CH632343A5 - Process for the detection or for the determination of polyiodothyronines - Google Patents

Description

632343

PATENT CLAIMS

1. A method for the detection or determination of the thyroid hormones triiodothyronine and thyroxine in a medium suspected of containing these hormones, characterized in that in a buffered aqueous liquid zone, the medium, an inhibited enzyme and one for the brings together the hormone-specific antibody to be determined, the enzyme being reversibly inhibited by direct binding or via a coupling component with an inhibitor which can be bound to the antibody, and the antibody being present in a concentration such that a substantial increase in the enzyme activity in Absence of free hormone occurs; and that the effect of the medium on the enzymatic activity of the reversibly inhibited enzyme is investigated in the said zone in the presence of enzyme substrates.

2. The method according to claim 1, characterized in that one detects or determines thyroxine.

3. The method according to claim 1 or 2, characterized in that the inhibited enzyme is inhibited malate dehydrogenase.

4. The method according to claim 2, characterized in that the inhibited enzyme is inhibited triose phosphate 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 7 to 10.5, and in that the inhibited enzyme is less than 50% of the enzyme activity before the inhibition, but not less than 0.5% of the enzyme activity before inhibition.

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

8. The method according to claim 3, characterized in that one combines the medium and the antibody before the addition of the inhibited malate dehydrogenase.

9. The method according to claim 3, characterized in that the inhibited malate dehydrogenase has 1 to 10, inhibitor molecules per malate dehydrogenase, the malate dehydrogenase is mitochondrial malate dehydrogenase and up to 65-99.5% of the enzyme activity of the malate dehydrogenase is deactivated before the inhibition.

10. The method according to claim 3, characterized in that the inhibited malate dehydrogenase is mitochondrial malate dehydrogenase.

11. The method according to claim 2, characterized in that the aqueous liquid zone is buffered at a pH in the range from 6 to 10.5, that the enzyme is a dehydrogenase enzyme and that the enzymatic activity with NAD or NADH determined.

12. The method according to claim 11, characterized in that the inhibited enzyme is inhibited malate dehydrogenase and the analysis is carried out with NAD and malate.

13- Method according to claim 12, characterized in that first the medium and the antibody and then the inhibited enzyme are brought together.

14. The method according to claim 1, characterized in that one of the formula as an inhibited enzyme

ENZ-

X Y C0oD

11 "x 11 I 2

C = A) -CNH-CHCH_ Z

a d.

n used, in which the symbols have the following meanings: ENZ is a reversibly inhibited enzyme; a is 0 or 1; n is on average at least 1 and not more than 10; X and Y, which are the same or different, are chalcogen io or imino; D is hydrogen or an alkyl group of 1 to 6 carbon atoms; Z is 3-iodo-4- (3 ', 5' -diod-4 '-hydroxyphenoxy-1') -phenyl-l, 3,5-diiod-4- (3'-iodo- or 3 ', 5'-diiodo-4'-hydroxyphenoxy-1 ') -phenyI-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 heterofunctional groups being between 0 and 4.

20th

The concentration of thyroxine in the blood is critical for the proper functioning of the body within relatively narrow limits. 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 Trijodthy-30 ronine differ from thyroxine (T-4) in that the iodine is missing in the 5- or 5'-position. It is believed that (T-3) has a marked effect on the overall metabolic interactions of the thyroid hormones.

One method for determining the thyroid hormones 35 is radioimmunoanalysis (radioimmunoassay). Although this 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 hormone. The amount of the "hot" hormone that is bound to the antibody or remains free is a function of the amount of the hormone in the serum. The amount of hormone, based on standards 45 with known amounts of hormone, can be calculated by determining the radioactivity of the antibody-freed solution.

Radio immunoanalysis requires a separation stage through which errors can be introduced and which can be very time-consuming. Furthermore, radioactive substances have to be used which decay 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 reduced to a minimum 55 while ensuring high sensitivity.

US Pat. No. 3,817,837 describes an enzyme analysis (enzyme assay) which has generally proven to be useful for a large variety of ligands. The invention now relates to a homogeneous enzyme immunoanalysis method with which the thyroid hormones (thyroid hormones) T-3 and T-4 can be detected or determined. The method according to the invention is defined in claim 1.

65 In the method, an enzyme reagent in the form of an inhibited enzyme is used, in particular an enzyme-bound T-3 or T-4 (EBP), in which the enzyme, after conjugation with the inhibitor, preferably to more than 50%

3 632343

tiviert and that after binding of the antibody, d. H. of an enzyme has this activity, especially malate dehydrogen receptor for T-3 and T-4, partially or completely reactivated nose, and again the mitochondrial, preferably that. The conjugated enzyme used thus has a low mitochrondic malate dehydrogenase (MDH) from swine conversion rate, which in the presence of a receptor, heart; and triose phosphate isomerase, particularly triose of an antibody to T-3 and T-4, increases significantly. 5 phosphate isomerase (TIM) from rabbit muscles. Ordinarily, the analysis is carried out, for example, by connecting the polyiodothyronine to the serum sample, the appropriate receptor, EBP and the enzyme enzyme via a connecting group which is connected to the amino group of the polyiodothyronine to be determined in a buffered aqueous zone , wherein the carboxy group is esterified, combined into the substrate and the speed of the particular one with a lower alkyl group, which usually determines 1 reaction catalyzed by the enzyme. Contains up to 3 carbon atoms. The poly can expediently be bound by one or more incubation periods between iodothyronine by a functional group which, seen after the addition of the individual reagents, contains two carboxyl functions. Be related in this context. By establishing standards with known amounts of carboxyl function, the oxygen-containing T-3, reciprocal T-3 or T-4, one can obtain a carboxy group calibration curve with which the unknown samples can be related.

