DE3833149A1 - Hapten derivatives and hapten-protein conjugates derived therefrom - Google Patents

Abstract

Hapten derivatives of the general formula I <IMAGE> in which Hap is the radical formed from a hapten which has keto or aldehyde groups by elimination of an oxo group, and R<1> and R<2> are identical or different and mean a lower alkyl group with 1 to 7, in particular 1 to 3, carbon atoms, and one radical R<1> or R<2> can also be a hydrogen atom, as well as hapten-protein conjugates of the formula (III) formed from these hapten derivatives (I) <IMAGE> in which E-NH- is the radical of a protein bonded via an amino group of a lysine residue, are described. The hapten-protein conjugates of the formula (III) are very suitable for use for immunisation of organisms suitable for antibody production, and as labelling enzyme in immunoassays.

Description

The invention relates to new hapten derivatives and the like derived hapten-protein conjugates.

In clinical diagnostics are increasing Determination method according to the principle of the immunoassay carried out by special sensitivity award. There are many for these immunoassays Process variants. For many purposes, e.g. B. competitive Assays performed. The compete to determining substance and one added to the sample Known amount of substance to be determined with an enzyme is labeled to an antibody.

There is therefore a need for conjugates from one Labeling enzyme and one with the substance to be determined or specifically bind an antibody Connection, whereby the conjugate only with the to be determined Substance or the antibody may react and no cross-reactivity with others in the test solution existing connections should have to the result not to falsify. Because the specifically binding Substance and the labeling enzyme are often not direct tied together, but via a link grouping (Spacer) can be coupled, the affinity the antibody to the spacer is causing problems.

There is still a great need for specific ones Antibodies. Antibodies are generally produced by using an immunogen like an antigen or hapten suitable form several times in one for antibody formation  qualified organism injected. Haptens are by definition Molecules with a molecular weight of less than 1000 Daltons, which alone are not immunogenically effective are immunogenic, however, by binding to a protein will. To immunize the hapten with a immunogenic molecule, such as. B. a serum protein, conjugated. If you inject such a conjugate, so the organism forms alongside anti-protein antibodies the desired anti-hapten antibodies. The educated Antibodies are then extracted from the organism. On in this way polyclonal antibodies can be obtained.

Also for the production of monoclonal antibodies must first immunization of a suitable organism in which Rule, mice. By the usual repeated injection of an antigen or hapten A B-line conjugate causes antibodies to synthesize against this antigen or hapten and to secrete. Screening then turns a B cell, which produces the desired antibodies from the spleen isolated and by fusion with a myeloma cell immortalized. This cell then constantly produces the same monoclonal antibodies.

To bind haptens that have keto or aldehyde functions to a protein, it is a common method to convert the carbonyl function with O- (carboxymethyl) hydroxylamine to the oxime and to couple it via the free carboxyl function to ε- amino groups of the high molecular weight carrier molecule (cf. PK Grover and WD Odell, J. Steroid, Biochem. 8 (1976) 121). The coupling products can then be used to generate antibodies against the hapten component or as a tracer in immunoassays (cf. K. Luebke and B. Nieuveboer, "Immunological Tests for Small Molecular Active Agents", Thieme Verlag, Stuttgart 1978).

In those commonly used for immunization Conjugates in which the hapten has a linking group (Bridge) bound to the immunogenic protein cross-reactions with the bridge molecule part now occur frequently on and show the antibodies obtained a sometimes very high affinity for those in the immunogen existing bridge structures. This results in immunological Tests often cause problems because part of the binding activity the antibody against the linker, d. H. against the bridge molecule between hapten and carrier protein in the immunogen. To get this effect, of the often adverse effects in development of immunoassays, especially for low-concentration ones Haptene, leads to bypass, one makes use of the Preparation of hapten-enzyme conjugates preferably the method of heterologous linkers (see e.g. B. K. Van Weemen and A. H. W. M. Schuurs, Immunochem. 12 (1975) 667; H. Hosada et al., Chem. Pharm. Bull. 34 (1986) 2105), d. that is, the linkage grouping in the conjugate is alienated from the immunogen, i. i.e., structurally modified.

