the following meanings:

ENZ, D and Z are as defined above;

n [high] 1 has the same limit values as n, but averages between 1 and 10, preferably between 1 and 8 and in particular between 2 and 6;

X [high] 1 and Y [high] 1 have the same limits as X and Y, respectively, where X [high] 1 is usually chalcogen and Y [high] 1 is preferably oxygen;

p, q and r are each 0 to 1, the sum of p, q and r preferably being at least = 1 (p + q + r> 1); s is 0 to 2, usually 0 to 1;

Small alpha and small gamma are hydrocarbon groups with 1 to 8, usually 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 p or r = 0 small alpha or small gamma preferably contain 1 to 8 carbon atoms, in particular 1 to 6 carbon atoms;

Small alpha and small gamma can be aliphatic, alicyclic, aromatic or heterocyclic; together with small beta and small delta they can satisfy the definition of A; preferably small alpha and small gamma are saturated aliphatic groups, in particular unbranched groups, e.g. methylene or polymethylene groups, or aromatic, in particular monocyclic groups, only one of small alpha and small gamma being an aromatic group;

Small beta is oxy (-O-), thio (-S-), sulfonyl (-SO [deep] 2-) or amino wherein T is an alkyl group having 1 to 6 carbon atoms, usually 1 to 3 carbon atoms, or hydrogen; if small beta is linked to a non-oxo-carbonyl group (p = O), then small beta is an amino or alkylamino group; and

is wherein T [high] 1 is hydrogen or a lower alkyl group having 1 to 3 carbon atoms.

In a preferred subgroup, the polyiodothyronine can be combined with a dibasic dicarboxylic acid 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 ethylenically unsaturated sites in the chain; it also contains 0 to 1 atoms with an atomic number of 7 to 8 (nitrogen or oxygen), the nitrogen or oxygen atoms being connected only to carbon atoms, i.e. in the form of tertiary amino groups or ether groups. Preferred dibasic acids form cyclic anhydrides with 5 to 7, in particular 5 to 6, ring members. To ensure that the carboxyl group of the dibasic acid conjugated to the thyronine is the one that reacts with the ENZ, the original carboxyl group of the thyronine is usually substituted with an alkyl group with 1 to 3 carbon atoms, preferably with one carbon atom (methyl) esterified.

The modified polyiodothyronine or its analog used for conjugation with the ENZ generally has the formula given below where the symbols have the following meanings:

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

D 'is hydrogen or an alkyl group with 1 to 3 carbon atoms, preferably with 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, the heteroatoms being completely substituted by carbon atoms and 1 to 6 carbon atoms being present; and

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

Another useful series of compounds has a carbocyclic or heterocyclic ring with 5 to 6 ring members in the chain, the heterocyclic ring containing 1 to 2, usually 1, heterocyclic member, which is O, S or N. These compounds preferably have an isocyanate or isothiocyanate group which is directly or indirectly linked to a ring member, especially if it is an aromatic ring. In most cases, these compounds have the formula given below where the symbols have the following meanings:

D and Z are as defined above;

X [high] 2 and Y [high] 2 are chalcogen (O or S), where X [high] 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 members, namely O, N and S, respectively, and with 4 to 10 carbon atoms, usually 4 to 8 carbon atoms, and when the compounds are heterocyclic, there are preferably 4 to 6 carbon atoms, and when the compounds are carbocyclic, there are preferably 6 to 8 carbon atoms.

The ENZ conjugate of the series of compounds given above has the formula wherein all symbols are as defined above.

The polyiodothyronine analog is used in an active or activated form so that it is able to react with available non-oxo-carbonyl recative groups of ENZ, e.g., amino and hydroxyl groups.

For the carboxy group, a mixed anhydride, an N-hydroxysucchinimide, 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 carried out at moderate temperatures, generally in the range of about -5 to 30 ° C, using a mixed aqueous buffer medium, usually having a mildly alkaline pH in the range of about 7-10. Hexamethylphosphoramide, for example, can be used as an auxiliary solvent. 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 worked up in the customary manner. The mixture is preferably chromatographed in order to separate off any unreacted analogs from the ENZ-bound T-3 or T-4. A convenient chromatographic material is Sephadex. The fractions can be isolated, their enzyme activity can be determined and the fractions with enzyme activity can be collected.

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

Desaminopolyiodothyronine and its monocyclic analog are defined by the formula given below:

HO [deep] 2CCH [deep] 2 (CH [deep] 2) [deep] tZ

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

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

ENZ- (COCH [deep] 2 (CH [deep] 2) [deep] tZ) [deep] n1

wherein all symbols are as defined above.

The ENZ-linked T-3 or T-4 is usually at least about 50% deactivated (compared to 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 with the addition of excess

Anti-T-3 or T-4 can be almost completely restored. However, it has been found that upon binding to the receptor, the conjugated enzyme is activated 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. 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. Excess is understood to mean that there are enough antibodies to bind virtually any available T-3 or T-4.

The enzyme is conveniently mitochondrial malate dehydrogenase from pig hearts or triose phosphate isomerase from rabbit muscles.