In the EBP compositions, on average, at least one inhibitor is bound to the enzyme via an aliphatic carbon atom or a carboxyl group (or a nitrogen or thio analogue thereof), 20 the nitrogen-containing imidine group being an amide (amidine or thioamide) and / or an ester is formed. IT

With the preferred embodiments described below, II

Forms of the process according to the invention can be poly- - C - OH

iodothyronine (T-3 and T4) at concentrations of only 10 ~ 7 "25

molar or below can be determined. Analogous to the invention described in the sulfur-containing thionocarboxy group of U.S. Patent 3,817,837

T-3 and T4 the ligands, the enzyme-bound ligand is e.g. B. S

enzyme-linked T-3 or T-4, and the anti-ligand is an anti

body for T-3 or T4. In particular, it is the 30-U-UH

Enzymes for malate dehydrogenase or triose phosphate isomerase. and their derivatives, e.g. B. amides, esters and anhydrides.

In a preferred enzyme immunoanalysis, the number of polyiodothyroid conjugated to the enzyme

Reagent inhibited enzyme (in particular T-3 and T4-inhibin is on average at least 1 and not more than beer), a receptor for polyiodothyronine, namely anti-T-3 and 35 about 10 per enzyme, usually 1 to 8 and usually 2 to 6. Anti-T4, substrates for the enzyme and an aqueous, normal- The polyiodothyronines can be bound to available sites as alkaline buffer medium, which is usually one or more (e.g. amino or hydroxy groups to form amino contains additional additives for improving the enzyme stability, for the and esters), by means of appropriate linking group determination of the product of the enzyme reaction or the same used by the amino group or the carboxy group (in particular. The order in which the reagents are added 40 particular from the amino group of the polyiodothyronine) to the aqueous analysis medium is not critical, although hen. Because the amino group is protected during conjugation, certain procedures are preferred. Furthermore, if the carboxyl group is involved in the incubation after the addition of certain reagents, the linking groups will be desirable in most cases. most cases appended to the amino site. Will be appropriate

After all of the reagents have been added, the reaction ester of the polyiodothyronine is used, and a group speed of the enzyme is followed, usually spectrophotometrically with a carboxyl functionality (see the definition above. By comparing that with the unknown substance), conjugation becomes to the amino and hydroxyl group results obtained using the known ones of the enzyme.

Results obtained from T-3 and T-4 can be used as a binding or a connecting or linking

Determine the amount of T-3 and T-4 in the unknown sample. 50 group can combine the polyiodothyronine with the enzyme.

The reagents are first described below. The connecting group as such is not the actual analytical method for the invention. critical, although certain classes of connecting groups are preferred. The number of atoms in the connecting reagent group (except hydrogen) is generally not

Inhibited enzymes 55 more than 20, usually not more than 16; the connecting

In the preparation of the inhibited enzyme one can use the group 0 to 7, usually 1 to 6 heteroatoms, in particular inhibitor directly, the carboxyl group, especially chalcogen atoms (O and S), which are normally only attached to the available groups of the enzyme , e.g. B. lysine and tyro-carbon are bound, and contain nitrogen, which is only sine, should be bound, activated, or preferably is bound to carbon and hydrogen and neutral to the available functionalities such. B. the amino group or 60, when bound to hydrogen, e.g. B. in the form of ami-carboxy group, especially the amino group, an extensible nitrogen. The specially used enzyme can be generated by a chain. The phenolic hydroxyl group will be reversibly deactivated as polyiodothyronine so that the conjugation site is not used. Enzyme in the absence of the polyiodothyronine receptor

Useful enzymes are those that, when conjugated, less than about 50%, usually less than about 35%, and practically reversibly deactivated with a polyiodothyronine 65 usually less than 25% of its original enzyme, so that when bound to an antibody for activity, of conjugation, but at least 0.5, polyiodothyronine activity is largely recovered, usually at least 1% of the original enzyme activity. It was found that only a certain group of pre-conjugates were present. With the addition of

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closed receptor for polyiodothyronine to the enzyme conjugate increases the enzyme activity by at least 50%, usually by at least 100% and often by 200% or more. The value of the increase in activity largely depends on the initial decrease in activity during conjugation. Other enzymes, especially dehydrogenases, which are inhibited by thyroxine are given in Wolff and Wolff, Biochimica et Biophysica Acta, 26, 387 (1957). These enzymes are e.g. B. to glutamine dehydrogenase, lac-tatdehydrogenase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase.

In general, enzyme-linked polyiodothyronine (EBP) has the formula below

ENZ =

'W 0CGHGH „Z l 2

(NHW)

m x

where the symbols have the following meanings:

ENZ is an enzyme that is particularly reversibly inhibited by T-3 and / or T4, so that the enzyme is activated when the inhibitor is activated by a receptor, i.e. H. an antibody is bound; the enzyme is generally a dehydrogenase or a triose phosphate isomerase; x is 0 or 1, Z is

J J

wherein m10 or 1; G and G1 are hydrogen or iodine, with the proviso that G1 is iodine when m10 and that there are at least 3 iodine atoms when m11 is; one of W and W1 is a connecting group, in particular an aliphatic group with 0 to 1 ethylenically unsaturated positions and 1 to 20 atoms other than hydrogen, which are carbon, chalcogen atoms or nitrogen, the chalcogen atoms being oxy- or oxo atoms (including the thio analogues) are only bonded to carbon, in particular car-boxyl; and wherein the nitrogen is only bound to carbon and hydrogen and is neutral when bound to hydrogen, e.g. B. as an amido nitrogen; the number of hetero atoms 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 normally no more than 5, usually no more than 4 and preferably 2 to 3 functionalities in the chain of the connecting group, e.g. B. tert-amino, oxy,

Amido etc., with the proviso that W1 can be a bond, especially for x - 0; if it is not a connecting group, W is hydrogen and W1 is a hydroxyl group or an alkoxy group with 1 to 3 carbon atoms, in particular the methyl group; and m is on average at least 1 and not more than 10, usually 1 to 8, usually 2 to 6.