Now an immunogen is used to generate antibodies used in a hapten with a keto or aldehyde function via O- (carboxymethyl) hydroxylamine to the Lysine residue of a carrier protein is coupled, so were for a heterologous linker with the same link of hapten in the immunogen and enzyme conjugate the following possibility is known so far:

• a) the use of glycine hydrazide as a bridge (see R. Mueller et al., Hoppe-Seylers Z. Physiol. Chem. 357 (1976) 1007). The hydrazide reacts Function to hydrazone and free The amino group of the linker then takes place Coupling of the hapten derivative to a carboxyl function of the enzyme.
• b) The conversion of the carbonyl function into a Hydroxyl function and coupling via an ether or ester function on the bridge molecule (see K. Luebke and B. Nieuweboer, l.c. and B.K. Van Weemen and A. H. W. M. Schuurs, l.c.).

However, both methods have disadvantages: the method a) is limited to enzymes, the free carboxyl groups own that are not at the reactive center of the Enzyme allowed to sit. The coupling reaction takes place using carbodiimide, it being generally to undesirable crosslinking reactions of the Enzyme can come. Method b) uses hapten chemically changed so much that the bond to Antibody is adversely affected.

The object of the present invention was therefore to To provide protein conjugates that are used for Formation of antibodies directed specifically against the hapten lead to and when used in immunoassays cause specific binding of antibodies to the hapten, without cross-reactivities with the bridge molecule part occur. This object is achieved with the present invention solved.  

The invention relates to hapten derivatives of the general Formula (I)

wherein Hap from a keto or aldehyde groups bearing hapten by splitting off an oxo group formed residue, and R¹ and R² are the same or different are and a lower alkyl group with 1 to 7, in particular 1 to 3 carbon atoms, and a radical R¹ or R² can also be a hydrogen atom, with the proviso that R¹ and R² are not simultaneously one Are methyl group if the hapten is oestradiol.

A lower alkyl group R1 and R2 with 1 to 7, preferably 1 to 3 carbon atoms can be straight-chain or to be branched; preferably the lower alkyl group straight chain, and is z. B. methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl or n-heptyl.

The hapten residue in the general formula (I) conducts preferably from a steroid hormone, such as. B. of testosterone, progesterone, oestradiol (via 6-keto Oestradiol) or from oestriol (via 6-keto-oestriol).

The hapten derivatives according to the invention of the general Formula (I) can be prepared by a hapten carrying a keto or aldehyde group a keto or aldehyde group with a compound of Formula (II)  

wherein R¹ and R² have the meaning given for formula (I) own, converted into the corresponding oxime derivative.

The conversion of the keto or aldehyde groups into that Oxime by reacting the hapten with a compound of the formula (II) can for such oxime formation generally known manner. The condensation to Oxime takes place e.g. B. by implementation with the corresponding Hydrochloride or hydrobromide of carboxyalkoxyamine of formula (II) under alkaline conditions.

If the hapten has no suitable functional group (Oxo group), such a condensation be introduced (e.g. in the 6-position of a steroid hormone); the condensation of the hapten with the compound The formula (II) must in any case be such that Epitope remains freely accessible after condensation. After the reaction with the hapten, if necessary protective groups introduced into the hapten in known per se Split off.

The invention further relates to hapten protein Conjugates of the general formula (III)

wherein Hap, R¹ and R² have the meaning given above for the formula (I), and E-NH- means the residue of a protein bound via an ε -amino group of a lysine residue.

In preferred embodiments of the hapten-protein conjugates of the formula (III) according to the invention, the hapten is a steroid hormone, particularly preferably testosterone, or else an enzyme, particularly preferably β- galactosidase or peroxidase.