Anti-T-3 or -T-4

The appropriate antibodies are generated by injecting an antigen bound to T-3 or T-4 into a vertebrate animal, usually a domestic animal such as a sheep, rabbit or goat. Since the antigen is usually a polypeptide or protein, which normally has a molecular weight of about 5,000 to 10 million, the conjugated antigen is formed by combining a T-3 or T-4 analog with the antigenic polypeptide or protein. Either the same or a different analog used for conjugation with ENZ can be used. In addition to the analogs used to make the conjugate, other analogs can also be used. Thyroglobulin can also be used to produce the antibodies.

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

The antigen is injected into the animal in the usual manner, although it may be advantageous to use Freund's complete Akjuvans with the main injection (booster injection).

A wide variety of proteins can be used as antigens such as albumins, globulins, keyhole limpet hemocyanin, and the like.

buffer

A wide variety of buffers can be used, e.g. phosphate, carbonate, glycinate, Tris and similar buffers. Phosphate buffers should not be used in high concentrations, preferably in concentrations less than about 0.1 molar. Certain buffers are preferred, such as glycinate or triethanolamine buffers.

Other accessories

Depending on the course of the reaction, the substrates for MDM are malate and NAD or oxaloacetate and NADH. For triose phosphate isomrase (TIM) the enzymatic reaction of the enzyme is coupled with a second enzyme so that a spectrophotometric determination is possible. That

Analysis medium thus contains the substrate for TIM, glyceraldehyde phosphate, NADH and small alpha-glycerophosphate dehydrogenase. The latter enzyme and NADH are used in a considerable excess so that they are not the rate-limiting substances.

A small amount of ethylenediaminetetraacetic acid is expediently added to reduce bacterial growth and to bind heavy metals. A protein and glycerine can also be added to the analysis medium in order to improve the enzyme stability. Further stabilizers such as dithioerythritol or other antioxidants can also be added. In addition, small amounts of sodium azide can be added as a preservative.

Analytical method

Reagent solutions

The buffer solution used normally has a concentration such that a concentration of about 0.001 to 0.5 molar, usually about 0.01 to 0.2 molar and preferably about 0.05 to 0.15 molar is obtained in the analysis medium. The added protein, which is expediently an albumin such as rabbit serum albumin and / or gelatin, is generally present in the finished analysis mixture in amounts of about 0.005 to 0.5% by weight, usually in amounts of about 0.01 to 0.2% by weight .-%, before. Glycerin can be present in amounts from 0.1 to 5, usually in amounts from 0.4 to 4% 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 NAD and malate or NADH and oxaloacetate can be used; it is preferably 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 [high] -5 to 10 [high] -12 molar, usually from about 10 [high] -7 to 10 [high] -11 molar. The antibody concentration depends on the ratio between the antibody binding sites and the concentration of the polyiodothyronines bound to ENZ. In general, the ratio of the 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 expediently about 1 to 25. A sufficient amount of antibody is usually added to recover from 25 to 75, preferably from 25 to 50% of the recoverable enzyme activity.

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

Other additives that may optionally be added in smaller amounts are ethylenediaminetetraacetic acid, which can be present in amounts of about 0.001 to 0.1% by weight, sodium azide, which can be present in amounts of about 10 [high] -5 to 10 [high ] -3 molar, 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 and NADH or oxaloacetate and NADH are used, depending on the direction of the reaction, the first-mentioned substances being preferred. The concentration of these substances directly affects the sensitivity of the analysis and must be determined empirically in order to determine the difference between the

Optimize reaction rates in the presence and in the absence of the added 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. Normally, the concentrations of enzyme, antibody and substrate are chosen to optimize the sensitivity of the analysis.

If TIM is used, the glyceraldehyde phosphate is generally present in a concentration in the range from about 10 [high] -1 to 10 [high] -4 molar; Small alpha-glycerophosphate dehydrogenase is generally used in amounts of about 10 [high] -5 to 10 [high] -9 molar, while the NADH concentrations are generally between about 10 [high] -2 and 10 [high] -4 molar lie.

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

The stages of analysis

The order in which the individual reagents are combined is not critical, although some orders are preferred and in some cases one order is preferred over another. An incubation can be carried out after adding a particular reagent or after adding all reagents. Usually, the analysis mixture is not incubated in the presence of the enzyme substrates for more than 10 minutes before the start of the speed measurement.

All reagents can be added at the same time. However, the sample, e.g. serum, buffer solution and antibody, can also be combined, which is followed by an incubation if necessary. In most cases the antibody and the conjugated ENZ will not be conjugated prior to adding the sample. However, if a monovalent antibody is used to avoid precipitin formation, it may be desirable for certain applications to store a mixture of antibody and enzyme.

Depending on the order of addition, the events proceed differently. When the sample and antibody are combined, the polyiodothyronine becomes bound to the antibody, reducing the available sites for binding 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. When the ENZ-linked T-3 or T-4 is added, the amount of antibody that binds to the conjugated T-3 or T-4 is a function of the T-3 or T-4 in the Sample. This procedure is less sensitive to differences in the binding constants between T-3 or T-4 and ENZ-bound T-3 or T-4.

However, one can also combine the sample, the suitable antibody and the conjugated ENZ and allow the free T-3 or T-4 to compete with the T-3 or T-4 conjugated to ENZ with regard to the available binding sites.

Optionally, the analysis mixture can be incubated after each addition of a reagent, either before or after the addition of the enzyme substrates or the other reagents. Incubation can last about 2 minutes to 1 hour and is generally carried out at temperatures in the range of about 15 to 40 ° C, usually about 25 ° C (room temperature) to 37 ° C.