The EBP used can in particular have the formula given below, in which the symbols have the following meanings: ENZ is an enzyme which is inhibited in particular by T-3 and T-4, so that the enzyme is activated when the inhibitor is bound by a receptor becomes; the enzyme is common to a dehydrogenase, especially malate dehydrogenase, or triosephosphate 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, are chalcogen (O or S) io 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- (hydroxyphenoxy-l ') phenyl-l or i5 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 not more than 12 carbon atoms, usually not more than 10 carbon atoms, with 0 to 12, 20 usually 1 to 10, usually 2 to 9 and preferably 3 to 7 atoms in the chain (in the case of cyclic groups, the larger number of ring atoms is included) are present between the carboxyl groups or analogs, and 0 to 4, usually 0 or 1 to 3, usually 1 to 2 heteroatoms in 25 the chain is present, 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, in particular 1 to 3, these being chalcogen 30 (O or S) (as carboxyl, thiocarboxyl, oxy, thio or Sulfonyl) or nitrogen (imino, tert-amino or amido); there are usually 2 carbon atoms in the chain between the heteroatoms; A can be a hydrocarbon group (an aliphatic, alicyclic, aromatic or a combination of these groups) or a non-hydrocarbon group (a substituted aliphatic, alicyclic, aromatic, heterocyclic or a combination of these groups), generally 0 to 1 cyclic groups present in the chain, usually 5 to 6 ring members 40 with 0 to 2 hetero ring members, usually with 0 to 1 hetero ring members, the substituents of the cyclic group usually being separated by 2 to 4, usually 3 to 4 ring members; normally the only aliphatic unsaturation in the hydrocarbon group is from 0 to 1 45 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.

so 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-linked T-3-55 or T4 compositions which have a di-carboxyl link with an aliphatic chain which is not interrupted by heteroatoms or which have about 1 to 2 heteroatoms, namely chalcogen and nitrogen. These compounds generally have the formula given below 6o

ENZ-

X Y C0oD II s II I 2 C = A) -CNH-CHCH0Z

n e? X1 Y1 C02D

ENZ- (C- d -oc - B - r - C-NHCHCH0Z) 1 s p 'q' r 2 n

5

632343

where the symbols have the following meanings: ENZ, D and Z are as defined above; n1 has the same limit values as n, but is on average between 1 and 10, preferably between 1 and 8 and in particular between 2 and 6; X1 and Y1 have the same meaning as X and Y, respectively, where X1 5 is normally chalcogen and Y1 is preferably oxygen; p, q and r are each 0 or 1, the sum of p, q and r preferably being at least = 1 (p + q + r ^ l); sistObis 2, usually 0 or 1; a and y 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 in particular 2 to 6 carbon atoms present; for p or r = 0, a or y preferably contain 1 to 8 carbon atoms, 15 in particular 1 to 6 carbon atoms; a and y can be aliphatic, alicyclic, aromatic or heterocyclic; together with ß and 8 they can meet the definition of A; preferably a and y are saturated aliphatic groups, especially unbranched groups, e.g. B. methylene or polyethylene, or aromatic, especially monocyclic groups, only one of a and y being an aromatic group; β is oxy (-0-), thio (-S-), sulfonyl (-SO2-) or

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 positions and 0 to 1 heteroatoms with the atomic numbers 7 to 8, the heteroatoms being only bonded to carbon atoms and 1 to 6 carbon atoms present; and E is hydroxyl or can form a double bond together with a terminal nitrogen atom of R, e.g. B. isocyanate or isothiocyanate.

Another useful series of compounds has a car-bocyclic or heterocyclic ring with 5 to 6 ring links in the chain, the heterocyclic ring containing 1 to 2, usually 1 hetero ring link, which is O, S or N. These compounds preferably have an isocyanate or isothiocyanate group which is directly or indirectly connected to a ring member, especially when it is an aromatic ring. In most cases, these compounds have the formula given below

Y2 CCLD 0 II I

X = C = NArGNHGHCH2Z

Amino (-N-), wherein T is an alkyl group of 1 to 6 carbon atoms, usually 1 to 3 carbon atoms, or hydrogen; if β is linked to a carboxyl function (p = 0), then β is an amino or alkylamino group; and ô is

0 II

-CH-NH-C-

I1

wherein T1 is hydrogen or a lower alkyl group having 1 to 3 carbon atoms.

In a preferred subset, the inhibitor can be combined with a dicarboxylic acid to form an amic acid (monoamide of a dicarboxylic acid). The dicarboxylic 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 atomic numbers 7 to 8 (nitrogen or oxygen), the nitrogen or oxygen atoms being connected only to carbon atoms, i. H. in the form of tertiary amino groups or ether groups. Preferred dicarboxylic acids form cyclic anhydrides with 5 to 7, in particular 5 to 6, ring members. In order to ensure that the carboxyl group of the dicarboxylic acid conjugated with the inhibitor is the one that reacts with the ENZ, the original carboxyl group of the inhibitor is normally esterified with an alkyl group with 1 to 3 carbon atoms, preferably with one carbon atom (methyl) .

The modified inhibitor used to conjugate with the ENZ generally has the formula given below

Z-GHo-CH-C0oD '

I 2 2

NH X (CY) (RG) aE

wherein the symbols have the following meanings: X, Y, Z and a are as defined above; D 'is hydrogen or one

25 where the symbols have the following meanings:

D and Z are as defined above; X2 and Y2 are chalcogen (O or S), with X2 preferably S; Ar is an arylene or aralkylene group, including carbocyclic and heterocyclic groups with 5 to 6 ring members, 1 to 30 2, preferably 1 hetero ring members, namely O, N and S respectively, and 4 to 10 carbon atoms, usually 4 to 8 carbon atoms, and if they are heterocyclic compounds, preferably 4 to 6 carbon atoms, and if they are carbocyclic compounds, 35 preferably 6 to 8 carbon atoms.