The hapten-protein conjugates of the formula (III) can are produced by using an inventive Hapten derivative of the formula (I) with a protein E-NH₂, in which -NH₂ is an -amino group of a lysine residue, implements.

The reaction of the hapten derivative of the formula (I) with the epsilon amino group can selectively on known per se Ways are carried out by transferring the carboxyl group of the hapten derivative of the formula (I) into one activated esters, especially in the corresponding ones N-hydroxysuccinimide esters (see K. Luebke and B. Nieuweboer, l.c.)

In the hapten-protein conjugates of formula (III) means the hapten residue hap preferably one of those for the hapten derivatives of the formula (I) are preferably mentioned Leftovers.

The preferred meaning of the protein residue E depends in particular on the intended use of the hapten-protein conjugate according to the invention. To immunize organisms suitable for antibody formation, the protein is an immunogen protein, in particular one of the commonly used immonogenic carrier proteins, such as. B. edestin or bovine serum albumin. For use in immunoassays, the enzymes usually used in immunoassays are preferably used, and in particular the marker enzymes such as e.g. B. β- galactosidase or peroxidase.

In one embodiment of the invention, hapten Protein conjugates are provided in which the Hapten is a steroid hormone. The detection of steroid hormones such as estrogens, testosterone, cortisone and Cardiac glucosides that all share the basic steroid structure is very important for diagnostics. The Providing antibodies against steroid hormones as well as steroid hormone-enzyme conjugates for the Performing immunoassays is therefore desirable. It has now proven very beneficial to use steroid hormones if necessary, to derivatize them to carry an oxo group to the C6 atom. This oxo group can then with the carboxyalkoxyamines of formula (II) to Hapten derivative (I) are implemented. This bond affects not the epitope of the steroid hormone and does not lead to any undesirable changes in the molecule. Depending on the intended use, the hapten derivative can then either an immunogenic protein or a Enzyme to be coupled.

When used for immunization is preferred uses a conjugate in which the protein is an immunogenic Carrier protein, e.g. B. edestin or bovine serum albumin, is. However, there are also conjugates that Enzymes included as protein for immunization suitable. The conjugate according to the invention is used in the Antibody suitable organism at intervals injected several times. The conjugate then causes the formation of antibodies that are in a very high percentage only are directed against the hapten and no cross-reactivity with the link grouping. The Antibodies can then be produced in a manner known per se the organism. The invention  Hapten-carrier conjugates used are both suitable for immunization for the production of polyclonal Antibodies as well as for the production of monoclonal antibodies.

In a further embodiment of the present invention becomes a hapten-protein conjugate according to the invention of the formula (III) from a hapten which has the Linking grouping according to the invention to an enzyme is used to perform immunoassays. The hapten-enzyme conjugate according to the invention can then as a marker (labeling enzyme) in determination procedures used according to the principle of the immunoassay will. The hapten-enzyme conjugate according to the invention can e.g. B. in a known amount of a sample solution and then competes for one against the hapten targeted antibodies.

Surprisingly, it works by using a hapten-protein conjugate according to the invention, antibodies to get the very little cross reactivity with the Have bridge connection. The antibodies obtained have a high affinity for the hapten. Using of the hapten-protein conjugates according to the invention Immunization will get high antibody titers. Furthermore, the hapten protein Conjugates particularly well suited for use in immunoassays. Because the antibodies to the bridge molecule are none or have very little affinity and therefore bind the hapten very specifically, you get with these conjugates when used in immunoassays very accurate results.

The hapten derivatives of the formula (I) according to the invention have the advantage that on the one hand they have the structure free hapten largely unchanged,  on the other hand, in comparison to derivatives which, for. B. analog were prepared with O- (carboxymethyl) hydroxylamine, have a characteristic change in the bridge molecule. The use of the hapten derivatives according to the invention therefore enables the implementation of heterologous Linker techniques in a simple and effective way: for the two uses as

• 1. Immunogen and
• 2. Conjugate in immunological analysis

are preferably different bridges in the invention Hapten-protein conjugate used (at least a radical R¹ or R² is different, for one of the two uses also derivatives that differ from Derive O- (carboxymethylene) hydroxylamine, i.e. H. R¹ and R² = H, can be used), but on the other Should be as similar as possible to the Maintain specificity.