After adding the mixture to the substrates for the enzyme, the mixture can be incubated for up to about 10 minutes before the initial reading; this reading can be taken either with a flow cell or with a cuvette. The rate is determined at a temperature in the range of about 20 to 40 ° C, usually about 25 to 37 ° C. Generally, the readings take about 0.1 to 45 minutes, usually about 0.5 to 2 minutes if a flow cell is used and about 5 to 45 minutes if a cuvette is used. Measurement periods of about 5 to 20 minutes are suitable for certain automated instruments.

Using standards with known amounts of T-3 or T-4 in the serum, a calibration curve can be set up which can be used to determine T-3 or T-4 in an unknown sample.

While spectrophotometric methods are most convenient for determining the course of the enzyme reaction (i.e., by determining the absorption spectrum), other methods such as fluorimetry, titrimetry, etc. can also be used.

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

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 x 10 [high] -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 μl of dry triethylamine were added.

The mixture was stirred for 15 minutes, after which 0.279 g (2.16 x 10 [high] -3 mol) of N-methyliminodiacetic anhydride in 2.5 ml of dry THF was added all at once. The reaction obviously started immediately. The volatiles were removed under vacuum in 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. After the organic layer was filtered off with suction, the solvent was removed on a rotary evaporator to obtain a solid white substance which was dissolved in 30 ml of THF has been resolved. 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 allowed to cool to room temperature and then in a freezer to give a white, fluffy product which was washed with 1-hexane and dried under vacuum over phosphorus pentoxide. 1.26 g (75%) of a white, flaky product were obtained.

Example 2

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

0.10 g (1.09 x 10 [high] -4 mol) T-4-MEMIDA and 0.013 g (1.1 x 10 [high] -4 mol) N- were placed in a reaction vessel with a stirrer and glass stopper with a membrane. Hydroxysuccinimide filled.

1 ml of dry THF and then 15 μl of dry triethylamine were added to the reaction mixture under an argon atmosphere. After cooling the mixture in an ice bath to 0 ° C, 0.024 g (1.25 x 10 [high] -4 mol) 1-ethyl-3- (3'-dimethylaminopropyl) -carbodiimide hydrochloride (ECDI) were in the form of a Powder added. The mixture was stirred at 0 ° C for 2 hours and then at 4 ° C for 12 hours. Then a solution of 0.100 g (1.55 x 10 [high] -6 mol) of bovine serum albumin (RSA) in 0.3 ml of sodium bicarbonate carbonate buffer (pH = 9.4) was prepared and the pH was adjusted to 6n -Sodium hydroxide solution adjusted to 0.5. The RSA solution was cooled to 0 ° C., 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.

A 3M hydroxylamine hydrochloride solution which had been neutralized to a pH of 8.9 with 6N sodium hydroxide solution was then added dropwise to the solution. After the mixture had been stirred for 10 hours at 4 °, it was placed in a dialysis bag and dialyzed for one day against two portions of 500 ml each of a Tris-HCl buffer (0.05 molar, pH = 7.8). The volume of the protein mixture was then reduced to 25 ml with Aquacide II (manufacturer: Calciochem), whereupon the mixture was subjected to gel filtration chromatography twice, in each case freshly packed Sephader G-15, which was originally stored in Tris-HCl buffer (0, 1 m, pH = 9.0) was used. The column size was 2.6 × 22.2 cm, the flow rate was 48 drops per minute, 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 x 2000 ml) for 3 days. The conjugate solution was then lyophilized to give 0.15 g of a white fluffy solid substance, which was dried under vacuum over P [deep] 2O [deep] 5 for 3 days to give 0.140 g of conjugate. UV analysis showed that 22 haptens were bound to each RSA molecule.

Example 3

Carboxymethoxyacetyl-thyroxine-methyl ester (DGMA)

To a solution of 1.65 g of the methyl ester of thyroxine hydrochloride in 80 ml of dry THF and 30 ml of chloroform in a flask protected from light, 300 µl of triethylamine was 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 purified on a 30 g Sephadex LH-20 column using a solution of 20% methanol in dichloromethane as the eluent. The clear fractions were collected, the solvent was removed and the residue was precipitated from methanol with water, 1.53 g of substance (84%) being obtained.

Example 4

DGMA conjugate with bovine serum albumin

25 ml of DMF were slowly added to a solution of 100 mg of BSA in 30 ml of aqueous 8m urea solution at 0 ° C., whereupon 453 mg (5.9 × 10 [high] -4 mol) of DGMA in 5 ml of DMF were added dropwise. 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 were added at half-hour intervals to the stirred solution, which was kept at 0 ° C. 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 was added 98 μl of oxalyl chloride (146 mg, 1.15 molar). A drop of dimethylformamide was first added to start the reaction and the reaction mixture was stirred for 30 minutes until foaming had stopped and the acidic starting material had dissolved. The solvent was then removed on a rotary evaporator and the remaining yellow oil was added to a suspension of 827 mg (1 mmol) of methyl thyroxinate hydrochloride in 10 ml of dry tetrahydrofuran containing 280 μl of triethylamine (2 mmol). The reaction was terminated after 2 hours after analytical thin-layer chromatography (silica gel, 25% ethyl ether in chloroform) could no longer detect any residues of the thyroxine ester starting material. The reaction mixture was poured into water covered with 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) removed a polar impurity. Yield 85%.