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

X

G0 "D I 2

ENZ- (C-NHA rGNHGHGH2 Z) 1

where all symbols are as defined above. 45 The modified inhibitor is used in an active or activated form so that it is able to interact with available carboxyl-reactive groups from ENZ, e.g. B. amino and hydroxyl groups to react.

For the carboxy group, a mixed anhydride, such as N-hydroxysuccinimide, a p-nitrophenyl, a phenylthio etc. ester derivative or a carbodiimide is used as a 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 55 in the range of about -5 to 30 ° C, using a mixed aqueous buffer medium which usually has a mild alkaline pH in the range of about 7 to 10. As an auxiliary solvent z. B. hexamethyl phosphoramide can be used. This is usually used in 60 amounts from about 10 to 40%, preferably from about 20 to vol%.

The molar ratio between the inhibitor and the ENZ molecules is generally between about 1: 1 to 20: 1, usually between about 3: 1 to 10: 1.

The inhibitor can be added in several 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 a conventional manner.

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6

The mixture is preferably chromatographed in order to separate all unreacted inhibitors from the ENZ-bound inhibitor. A convenient chromatographic material is Sephadex. The fractions can be isolated, their enzyme activity determined and the fractions with enzyme activity can be collected. Instead of the modified T-3 or T-4, which is formed by combining the T-3 or T-4 with a dicarboxylic acid, Desamino-T-3 or -T-4 can be conjugated with ENZ. With the T-3 and T-4, the amino group must be protected when ENZ is conjugated with the amino acid, while with de-aminopolyiodothyronine no protective group is required and the carboxy group can be activated in the same way as the other car-boxy groups.

The desaminopolyiodothyronine or its monocyclic analog are defined by the formula given below:

H02CCH2 (CH2) tZ

wherein Z has the meaning given above and is 10 or 1; the ENZ conjugate of these compounds has the formula given below:

ENZ- (COCH2 (CH2) tZ) nl where all symbols are as defined above.

The ENZ-linked T-3 or T-4 is normally at least about 50% deactivated (compared to pre-conjugate enzyme activity), usually at least about 65% and not more than about 99.5%. Appropriately, the enzyme should be almost completely deactivated and its activity should be restored almost completely when excess anti-T-3 or -T4 is added. However, it has been found that the conjugated enzyme is activated 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. The main issue is the range of the measured units between ENZ-bound-T-3 or -T4 in the absence of the antibody or in the presence of the excess antibody. An excess is understood to mean that there are sufficient antibodies to bind practically all available T-3 or T4.

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

Anti-T-3 or -T-4

The appropriate antibodies are raised by injecting an antigen bound to T-3 or T-4 into a vertebrate, usually a pet, e.g. B. a sheep, rabbit or goat. Since the antigen is usually a polypeptide or protein, which usually has a molecular weight of about 5000 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 analogue as used for conjugation with ENZ can be used. In addition to the analogs used to prepare 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 for producing the antigens can be used. The antigens have at least one analog molecule, preferably one molecule to about 2000 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 complete Freund's adjuvant with the booster injection.

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

buffer

A wide variety of buffers can be used, e.g. B. 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 additives

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

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

Analytical procedure reagent solutions

The buffer solution used normally has a concentration such that the concentration in the analysis medium is 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 expediently an albumin, such as rabbit serum albumin and / or gelatin, is in the finished analytical 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 .-%, in front. 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 IO-5 to 10 ~12 molar, usually about 10 107 to 10 1011 molar. The antibody concentration depends on the relationship between the antibody binding sites and the concentration of inhibitors bound to ENZ. In general, the ratio of the binding sites to T-3 or T-4 (as ENZ-linked 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 account for 25 to 75, preferably 25 to 50% of the recoverable enzyme activity

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

7

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win.

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 may be present in amounts of about 0.001 to 0.1% by weight, sodium azide, in amounts of about 10 -5 to 10 -3 molar and a surfactant such as Triton X-100, which can be present in amounts from about 0.0001 to 0.03% by weight.

If MDH is used, depending on the direction of the reaction, either malate and NAD or oxaloacetate and NADH are used, the former substances being preferred. The concentration of these substances directly affects the sensitivity of the analysis and must be determined empirically in order to optimize the difference in 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. Usually the concentrations of enzyme, antibodies and substrates are chosen to optimize the sensitivity of the analysis.

When TIM is used, the glyceraldehyde phosphate is generally present in a concentration in the range of about 10 "1- to 10 ~ 4 molar; a-glycerophosphate dehydrogenase is generally used in amounts of about 10 ~ 5 to 10 ~ 9 molar while the NADH concentrations are generally between about 10-2 and 10_4 molar.

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

The analysis levels

The order of combination of the individual reagents is not critical, although some orders are preferred and in some cases a particular order is preferred over another. After adding a certain reagent or after adding all reagents, an incubation can be carried out. The analysis mixture is usually incubated no more than 10 minutes in the presence of the enzyme substrates before the start of the speed measurement.

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

Depending on the order of addition, the events are different. If the sample and the antibody are combined, the hormone to be determined is bound to the antibody, the available sites for binding to the ENZ-bound T-3 or -T4 being reduced. 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 T-3 or T-4 in that 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 analytical mixture can be incubated after each addition of a reagent, either before or after the addition of the enzyme substrates or other reagents. The incubation can last from about 2 minutes to 1 hour and is generally carried out at temperatures in the range from about 15 to 40 ° C, usually at about 25 ° C (room temperature) to 37 ° C.

After adding the mixture to the substrates for the enzyme, the mixture can be incubated up to about 10 minutes before the initial reading; this reading can be done 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. Measuring periods of around 5 to 20 minutes are suitable for certain automated instruments.