The invention therefore also relates to the use of a hapten-protein conjugate according to the invention of the formula (III) for the immunization of for antibody formation suitable organisms, and as a labeling enzyme in an immunoassay.

The following examples illustrate the invention in Link with the figure closer.

Fig. 1 shows the results of the determination of testosterone with the inventive testosterone-3-DMC POD conjugate as compared to testosterone-3-CMO-POD.

The compounds of formula (II) are known or can be analogous to a process for producing the Establish known connections (cf. K.S. Suresh and R.K. Malkani, Ind. J. Chem. 10 (1972) 1068).

Examples Example 1

Production of testosterone-3-dimethylcarboxymethoxime POD conjugate (testosterone-3-dmc-POD, formula (III), R¹ = R² = methyl, Hap-testosterone residue, E-NH = peroxidase residue).

The preparation was carried out according to the following reaction scheme:

1. Testosterone-3-dimethylcarboxymethoxime (3)

2.88 g (10 mmol) of testosterone (1) are dissolved in 50 ml of methanol, 1.65 ml (20 mmol) of pyrrolidine are added and the mixture is stirred at 20 ° C. Precipitation occurs in the yellow solution after a short time. After 10 minutes, 2.1 g (10.05 mmol) of 2-aminooxy-isobutyric acid hydrobromide (2) (prepared according to the instructions from KS Suresh and RK Malkani, Ind. J. Chem. 10 (1972) 1068) are added ) and heated to 65 ° C for 5 minutes. The solution is then evaporated in a water jet vacuum, 50 ml of 1 mol / l NaOH are added to the residue and then dissolved in 2 l of water. The almost clear solution is washed twice with 250 ml of ethyl acetate. The alkaline phase is adjusted to pH 1 with concentrated HCl and extracted twice with 200 ml of ethyl acetate. The ethyl acetate extract is then washed with 0.5 l of water, dried with 20 g of Na₂SO₄ and evaporated in vacuo. The mixture is digested with petroleum ether, the suspension is left under stirring for about 2 hours and the solid product (3) is filtered off with suction. It is dried in a desiccator over phosphorus pentoxide. Yield: 1.8 g (46% of theory).
TLC: silica gel; Chloroform / methanol / glacial acetic acid = 66/33/1;
R _f = 0.69.

2. Testosterone-3-dimethylcarboxymethoxime-N-hydroxysuccinimide ester (5)

0.97 g (2.5 mmol) of the carboxylic acid (3) are dissolved with 0.35 g (3 mmol) of N-hydroxysuccinimide (4) in 20 ml of tetrahydrofuran (THF) and with 0.62 g (3 mmol) of dicyclohexylcarbodiimide added in 10 mml THF. The mixture is stirred for 3 hours at 20 ° C., then the precipitated dicyclohexylurea is filtered off and the solution is evaporated in a water jet vacuum. The residue is dissolved in 50 ml of dioxane and the solution is cooled at 4 ° C. for 16 hours. Then it is filtered and the solvent is removed in vacuo. The oily residue is digested with 50 ml of diisopropyl ether until it has completely solidified. The product (5) is suctioned off and dried over phosphorus pentoxide in a desiccator.
Yield: 0.86 g (70% of theory).
TLC: silica gel; Chloroform / methanol / glacial acetic acid = 80/19/1;
R _f = 0.70.

3. Testosterone-3-dimethylcarboxymethoxime-POD conjugate (7)

35 mg peroxidase (POD) (6) (EC 1.11.1.7) are in 3.5 mml 0.1 M potassium phosphate (pH 8.0) dissolved Cooled to 4 ° C. and slowly with 3.3 ml of DSMO (dimethyl sulfoxide) transferred.