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 which was provided with a serum stopper. A solution of sodium methoxide in methanol was then added (1.1 base equivalents) and the reaction mixture was stirred in the dark overnight. After 20 hours, thin layer chromatography (25% ethyl ether in dichloromethane, silica gel) showed practically complete conversion, with only a few colored impurities being 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 mTris buffer pH = 8.5 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 x 10 [high] 3M [high] -1cm [high] -1. 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-3-aza-3-methylglutaramic acid amide of methylthyroxinate) conjugate to MDH.

A. A stirred solution of 0201 g (0.22 mmol) T-4-MEMIDA, 1 ml dry THF, 25 mg (0.22 mmol) N-hydroxysuccinimide and 30 µl dry triethylamine at 0 ° was added 0.048 g (0, 25 mmol) ECDI added. 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 1N 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 poured into 15 ml of deionized water and extracted 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 [deep] 4.

The volatile substances were removed in vacuo, and the residue was dissolved in 2 ml of methanol and chromatographed on silica gel (Et [deep] 3N: CH [deep] 3OH: CH [deep] 2Cl [deep] 2-2.1: 10: 90). The product was desorbed with CH [deep] 3CH / CH [deep] 2Cl [deep] 2 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 / HCCl [deep] 3 / cyclohexane, a yield of 0.046 g (21.6%) being obtained.

B. 4.9 mg of N-carboxymethyl-3-aza-3-methylglutaramic acid amide of methylthyroxinate, which had been labeled with radioactive C [high] 14, dissolved in 39 μl of DMF and cooled to 4 ° C. were poured into a 1 ml vessel. 51 μl of a 0.11 molar ECDI solution in DMF of 4 ° and 10 μl of 0.5 molar N-hydroxysuccihimide solution in DMF of 4 ° were added to the DMF solution with stirring, and the mixture was im Dark stirred.

C. Pig mitochondrial heart MDH was dialyzed against 0.05 M carbonate buffer (pH = 9.0) to give 4 ml of a 1.31 x 10 [high] -5 molar solution of MDH. 445 μl of DMF were added to the solution at a rate of 15 μl / min. admitted. The ester prepared above was added in 2 µl portions and the enzyme activity was determined after waiting 5 minutes. In a first reaction, 5 μl of ester were used, while in a second reaction 10 μl were added, with a ratio of thyroxine to MDH of 4.1 and 7.4, respectively, being set.

Both reaction mixtures were three times against 300 ml of 1 molar K [deep] 2HPO [deep] 4 solution (pH = 9.8) once against 300 ml of 0.05 molar carbonate buffer (pH = 9.0) and then twice against the Dialyzed phosphate buffer. The two reaction mixtures were then chromatographed at 4 ° on a Sephadex G-50M column (0.9 x 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 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 µmol) of T-4-MEMIDA and 1.3 mg of N-hydroxysuccinimide were dissolved in 250 μl of dry DMF. The reaction mixture was kept at 0 ° under a nitrogen atmosphere with stirring, after which 2.3 mg of ECDI was added. The mixture was held at 0 ° until all of the ECDI had dissolved. The solution was left to stand at 4 ° overnight.

A general procedure is given for preparing the conjugate with greater or lesser hapten numbers, depending on the amount of T-4-MEMIDA hydroxysuccinimide ester relative to MDH. 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 1 ml of DMF. The subsequent additions of the T-4-MEMIDA ester were made at intervals of about 60 to 90 minutes, with aliquots being removed and examined for enzyme activity. The table below shows the order of addition, the amount of the additives, the time of addition, the ratio between added T-4 and MDH and the observed percentage deactivation. To determine the conjugate numbers, 2 to 20 μl of the conjugation mixture were added to 0.5 ml of 1M potassium monohydrogen phosphate at 0 °. To determine the enzyme activities, 2 to 20 µl aliquots of the diluted conjugate with 0.1% rabbit serum albumin in glycine buffer (0.1m, pH = 9.5), 100 µl 0.108m NAD and 100 µl 2m sodium malate solution (pH = 9 , 5) were added, diluted to 0.8 ml. Velocities were measured between 60 and 120 seconds after insertion into a Gilford Model 300-N Spectrophotometer at 30 °.

1) Samples were taken periodically to determine enzyme activity, which is not specified, which affects the total volume reported.

2) Number of T-4 units bound to MDH

Example 8

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

6.0 mg (6.8 µmol) of T-4-MEMIDA and 0.8 mg (7.3 µmol) of N-hydroxysuccinimide were placed in 500 µl of dry DMF in a glass tube; the tube was purged and covered with dry argon and the mixture was cooled in an ice bath. 1.5 mg ECDI was then added to the stirred mixture, and the tube was purged with dry argon and stirred, until everything was resolved. The tube was wiped dry, placed in a covered plastic container with a desiccant (Drierite [high] R), 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. When the enzyme was about 42% deactivated, DMF was added to make a 50% volume DMF solution.