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

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

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

example 1

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

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

The mixture was stirred for 15 minutes after which 0.279 g (2.16 x 10 -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 substance 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 suction filtered, the solvent was removed in a rotary evaporator, whereby a solid white substance was obtained, which was dissolved in 30 ml of THF. 35 ml of chloroform was added to the THF, the solution was heated under reflux and n-heptane was slowly added. The volume of the solution was reduced until a permanent turbidity appeared.

5

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35

40

45

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55

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632 343

8th

The solution was allowed to cool to room temperature and then freeze 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. 5

Example 2

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 light-protected flask was injected with 300 μl of triethylamine 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 15 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 with water from methanol, giving 1.53 g of substance (84%).

Example 3

(N-carboxymethyl-3-aza-3-methylglutaramic acid amide of 25 methylthyroxinate) conjugate to MDH.

A. A stirred solution of 0.201 g (0.22 mmol) of T-4-MEM1DA, 1 ml of dry THF, 25 mg (0.22 mmol) of N-hydroxysuccinimide and 30 ml of dry triethylamine from

0.048 g (0.25 mmol) of ECDI was added at 0 °. The mixture was stirred at 0 ° for 35 minutes, then at 4 ° for 14 hours.

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, the above-mentioned solution was vigorously at 0 ° C. Stir added dropwise. After stirring had continued at 4 ° C. in the dark for 36 hours, the solvent was removed in vacuo, the residue was dissolved in 5 ml of abs. Dissolved ethanol and evaporated the ethanol, the treatment with ethanol was repeated three times to obtain a white foamy solid. 40

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 each of ethyl acetate. The ethyl acetate layers were combined, extracted with 25 ml of saturated saline and dried over MgSC> 4. 45

The volatile substances were removed in vacuo and the residue was dissolved in 2 ml of methanol and chromatographed on silica gel (Et3N: CH30H: CHzCl2-2, 1:10:90). The product was desorbed with CH3OH / CH2CI2 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 / HCCla / cyclohexane, a yield of 0.046 g (21.6%) being obtained. 55

B. 4.9 mg of radioactive C14-labeled N-carboxymethyl-3-aza-3-methylglutaramic acid amide of methylthyroxinate, dissolved in 39 ml of 1 DMF, were introduced into a 1 ml vessel and cooled to 4 ° C. The DMF solution was stirred with 51

It was added to a 0.11 molar ECDI solution in DMF of 4 ° and 10 ^ 1 6o 0.5-molar N-hydroxysuccinimide solution in DMF of 4 °, and the mixture was stirred overnight in the dark.

C. Swine heart mitochondrial MDH was dialyzed against 0.05 M carbonate buffer (pH = 9.0) to obtain 4 ml of a 1.31 x 10 -5 molar solution of MDH. 445 ul of DMF was added to the solution at a rate of 15 ul / min. The ester prepared above was added in 2 jo.1 portions and the enzyme activity was determined after a waiting time of 5 minutes. In a first reaction 5 | il esters were used, in a second reaction 10jj.1 were added, whereby a ratio of thyroxine to MDH of 4.1 and 7.4 was set.

Both reaction mixtures were dialyzed three times against 300 ml of 1 molar KzHP04 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 (0.9x54 cm) which had been equilibrated with the phosphate buffer and eluted with the same phosphate buffer at a flow rate of 4 drops / min and in Fractions of 20 drops each were collected. 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 ali quota was determined and, assuming that no protein was lost, the number of haptens per protein molecule was 2.0 and 3.0, respectively.

Example 4

T-4-MEMIDA conjugate to malate dehydrogenase

10 mg (11 jiMol) of T-4-MEMIDA and 1.3 mg of N-hydroxysuccinimide were dissolved in 250 ml of dry DMF. The reaction mixture was kept under stirring under a nitrogen atmosphere at 0 °, after which 2.3 mg of ECDI were added. The mixture was kept at 0 ° until all of the ECDI had dissolved. The solution was left at 4 ° overnight.

A general procedure for the preparation of the conjugate with higher or lower hapten numbers is given, which depends on the amount of T4-MEMIDA-hydroxysuccin imide ester in relation to MDH. 1 ml of DMF was added to 4 ml of a stirred solution of MDH (from pig hearts, mitochondrial, Miles, 5.0 mg / ml) in carbonate buffer (pH = 9.2). Subsequent additions of the T-4-MEMIDA ester were made at approximately 60 to 90 minute intervals, aliquots being removed and assayed for enzyme activity. The table below shows the order of addition, the amount of additions, the time of addition, the ratio between T-4 and MDH added and the percent deactivation observed. For the determination of the conjugate numbers, 2 to 20 jj.1 of the conjugation mixture were added to 0.5 ml of 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 (0.1 m, pH = 9.5), the 100 ml 0.108 m NAD and 100 μl 2 m sodium malate solution ( pH = 9.5) were added, diluted to 0.8 ml. The speeds were measured between 60 and 120 seconds after introduction into a Gilford spectrophotometer, model 300-N at 30 °.

Time of addition of total T4 / MDH T4-2%

of lumen1 conjugate deactivation

T-4 esters | xl

Ml

10:34 13 4996 0/1 0

11:24 5007 2/1

5003 2/1 30.1

1 ml taken as conj.c 1.3 11:30 10.5 4011 4/1

4009 4/1 85.7

1 ml taken as conj. 2.8 13:10 8 3001 6/1

9

632343

Time addition of total T-4 / MDH T-4-2%

from lumen 'conjugate deactivation

T-4 esters p.1

13:37

2991 6/1

94.0

1 ml taken as conj

3.7

13:58

5.5 1977 8.1 / 1

14:49

4 1941 9.6 / 1

98.8

1 ml taken as conj.f

5.6

1 Samples were taken periodically to determine enzyme activity, which is not listed, which affects the total volume shown.