5 mg of the testosterone 3-dimethylcarboxymethoxime-N-hydroxysuccinimide esters (5) are in 200 µl DMSO at room temperature solved and in 6 equal portions at a distance of about 30 minutes added to the cold POD solution. The mixture was stirred at 4 ° C. overnight.

For the separation of the free ester a Column (2 cm in diameter, 16 cm in height) with Sephadex G 25, fine, (Pharmacia) packed and at 4 ° C with  40 mmol / l potassium phosphate (pH 6.5) equilibrated. The entire conjugate solution is at 4 ° C with 1 column volume / Hour on the column and at same flow eluted with equilibration buffer. The Eluate is collected in fractions and the reddish brown colored fractions collected.

The combined conjugate fractions are with Thymol saturated and stored at 4 ° C.

Example 2 Testosterone determination with Testosterone-3-carboxymethoxime (cmo) -POD

Reagents:

Test buffer:
400 µg / ml monoclonal antibody against testosterone (ECACC 8 51 21 701)
40 mmol / l sodium phosphate, pH 6.8

Loading solution:
10 µg / ml polyclonal sheep antibody (IgG) against mouse Fc-gamma
20 mmol / l sodium carbonate buffer, pH 9.6

Wash solution:
250 mg / 100 ml sodium chloride
1 mg / 100 ml copper sulfate

Substrate solution:
1.9 mmol / l ABTS® (2.2 ′ ′ - azino-di- [3-ethyl-benzothiazolinesulfonic acid (6)]) - diammonium salt
100 mmol / l phosphate citrate buffer, pH 4.4
3.2 mmol / l sodium perborate

Sample:
Testosterone in various concentrations in buffer solution was used as a sample:
40 mmol / l sodium phosphate, pH 7.4
0.9% sodium chloride
0.3% RSA (bovine serum albumin)
0.2% Pluronic PF68

1 ml of coating solution was used to carry out the determination 30 minutes at room temperature in one Luran tubes incubated. Then was twice washed with washing solution. It became 1 ml of test buffer added, incubated for 60 minutes at room temperature and washed twice with washing solution. There were 50 ul Sample and 1 ml testosterone 3 cmo-POD (200 mU / ml) in 40 mmol / l sodium phosphate buffer, pH 6.8, with 0.2% Pluronic PF68 added, 30 minutes at room temperature incubated and washed twice with washing solution. After that 1 ml of substrate solution was added for 30 minutes Incubated at room temperature and the absorbance at 405 nm measured.  

Example 3 Testosterone determination with testosterone 3-dmc-POD conjugate

The determination was as described in Example 2 carried out using 3-cmo-POD- instead of testosterone Conjugate Testosterone 3-dmc conjugate was used.

The results of Examples 2 and 3 can be seen in FIG. 1. It can be seen from this that with the conjugate according to the invention a calibration curve (curve 2) which is substantially steeper in the lower concentration range is obtained than with a cmo conjugate (curve 1), which means that a testosterone test using the conjugate according to the invention has a significantly higher sensitivity is obtained.

Example 4 Production of oestradiol-6-dimethylcarboxymethoxim- POD conjugate

The preparation was carried out according to the following reaction scheme:
Reaction scheme:

a) Oestradiol-6-dimethylcarboxymethoxime (9)

2.86 g (10 mmol) 6-oxoestriol (8) were dissolved in 130 ml Dissolved ethanol and with 1.7 g (20 mmol) NaHCO₃ and 2.1 g (10.5) 2-amino oxide iso-butyric acid hydrobromide (2) offset. The mixture was heated to 65 ° C. for 30 minutes, then the solution was evaporated in a water jet vacuum, 50 ml of 1 N NaOH were added to the residue and then dissolved in 1 liter of water. The almost  clear solution was twice with 150 ml of ethyl acetate washed. The alkaline phase was isolated adjusted to pH 1 with concentrated HCl and extracted with two 150 ml portions of ethyl acetate. The ethyl acetate extract was washed with 0.25 l of water, dried with 10 g Na₂SO₄ and in vacuo evaporated. It was digested with petroleum ether, let stir the suspension for about 2 hours and suck the solid product 9. It was in the desiccator dried over phosphorus pentoxide.