The cold reaction mixture was passed through a Sephadex G-25 column (middle column) equilibrated with 0.1 M carbonate buffer (pH 9.2). The column was a 50 cc burette with a diameter of 1.1 cm and a bed volume of about 19 ml. The 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 , 3m ammonium sulfate and 40 parts of 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 with 0.02m triethanolamine (TEA) (pH = 7.9) and three times against 250 ml of aqueous DMF each time 0.02 molar TEA (pH = 7.9) dialyzed. The ratio of conjugated T-4 to enzyme was approximately 6: 1.

Example 9

Desaminothyroxine conjugate to MDH

9.1 x 10 [high] -3 g desaminothyroxine (1.2 x 10 [high] -5 moles), 1.4 x 10 [high] -3 g (1.2 x 10 [high] -5 moles) of NHS and 0.25 ml of dry DMF were successively charged into a 1 ml reaction vessel. The reaction mixture was cooled on an ice bath and 2.5 x 10 [high] -3 g (1.3 x 10 [high] -5 moles) of ECDI was added under a nitrogen blanket. The reaction mixture was stirred at 4 ° overnight.

With cooling on an ice bath and with stirring, 0.23 ml of DMF was slowly added to 1.9 x 10 [high] -3 g (2.8 x 10 [high] -8 mol) of MDH in 0.83 ml of 0.05 M NaHCO 3 [deep] 3-Na [deep] 2CO [deep] 3 (pH = 9.0) added. Then the above ester solution (5.8 µl) was added with stirring, and stirring was continued for an additional hour while maintaining the indicated temperature. The conjugation mixture was subjected to gel filtration on three Sephadex G-50M columns measuring 0.9 x 13 cm, a 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 [high] R was filled with 0201 g (2.18 x 10 [high] -4 moles) of T-4-MEMIDA and 0.025 g (2.17 x 10 [high] -4 moles) of N -Hydroxysuccinimide (NHS) charged. Then 2 ml of dry THF and 30 μl (2.15 × 10 [high] -4 mol) of dry triethylamine were added and the reaction mixture was cooled to 0 ° with the aid of an ice bath. Then 0.048 g of ECDI (2.50 x 10 [high] -4 mol) added in powder form, and the reaction 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 [low] 1 values of 0.06 (T-4-MEMIDA) and 0.60 (T-4-MEMIDA-NHS ester) on an analytical silica gel plate, where 10% methanol in dichloromethane was used as a rinse aid. A 25 ml flask with a stir bar and membrane stopper was filled with 0.033 g (4.39 x 10 [high] -4 mol) glycine, then with 1.50 ml distilled water, 0.50 ml pyridine and 100 µl (1.00 x 10 [high] -4 moles) 1.0 n-NaCH charged. The reaction mixture was cooled to 0 ° with the aid of an ice bath. The T-4-MEMIDA-NHS ester solution prepared above was added dropwise with vigorous stirring, and after the addition was complete, the reaction mixture was stirred in a cold (2 °) 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 [deep] 2O, 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 [deep] 4. After filtering, the ethyl acetate was removed 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 desorbed with methanol / dichloromethane (1: 1). The silica gel was filtered off and the filtrate concentrated to 2 ml in vacuo. A thin-layer chromatogram of the product in THF showed only one spot with an R [fief] f value of 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 MEMIDA glycylglycine

A 3 ml Pierce Reacti vial was charged 0.202 g (2.20 x 10 [high] -4 moles) of T-4-MEMIDA and 0.025 g (2.17 x 10 [high] -4 moles) of NHS. Then 2 ml of THF and 31 μl of dry triethylamine were added and the reaction mixture was cooled to 0 °. Then 0.051 g (2.66 x 10 [high] -4 mol) ECDI was added and the reaction mixture was stirred in a cold room (2 °) for 8.25 hours. A thin-layer chromatogram indicated the formation of the T-4-MEMIDA-NHS ester (R [low] f 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 x 10 [high] -4 mol) glycylglycine and then with 1.50 ml H [low] 2O and 0.50 ml pyridine and 100 μl (1st , 0 x 10 [high] -4 moles) 1.0N NaOH. 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 way 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 R [low] f value was 0.81 when the plate was developed twice.

Example 12

T-4 MEMIDA glycine / MDH conjugate

A 1 ml Pierce Reacti flask with stir bar was charged with 0.049 g (5.0 x 10 [high] -6 mol) T-4-MEMIDA and 39 µl dry DMF. The reaction mixture was cooled to 0 °, whereupon 10 µl (5.2 x 10 [high] -6 mol) of a 5.0 x 10 [high] -1m NHS solution in DMF at 0 ° and 51 µl (6.1 x 10 [high] -6 mol) of a 1.2 x 10 [high] -1m ECDI solution in DMF at 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 R [low] f values of 0.49 (T-4-MEMIDA-glycine) and 0.68 (T-4-MEMIDA-glycine-NHS-ester) on analytical results Silica gel plates developed with triethylamine / methanol / dichloromethane in a ratio of 2.1: 10: 90. MDH (4.0 ml, 1.3 x 10 [high] -5 mol, 0.05 M NaHCO [deep] 3-Na [deep] 2CO [deep] 3, pH 9.2) was added to a 10 ml Round bottom flasks with stir bar and membrane stopper were filled at 0 ° and the enzyme activity was determined in the manner described above. 445 μl of dry DMF were added to the reaction mixture at a rate of 15 μl / min. added until a reaction mixture containing 10% DMF was obtained. The enzyme activity was then redetermined. The 4.3 x 10 [high] -2 molar T-4-MEMIDA-glycine-NHS ester solution was added to the reaction mixture in aliquots of 1 to 2 μl, 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 exhaustively dialyzed against 0.1 m K [low] 2HPO [low] 4 (with 1.0 × 10 [high] -3m NaN [low] 3) at 2 °. After dialysis, the enzyme conjugate was carefully removed from the dialysis bag and replaced by two