1 Number of T-4 units bound to MDH.

Example 5

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

In 500 ml of dry DMF in a glass tube

6.0 mg (6.8 (iMol) T-4-MEMIDA and 0.8 mg (7.3 jiMol) N-hydroxysuccinimide were added, the tube was purged with dry argon and covered, and the mixture was cooled in an ice bath 1.5 mg of ECDI was then added to the stirred mixture and the tube was flushed with dry argon and stirred until everything dissolved. The tube was wiped dry in a covered plastic container with a desiccant (Drierite®)

provided, wrapped in foil and left overnight at 4 ° with stirring.

0.3 ml of DMF was slowly added to an injection syringe to 1 ml of triose phosphate isomerase (2 mg) in aqueous carbonate buffer (0.1 molar, pH = 9.2) at 4 °. The ester solution was slowly added in portions using a syringe and the enzyme activity was monitored. When the enzyme was approximately 42% deactivated, DMF was added to produce a 50 volume% DMF solution.

The cold reaction mixture was passed through a Sephadex G-25 column (middle column) which had been 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. 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 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 approximately 100 ml of aqueous, 20% by volume DMF with 0.02M triethanolamine (TEA) (pH = 7.9) and three times against 250 ml of aqueous each Dialyzed 0.02 molar TEA (pH = 7.9). The ratio of conjugated T-4 to enzyme was about 6: 1.

Example 6

Desaminothyroxine conjugate to MDH

9.1 x 10 ~ 3 g desaminothyroxine (1.2 x 10 ~ 5 mol), 1.4 x 10-3 g (1.2 x 10 "5 mol) NHS and 0.25 ml dry DMF were successively placed in a reaction vessel The reaction mixture was cooled on an ice bath, 2.5 × 10 -3 g (1.3 × 10 -5 mol) ECDI was added under a nitrogen blanket and the reaction mixture was stirred overnight at 4 °.

With cooling on an ice bath and with stirring, 0.23 ml DMF slowly became 1.9 x 10-3 g (2.8 x 10 ~ 8 mol) MDH in 0.83 ml 0.05m NaHC03-Na2CCb (pH = 9 , 0) added. Then the above-mentioned ester solution (5.8 ml) was added with stirring, and stirring was continued for 1 hour while the specified temperature was maintained. The conjugation mixture was subjected to gel filtration on three Sephadex G-50M columns with the dimensions 0.9 × 13

cm to give a desaminothyroxine / MDH conjugate which was 91% deactivated and which had a hapten number of 2.5 (according to iodine analysis). Enzyme activity of the conjugate was activated by 30% when treated with anti-T-4 serum.

Example 7

T-4-M EM ID A-Gly

A 3 ml Pierce Reacti-Vial® was charged with 0.201 g (2.18 x 10 ~ 4 mol) T-4-MEMIDA and 0.025 g (2.17 x IO "4 mol) N-hydroxysuccinimide (NHS). Then were 2 ml of dry THF and 30 (il (2.15 x 10 ~ 4 mol) of dry triethylamine were added and the reaction mixture was cooled to 0 ° using an ice bath. Then 0.048 g of ECDI (2.50 x 10-4 mol) were in powder form was added 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 Ri values of 0.06 ( T-4-MEMIDA) and 0.60 (T-4-MEMIDA-NHS ester) on an analytical silica gel plate using 10% methanol in dichloromethane as a rinse aid, and a 25 ml flask with a stir bar and membrane stopper was charged with 0.033 g (4.39 x 10 ~ 4 moles) of glycine, then 1.50 ml of distilled water, 0.50 ml of pyridine and 100 µl, 1 (1.00 x 10 ~ 4 moles) of 10-n-NaOH tion mixture was cooled to 0 ° using an ice bath. With vigorous stirring, the T-4-MEMIDA-NHS ester solution prepared above was added dropwise, and after the addition was completed, the reaction mixture was stirred in a cold room (2 °) for 36 hours. The solvents were then removed on a rotary evaporator to give an oily pyridine-smelling solid. 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 and then dried over anhydrous MgSOt. 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 was concentrated to 2 ml in vacuo. A thin layer chromatogram of the product in THF showed only one spot with an Rp 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 8

T-4-MEMIDA glycylglycine

In a 3 ml Pierce Reacti jar were placed 0.202 g (2.20 x 10-4 moles) of T-4-MEMIDA and 0.025 g (2.17 x 10 "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 °, 0.051 g (2.66 × 10-4 mol) of ECDI were then 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 (Rr value 0.63 on an analytical silica gel plate with triethylamine / methanol / dichloromethane in the ratio 2.1: 10: 90). Flask with stir bar and membrane stopper was filled with 0.058 g (4.39 x 10 ~ 4 mol) glycylglycine and then with 1.50 ml H2O and 0.50 ml pyridine and 100 (il (1.0 x 10-4 mol) 1.0n The reaction mixture was cooled to 0 ° and the T4-MEMIDA-

5

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30th

35

40

45

50

55

60

65

632343 10

NHS ester solution was added dropwise with stirring. After 2.1: 10: 90 were developed. 4.0 ml MDH in a concentration

Addition of the activated ester, 1.0 ml deionized ion of 1.5 x 10-5 mol in 0.05m NaHCC> 3-Na2CC> 3 solution (pH water and 0.50 ml pyridine was added. The reaction mixture = 9.2 ) were placed in a 10 ml round-bottomed flask with a stir bar and was stirred in a cold room (2 °) for 93 hours in which the membrane stopper was placed. The reaction mixture was worked up in the same manner as T-4-MEMIDA-glycine, with 5 being cooled to 0 ° in an ice bath, after which 440 µl of dry DMF 0.018 g (yield 9%) of a brown gold solid substance was added at a rate of 50 ml . Which were received. The product was after the thin-layer micro-enzyme activity was determined before and after the addition of DMF matography on silica gel with triethylamine / methanol / dichloro. The 1.9 x 10_2 molar T-4-MEMIDA-GlycyIglycin-methane (2.1: 10: 80) homogeneous; The Rr value was 0.81 when the NHS ester solution was developed twice in the reaction mixture in aliquots plate. io added from 3-10 jxl, after which the enzyme activity after each

Addition was determined. When 29 | il activated ester example 9 solution was added, an 82% deactivated enzyme conjugate was obtained.