Yield: 3.10 g (80% of theory).
TLC: silica gel, ethyl acetate; R _f = 0.58.

Oestradiol-dimethylcarboxymethoxime-N-hydroxysuccinimide ester (10)

970 mg (2.5 mmol) of the carboxylic acid (9) were mixed with 350 mg (3 mmol) N-hydroxysuccinimide (4) in 30 ml dissolved dry THF and with 620 mg (3 mmol) Dicyclohexylcarbodiimide added in 10 ml THF. Man allowed to stir at 20 ° C for 3 hours, then from filtered out precipitated dicyclohexylurea and evaporated the solution in a water jet vacuum. The residue was dissolved in 25 ml of THF and the Cooled solution at 4 ° C for 16 hours. Then was filtered and the solvent removed in vacuo. The oily residue was digested along with it 20 ml isopropanol until it solidifies completely would have. The product (10) was suctioned off with Washed 50 ml of diisopropyl ether and in a desiccator dried over phosphorus pentoxide.

Yield: 940 mg (78% of theory).
TLC: silica gel RP-18, nitromethane / ethanol 9: 1;
R _f = 0.70.

c) Oestradiol-6-dimethylcarboxymethoxime-POD conjugate (11)

Production analogous to (7) (see example 1, 3).

Claims (13)

1. hapten derivatives of the general formula (I) wherein hap is the radical formed from a heto bearing keto or aldehyde groups by cleavage of an oxo group, and R¹ and R² are identical or different and are a lower alkyl group having 1 to 7, in particular 1 to 3, carbon atoms, and a radical R¹ or R² is also a Can be hydrogen provided that when the hapten is oestradiol, R1 and R2 are not simultaneously a methyl group. 2. hapten derivatives according to claim 1, characterized, that the hapten is a steroid hormone. 3. hapten derivatives according to claim 1 or 2, characterized, that the hapten is testosterone. 4. A process for the preparation of hapten derivatives of the general formula (I) according to one of claims 1 to 3, characterized in that in a hapten carrying a keto or aldehyde group a keto or aldehyde group with a compound of the formula (II) wherein R¹ and R² have the meaning given for formula (I), converted in a manner known per se into the corresponding oxime derivative. 5. Hapten-protein conjugates of the general formula (III) wherein Hap, R¹ and R² have the meaning given in claim 1, an E-NH- means the residue of a protein bound via an ε- amino group of a lysine residue. 6. hapten-protein conjugate according to claim 5, characterized, that the hapten is a steroid hormone. 7. hapten-protein conjugate according to claim 5 or 6, characterized, that the hapten is testosterone. 8. hapten-protein conjugate according to one of claims 5 to 7, thereby characterized that the protein is an enzyme.   9. hapten-protein conjugate according to claim 8, characterized in that the enzyme is β- galactosidase or peroxidase. 10. A process for the preparation of a hapten-protein conjugate of the formula (III) according to one of claims 5 to 9, characterized in that a hapten derivative of the formula (I) according to one of claims 1 to 3 with a protein E-NH₂, wherein -NH₂ is a ε- amino group of a lysine residue, implemented in a manner known per se. 11. Use of a hapten derivative of the formula (I) according to one of claims 1 to 3 for the production of hapten-protein conjugates of the formula (III) according to one of claims 5 to 9. 12. Use of a hapten-protein conjugate after one of claims 5 to 9 for the immunization of organisms suitable for antibody formation. 13. Use of a hapten-protein conjugate after Claim 8 or 9 as a labeling enzyme in one Immunoassay.