Sephadex-G-50M columns (in 1.0m K [deep] 2HPO [deep] 4, pre-swollen with 1.0 x 10 [high] -3M NaN [deep] 3) passed through. The sizes of the two columns were 0.9 x 54.0 cm and 0.9 x 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 x 10 [high] -5 mol) ECDI were dissolved in 0.50 ml of dry DMF to form a 1.6 x 10 [high] -1 molar solution. 0.75 g (6.5 × 10 [high] -4 mol) of NHS were dissolved in 1.0 ml of dry DMF to form a 6.5 × 10 [high] -1 molar solution. Both solutions were cooled to 0 ° in an ice bath before use. A 1 ml Pierce Reacti vessel with stir bar was charged with 2.4 x 10 [high] -3 g of T-4-MEMIDA-glycylglycine and then with 78 μl of ice-cold dry DMF (cooled in an ice bath), whereupon 4 μl of a 6 , 5 x 10 [high] -1m NHS solution in DMF at 0 ° and 18 µl of a 1.6 x 10 [high] -1m ECDI solution in DMF at 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 at R [low] f values of 0.25 (T-4-MEMIDA-glycylglycine) and 0.59 (T-4-MEMIDA-glycylglycine-NHS ester) on analytical silica gel plates, which were developed in triethylamine / methanol / dichloromethane in a ratio of 2.1: 10:90. 4.0 ml of MDH in a concentration of 1.5 x 10 [high] -5 mol in 0.05m NaHCO [deep] 3-Na [deep] 2CO [deep] 3 solution (pH = 9.2) were in placed a 10 ml round bottom flask with stir bar and membrane stopper. That

The reaction mixture was cooled to 0 ° with an ice bath and 440 µl of dry DMF was added at a rate of 50 µl. The enzyme activity was determined before and after the addition of the DMF. The 1.9 × 10 [high] -2 molar T-4-MEMIDA-glycylglycine-NHS ester solution was added to the reaction mixture in aliquots of 3-10 μl, whereupon the enzyme activity was determined after each addition. With the addition of 29 μl of activated ester solution, an enzyme conjugate which was 82% deactivated was obtained. The reaction mixture was then exhaustively dialyzed against 1.0m K [deep] 2HPO [deep] 4 (with 1.0 × 10 [high] -3M NaN [deep] 3) at 2 °. After dialysis, the conjugate was passed through three Sephadex G-50M columns (pre-swollen in 1.0m K [deep] 2HPO [deep] 4 with 1.0 x 10 [high] -3M NaN [deep] 3) and with eluted in the same buffer. The column dimensions were 0.9 x 55 cm, 0.9 x 56 cm and 0.9 x 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. First, 2 mg conjugate was injected. Then 0.25 mg conjugate was injected every two weeks. In sheep, the total single injection was 2 ml, of which 0.5 ml was the conjugate dissolved in saline, and 1.5 ml was Freund's complete acjuvant. 0.25 ml aliquots were injected subcutaneously in 4 sites each and 0.5 ml aliquots were injected intramuscularly in each hind leg.

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

The animals are normally bled 5 to 7 days after each injection and the antibodies are isolated by methods known in the art.

To demonstrate the utility of the thyroxine-MDH conjugate for T-4 analysis, a number of analyzes were performed. In the first series of analyzes, the analyzes were carried out with varying amounts of antibody in order to show the increase in the activity of the enzyme conjugate with increasing amounts of antibody. In a second series of analyzes, varying amounts of T-4 were added to the antibody in order to change the effective concentration of the antibody which is available for binding to the MDH-bound thyroxine. These results show that by establishing a standard curve from samples with known amounts of thyroxine, one can determine the amount of free thyroxine in serum by relating the observed levels of enzyme activity to the standard curve.

The analytical procedure is as follows:

0.8 ml of a solution of 0.1% by weight rabbit serum albumin (KSA) in a 0.1 molar glycinate buffer (pH = 9.5) containing 10 [high] -3 mol EDTA is made from the antibody solution and the MDH -bound-T-4 made. The solution is incubated for 45 minutes, after which substrates (100 μl 2m malate and 100 μl 0.108m NAD) are added; the solution is in Brought to a spectrophotometer and read the values 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

Conjugate of Example 7

1. About 9.0 x 10 [high] -6m Ab [low] T-4, based on the binding sites K = 1.12 x 10 [high] -9

2. Molar concentration of enzyme

c - 5 x 10 [high] -10

d - 2.56 x 10 [high] -9

e - 1.4 x 10 [high] -8

f - 3.7 x 10 [high] -8

A series of analyzes has now been 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 carried out by combining 600 μl of 0.1% rabbit serum albumin, 100 μl of antibody in 0.1m glycine buffer (pH = 9.5), 10 [high] -3m EDTA and 100 μl of the T-4 solution, 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 III [high] 3

1. Volume and concentration of the enzyme conjugate used

c - 15 µl 1.11 x 10 [high] -7 molar

d - 5 µl 5.12 X 10 [high] -7 molar

e - 5 µl 2.81 - 10 [high] -6 molar

2. Anti-thyroxine 9 x 10 [high] -6 molar at binding sites, diluted 1:33 before adding to 100 µl

3.