T-4-MEMIDA-glycine / MDH conjugates. The reaction mixture was then exhausted against

A 1 ml Pierce Reacti vessel with a stir bar was dialyzed with 1.0m K2HPO4 (with 1.0x 10-3M NaN3) at 2 °. After

0.049 g (5.0 x 10 6 mol) of T-4-MEMIDA-glycine and 39 µl dry-15 dialysis, the conjugate was charged with three Sephadex-G-50M acids DMF. The reaction mixture was stripped to 0 ° (pre-swollen in 1.0m K2HPO4 with 1.0x 10 "3M NaNs), followed by 10 µl (5.2 x 10-6 mol) of 5.0 x 10 ~ 1 m -NHS soldering and eluting with the same buffer The column dimensions in DMF of 0 ° and 51 (il (6.1 x 10 ~ 6 mol) of a l, 2x 10_1m gene were 0.9x55 cm , 0.9x56 cm and 0.9x56 cm, the Strö-ECDI solution in DMF of 0 ° was added, the reaction speeds 4 to 5 drops per minute, and it was mixed in a cold room (2 °) Stirred for 49 hours, collected 20 by 20 drop fractions The protein fractions A thin layer chromatogram of the reaction mixture using a collodion bag device after 49 hours showed two spots with Rf values of 0.49 in a cold room. The hapten number was (T-4-M EM IDA-Glycine) and 0.68 (T-4-MEMIA-Glycine-NHS- determined according to the method described above to 3.9. Ester) on analytical silica gel plates which were treated with triethylamine / under use antigen conjugates, antibodies

Methanol / dichloromethane in a ratio of 2.1: 10: 90 developed 25 per manufactured. First 2 mg of conjugate was injected. Then were. MDH (4.0 ml, 1.3 x 10-5 mol, 0.05 m NaHCCh-NazCCh, 0.25 mg conjugate at two-week intervals inji-pH = 9.2) was placed in a 10 ml round bottom flask with stir bar and decorates. In sheep, the single injection totaled 2 ml, membrane stopper filled at 0 °, and the enzyme activity of which 0.5 ml on the conjugate, dissolved in saline,

was determined in the manner described above. Dem and 1.5 ml were used for Freund's complete adjuvant. The reaction mixture was mixed with 445 ml of dry DMF at a rate of 30, aliquots of 0.25 ml were subcutaneously added at 4 locations and a rate of 15 μl / min, until a reaction gel aliquot of 0.5 ml was injected intramuscularly into each hind leg. mixed with 10% DMF The enzyme activity was determined again in rabbits the total volume of the injection was then 4.3 x 10 ~ 2-molar T-4-MEMIDA-Gly-0.75 ml, 25 mg of the Conjugate in 0.25 ml saline cin-NHS ester solution was dissolved in the reaction mixture in aliquots and 0.5 ml complete Freund's adjuvant from 1 to 2 years 1 was added and the enzyme activity was added after 35. Injections of 0 , 09 ml was determined subcutaneously with each addition, 9% of the above activated ester solution at 4 sites and 0.19 ml injections into each hind leg gave an 82% deactivated enzyme conjugate.

The last addition of activated ester was the enzyme. The animals are usually 5 to 7 days after each

Reaction mixture exhaustively against 0.1 m K2HPO4 (bled with injection, and the antibodies are dialyzed per itself 1.0 x 10 ~ 3m NaN3) at 2 °. After dialysis, the 40 known methods were isolated.

Enzyme conjugate carefully removed from the dialysis bag and to demonstrate the usefulness of the thyroxine-MDH conjugate for through two Sephadex-G-50M columns (in 1.0m K2HPO4, using the T-4 analysis, a number of analyzes 1, 0 x 10-3M NaN3 pre-swollen). The sizes performed. In the first series of analyzes, the analy- s of the two columns were 0.9x54.0 cm and 0.9x51.0 cm, which were carried out with varying amounts of antibody, at flow rates of 4-5 drops per minute, 45 the increase in activity of the enzyme conjugate with increasing and 20 drop fractions were collected. To show the protein amounts of antibodies. In a second series of analyzes, using a collodion bag, varying amounts of T-4 were added to the antibody to concentrate in a cold room. The hapten - the effective concentration of the antibody, the number of binding was determined to be 3.0. to the MDH-bound thyroxine is available to change so. These results show that by setting up Example 10 a standard curve of samples with known amounts is shown

T-4-MEMIDA-Glycylglycine / MDH conjugate thyroxine determine the amount of free thyroxine in the serum

0.015 g (7.8 x 10 -5 mol) of ECDI were dry in 0.50 ml by dissolving the observed values of the enzyme activity DMF into a 1.6 x 10-1 molar solution. Relates 0.75 g to the standard curve.

(6.5 x 10 ~ 4 mol) NHS were dissolved in 1.0 ml of dry DMF to a 55. The analytical procedure is as follows: 0.8 ml of a solution of 6.5x 10-molar solution. Both solutions were cooled to 0 ° in one use in an ice bath prior to 0.1% by weight rabbit serum albumin (KSA). A 1 ml Pierce 0.1 molar glycinate buffer (pH = 9.5) of the 10 ~ 3 mol EDTA ent-Reacti vessel with stirring rod was held with 2.4 x 10-3 g, is from the antibody solution and the MDH-bound

T-4-MEMIDA-glycylglycine and then made with 78jj.1 ice-cold dry-T-4. The solution is incubated for 45 minutes, no DMF (cooled in an ice bath) is added, followed by 4 ul 60 ml of substrates (100 ul 2m malate and 100 (il 0.108m NAD) a 6.5x I0 ~ 1m NHS solution in 0 ° DMF and 18 (il one are added; the solution is added to a spectrophotometer 1.6x 10-1m ECDI solution in 0 ° DMF. and the values are at 30 ° C as a change in

The reaction mixture was stirred in a cold (2 °) 20 optical density between 120 and 60 seconds after the insertion hours. Then a thin layer chromatography showed in the spectrophotometer read. For the conjugate grams of the reaction mixture two spots at Rp values 65 according to Example 4 with sheep antibodies, the results are given in the table below of 0.25 (T-4-MEMIDA-glycylglycine) and 0.59 (T-4-MEMIDA).