Following the procedure of Example 7, another conjugate was prepared which was chromatographed on a Sephadex G-50 column equilibrated with 1 molar potassium monohydrogen phosphate solution. The flow rate was slow and fractions of 20 drops were collected to tube 39, whereupon fractions of 99 drops were collected. The procedure was repeated with a second G-50 column, each time concentrating the collected fractions with Sephadex G-200 by ultrafiltration. The number of T-4 groups per enzyme was determined by UV analysis to be 4.3 to 5.3 (average 4.8).

A series of analyzes were performed using various sources of antiserum and varying concentrations of thyroxine. As described in the procedure given above, the antibody and the thyroxine were combined in 0.1%, glycine-buffered KSA solution and incubated for 15 minutes, after which the indicated amount of the enzyme solution was added; the analysis mixture was incubated again for 10 minutes, the substrates added and the mixture was then introduced into a spectrophotometer, the change in the optical density units being read off in the second minute. The volume of the added antibody is 1 µl, and the volume of the added enzyme is also 1 µl. The results are given in the table below.

Table IV [high] -1

1.

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

Reagents for TIM-T-4 analysis

Buffer: 0.02 M triethanolamine (TEA) -HCl, pH = 7.9

0.0054m ethylenediaminetetraacetic acid (EDTA)

Rabbit serum albumin solution: 0.2% by weight KSA, in buffer solution

TIM-T-4 solution: About 7 x 10 [high] -3 µmol T-4 than TIM-T-4,

in KSA solution (results in an analysis speed of approx

200 OD units / min).

Anti-T-4 solution: About 4 x 10 [high] -5m Anti-T-4, based on 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 weight ratio

1: 7 with buffer solution)

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

DL-glyceraldehyde-3-phosphate diethylacetal, Ba salt from Sigma

Chemical Co., St. Louis, Mo .: 1.5 grams of salt was treated so that

a final volume of 10 ml was obtained. small alpha-glycerophosphate dehydrogenase (small alpha-GPDH) solution:

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

Thyroxine (T-4 solution): 2.57 x 10 [high] -4m 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 were mixed with one another and placed in a water bath at 30 ° C. for 12 minutes. 200 µl of the NADH solution, 25 µl of the small alpha-GPDH solution and 100 µl of the GAP solution were then added to the 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 366nm and 30 ° C. The remainder of the analysis solution was kept in a bath at 30 ° C. and read off after 13 minutes. The results are given in the table below.

Table V

The results of the above tables show that extremely low concentrations and extremely low amounts of thyroxine can be detected or determined by the method according to the invention. The process is very simple and requires only a few handling steps. By combining the reagents in a buffered medium and, if necessary, incubating the mixture and then adding the enzyme substrates, the T-4 can be determined by a spectrophotometric reading over a short period of time. The system can be automated so that the samples and reagents can be mixed automatically and the readings taken automatically.

Claims (27)

1. A method for detecting or determining a ligand, which is a polyiodothyronine with 3 to 4 iodine atoms, in a medium which is suspected to contain the ligand, characterized in that in an aqueous liquid zone (1) the medium; (2) a soluble ENZ-linked ligand; and (3) brings together an antiligand, the antiligand having sites that are common to the ligand and the ENZ-bound ligand and can bind to them, where ENZ means an enzyme that is reversibly inhibited by the ligand, and where the Anitligand is present in such a concentration that a substantial increase in enzyme activity occurs in the absence of the ligand; and that the effect of the medium on the enzymatic activity of the ENZ-bound ligand is investigated in said zone. 2. The method according to claim 1, characterized in that the ligand used is thyroxine. 3. The method according to claim 1 or 2, characterized in that malate dehydrogenase is used as ENZ to generate MDH-bound ligands. 4. The method according to claim 2, characterized in that once used as ENZ triosephosphate isomerase. 5. The method according to any one of claims 1 to 4, characterized in that the aqueous liquid zone is buffered at a pH in the range from about 7 to 10.5, and that the ENZ-bound ligand is less than about 50% of the enzyme activity of ENZ prior to conjugation to form the ENZ-bound ligand, but not less than about 0.5% of the enzyme activity of the ENZ prior to conjugation. 6. The method according to claim 5, characterized in that the ENZ used is malate dehydrogenase and the enzymatic activity is determined by adding NAD and malate to the aqueous liquid zone at a pH in the range from 8.5 to 10.5. 7. The method according to claim 5, characterized in that there is used as ENZ triosephosphate isomerase and the enzymatic activity by adding glyceraldehyde-3-phosphate, small alpha-glycerophosphate dehydrogenase and NADH to the aqueous liquid zone at a pH in the range from 7 to 9 determined. 8. The method according to claim 3, characterized in that the medium and the antiligand are combined before the addition of the MDH-bound ligand. 9. The method according to claim 3, characterized in that the MDH-bound ligand has about 1 to 10 ligands per MDH, the MDH is mitochondrial MDH and up to about 65-99.5% of the enzyme activity of the MDH before conjugation for formation of the MDH-bound ligand is deactivated. 10. The method according to claim 3, characterized in that the MDH-bound ligand is obtained from mitochondrial MDH. 11. A method for detecting or determining a ligand which is thyroxine in a medium which is suspected of containing this ligand, characterized in that in an aqueous buffered liquid zone at a pH in the range of 6-10.5 (1) the medium; (2) ENZ-linked ligands; (3) Antiligand brings together, wherein the antiligand has sites which are common to the ligand and the ENZ-bound ligand and which are able to bind to them, where ENZ is a dehydrogenase enzyme which is reversible by the ligand is inhibited, and wherein the antiligand is present in a concentration which causes a substantial increase in enzyme activity in the absence of the ligand; and that the influence of the medium on the enzymatic activity of the ENZ-bound ligand is analyzed in this zone with NAD or NADH. 12. The method according to claim 11, characterized in that ENZ represents malate dehydrogenase and the analysis is carried out with NAD and malate. 13. The method according to claim 12, characterized in that first the medium and the antiligand and then the ENZ-bound ligand are brought together. 14. ENZ-bound ligand with the formula where the symbols have the following meanings: ENZ is an enzyme reversibly inhibited by the ligand; a is 0 or 1; n averages at least 1 and no more than about 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-1') -phenyl-1, 3,5-diiodo-4- (3'-iodo- or 3 ', 5' -diiodo-4´-hydroxyphenoxy-1´) -phenyl-1 or 3,5-diiodo-4-hydroxyphenoxy; 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 being between about 0 and 4. 15. ENZ-bound ligand according to claim 14, characterized in that ENZ is triose phosphate isomerase. 16. ENZ-bound ligand according to claim 14, characterized in that ENZ is a dehydrogenase. 17. ENZ-bound ligand according to claim 16, characterized in that the dehydrogenase is malate dehydrogenase. 18. ENZ-bound ligand according to claim 17, characterized in that n 1 to 8, D is an alkyl group with 1 to 3 carbon atoms, Z 3,5-diiodine-4- (3 ', 5' -diiodo-4 '- hydroxyphenoxy-1´) -phenyl-1 and A denotes an aliphatic organic divalent radical with 1 to 10 carbon atoms and 0 to 2 heteroatoms in the chain between the non-oxo-carbonyl groups, the heteroatoms being oxygen and nitrogen, which are known as oxy- , tert-amino or amido groups are present and a total of 0 to 4 heteroatoms, namely oxygen or nitrogen, are present. 19. ENZ-bound ligand according to claim 14, characterized in that n an average of 1 to 8, X and Y chalcogen, D 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. 20. MDH-bound ligand which is reversibly inhibited by the ligand and has the following formula where the symbols have the following meanings: MDH is malate dehydrogenase; n [high] 1 is average in the range from about 1 to 8; X [high] 1 and Y [high] 1 which are the same or different from one another, are oxygen, sulfur or imino; p, q and r are each 0 or 1; s is 0 to 2; Small alpha and small gamma are hydrocarbon groups with 1 to 8 carbon atoms, small beta is an oxy, thio, sulfonyl or alkylamino group, the alkyl group containing 1 to 3 carbon atoms, with the proviso that small beta is an amino or alkylamino group when p is 0; is wherein T [high] 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 or 3,5-diiodo-4-hydroxyphenoxy-1; and D is hydrogen or an alkyl group having 1 to 6 carbon atoms. 21. MDH-bound ligand according to claim 20, characterized in that s = 0, small beta an alkylamino group, small alpha and small gamma 1 to 3 carbon atoms and p, q and r each denote 1. 22. MDH-bound ligand according to claim 20, characterized in that small beta is an oxy group, small alpha and small beta 1 to 3 carbon atoms, s = 0 and p, q and r are each 1. 23. MDH-bound polyiodothyronine with 3 to 4 iodine groups per thyronine and 1 to 10 thyronine groups that are bound to MDH. 24. MDH-bound polyiodothyronine according to claim 23, characterized in that the polyiodothyronine is thyroxine and 2 to 6 thyroxine groups are bound to MDH. 25. MDH-bound polyiodothyronine which is reversibly inhibited by the polyiodothyronine and an average of about 1 to 10 polyiodothyronines of the formula which is bonded to the amino group through a non-oxo-carbonyl group, including its nitrogen and sulfur analogues, to MDH, 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-iodine-4- (3´, 5´-diiodo-4´-hydroxyphenoxy-1´) -phenyl-1 or 3,5-diiodo-4- (3´-iodine- or 3´, 5´ -diiodo-4´-hydroxy-phenoxy-1´) -phenyl-1. 26. MDH-bound polyiodothyronine, which is reversibly inhibited by the polyiodothyronine, with the formula in which the symbols have the following meanings: t is 0 or 1; n [high] 1 is an average of 1 to 10; MDH 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´-hydroxy-phenoxy-1´) -phenyl-1. 27. TIM-bound polyiodothyronine, which is reversibly inhibited by the polyiodothyronine, with the formula where the symbols have the following meanings: TIM is triosephosphate isomerase; a is 0 or 1; n averages at least 1 and no more than about 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-iodine-4- (3´, 5´-diiodo-4´-hydroxyphenoxy-1´) -phenyl-1 or 3,5-diiodo-4- (3´-iodine 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, the total number of heterofunctionalities being in the range from 0 to 4.