Glycylglycine-NHS-Ester) on analytical silica gel plates in the ratio of triethylamine / methanol / dichloromethane

11

632343

Table II

Conjugate from Example 4

AbjV

c2

d2

e2

f2

St.

%

%

%

%

modification

modification

modification

modification

0

1

+ 17

113

246

34

2nd

+ 17

124

358

111

3rd

+ 17

130

389

190

4th

+ 17

126

399

290

5

+ 17

129

427

376

10th

+ 17

130

467

665

15

+ 17

136

471

725

20th

+ 17

139

503

829

25th

+ 17

144

485

825

1. About 9.0 x 10 ~ 6m AbT4, based on the binding sites K = 1, 12xl0 "9

2. Molar concentration of enzyme c-5X10 "10 e - l, 4x 10-8 d-2.56X10" 9 f-3.7xl0 "8

A series of analyzes was now carried out in which a thyroxine solution of 9.7 mg in 25 ml of 0.05 N sodium hydroxide solution was successively diluted with 0.05 N sodium hydroxide solution. The analyzes were carried out by combining 600 μl, 0.1% rabbit serum albumin, 100 μl antibody in 0.1 m glycine buffer (pH = 9.5), 10-3 m EDTA and 100 μl of the T-4 solution and the mixture was incubated for 15 minutes at room temperature. A certain 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 the readings were taken in a spectrophotometer at 30 ° C in the manner described above. The results are shown in the table below:

Table III3

T-4 sample

Conjugates1'2

molar conc.

AODc

AODd

AODe

91

81

248

5xlO "10

89

83

254

5xl0 "9

88

82

255

5xl0 "8

90

81

249

5xl0 "7

88

77

212

5xl0 "6

95

54

93

5x10-5

99

56

85

1. Volume and concentration of the enzyme conjugate used c - 15 jal l, llxl0 "7-molar d- 5 jxl 5.12x10 ~ 7-molar e - 5 jil 2.81 x 10" 6-molar

2. Anti-thyroxine 9x 10-6 molar at binding sites, diluted to 1:33 before addition to 100 yj. 1

3. AODxlO3

Following the procedure of Example 4 was another

Conjugate prepared on a Sephadex G-50 column with 1-molar potassium monohydrogen phosphate solution ins

Equilibrium had been brought, was chromatographed. The flow rate was slow and fractions from 20 drops to tube 39 were collected, after which fractions of 99 drops were collected.

The procedure was repeated with a second G-50 column, each time collecting the fractions with Sephadex

G-200 were concentrated by ultrafiltration. The number of T-4 groups per enzyme was determined to be 4.3 to 5.3 (average 4.8) by UV analysis.

A number of analyzes were performed using different sources of antiserum and varying concentrations of thyroxine. As described in the above procedure, the antibody and 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 were added and the mixture was then introduced into a spectrophotometer, the change in the optical density units being read 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 shown in the table below.

Table IV1

T-4 analysis molar conc.

Antisera AODA

AODB

AODc

76

61

87

5x 10 "5

20th

24th

26

5xl0 "6

28

29

29

5X10-7

30th

39

32

5X10-8

38

50

62

5X10 "9

73

59

84

5X10-10

81

61

94

5X10- »

78

63

92

1. AODxlO3

The following reagents were used to perform the T-4 analysis using TIM-T-4 conjugate:

Reagents for TIM-T-4 analysis

Buffer: 0.02mtriethanolamine (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 7x 10 "3 jiMolT-4 as TIM-T-4, in KSA solution (gives analysis speed of about 200 OD units / min).

Anti-T-4 solution: About 4 x 10_5m Anti-T-4, based on binding points 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 salt from Sigma Chemical Co., St. Louis, Mo .: 1.5 g of salt was treated so that a Final volume of 10 ml was obtained.

a-Glycerophosphate dehydrogenase (a-GPDH) solution: 0.2 mg / ml in buffer solution (Boehringer-Mannheim).

Thyroxine (T-4 solution): 2.57 x 10-4 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 together and placed in a water bath at 30 ° C. for 12 minutes. Then 200 μl of the NADH solution, 25 μl of the a-GPDH solution and 100 μl of the GAP solution were added to the solution. The analytical tube was covered with parafilm and an aliquot

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

632343

12

was then read in a thermal cuvette in a 300N Gilford and 30 ° C. The rest of the analytical solution was in

Spectrophotometer sucked in. The first reading was held in a bath at 30 ° C and read after 13 minutes,

after 30 seconds at a temperature of 30 ° C in the pre direction. The results are given in the table below, direction. The optical density was at 366nm

Table V

Sample No.

T-4 solution concentration

Decrease in optical density in 13 min

1

0

0.543

2nd

3.84xl0-6

0.550

3rd

7.68xl0-6

0.550

4th

l, 54xl0-5

0.490

5

6.14x10-5

0.448

6

l, 29xl0-4

0.422

7

5.14x 10-4

0.409

The results of the above table show that the enzyme substrates are incubated if necessary and then only very low concentrations and extremely small amounts are added to the T4, the T4 can be read by a spectrophotometric reading over a short time using a thyroxine method according to the method of the invention Period indicated or can be determined. The procedure is very definite. The system can be automated, making it simple and requiring only a few handling steps. By automatically mixing samples and reagents and combining the reagents in a buffered medium 25 readings can be made automatically.