WO1996023224A1

WO1996023224A1 - Stabilization of peptides and polypeptides in immunological tests by addition of small thermal shock proteins

Info

Publication number: WO1996023224A1
Application number: PCT/EP1996/000305
Authority: WO
Inventors: Christoph Hergersberg; Dorothee Ambrosius; Alfons Nichtl; Ursula-Henrike Wienhues-Thelen
Original assignee: Boehringer Mannheim Gmbh
Priority date: 1995-01-26
Filing date: 1996-01-25
Publication date: 1996-08-01
Also published as: DE19502386A1; EP0871882A1; JPH10512676A

Abstract

In immunological tests for the classification of an analyte in aqueous solution, good test linearity and a good signal/noise ratio can be achieved by the stabilization of immunologically reactive polypeptides (antigens) by the addition of a stabilizer comprising one or more proteins of the small thermal shock protein family. The stabilization process is suitable for improving the storage life of aqueous solutions containing peptides and can be used in conventional immunological test systems.

Description

Stabilization of peptides and polypeptides in immunological tests by adding small heat shock proteins

DESCRIPTION

The present invention relates to a method for determining an analyte in aqueous solution using immunologically reactive peptides or polypeptides and a stabilizer therefor, and the stabilization of peptides in aqueous solution.

The use of antigens and in particular of recombinant or chemically synthesized peptide or polypeptide antigens in immunological determination methods is known from the prior art. When peptide or polypeptide analytes are detected, solutions which contain a known concentration of the analyte are required, for example, for calibration. In test systems for the determination of specific antibodies, immunologically reactive peptides or polypeptides with the antibody to be determined are used for the qualitative and / or quantitative detection of the antibody. In such immunological determination systems, the stability of the peptides or polypeptides is of essential importance, on the one hand to ensure the linearity of the test and a good signal-to-noise ratio and on the other hand to ensure reliable reproducibility of the determination results over a longer period of time.

From the prior art is the stabilization of peptides and polypeptides in immunological test solutions by addition of non-specific proteins (eg RSA) or buffer systems (salt, pH etc. ^■) are known. However, these stabilization methods generally do not lead to satisfactory results, and in particular recombinant or chemically synthetic Tended peptides or polypeptides show a tendency to aggregate and adsorb, which cannot be sufficiently eliminated by the stabilization processes mentioned.

It is also known from the prior art that heat shock proteins can bind denatured proteins (Ang et al., J. Biol. Chem. 266 (1991), 24244-24236). It is also known that protein solutions can be stabilized by adding heat shock proteins, this stabilizing effect being based in particular on preventing the formation of insoluble aggregates.

However, it is assumed that proteins which are bound to heat shock proteins are generally not in their native conformation. Hayer-Hartl et al., EMBO J. 13 (1994), 3192-3202 report, for example, that proteins bound to GroEL (hsp60 from E. coli) are present in an open structure, the so-called "molten globule" structure. This change in conformation does not guarantee that peptides and polypeptides which are stabilized by binding heat shock proteins have their structural features which are necessary for use in immunological test methods. There is also the possibility that immunologically reactive areas on peptides and polypeptides are masked by the presence of heat shock proteins.

DE-OS 42 39 969 discloses a method for stabilizing proteins in aqueous solution by adding one or more proteins from the family of small heat shock proteins (small hsp's). However, there is no data on the immunological properties of proteins bound to small hsp's.

It was therefore the object of the present invention to provide an improved method for stabilizing antigens in aqueous solution, in which the abovementioned Disadvantages of the methods known from the prior art are largely eliminated.

This object is achieved by a method for determining an analyte in aqueous solution using immunologically reactive peptides or polypeptides and a stabilizer therefor, which is characterized in that the stabilizer comprises one or more proteins from the "small" family Heat shock proteins (small hsp) included.

It has surprisingly been found that the small hsp are capable of stabilizing peptides and polypeptides in solution without impairing their immunological properties.

The family of small heat shock proteins is a heterogeneous group of proteins with one or more representatives in yeast (hsp 26), vertebrates (hsp 24, hsp 28), Drosophila (hsp 22, 23, 26, 28), mouse ( hsp 25) and over 20 proteins in plants (Nover & Schaft, Eur. J. Biochem. 139 (1984), 303-313; Lindquist & Craig, Ann. Rev. Genet. 22 (1988), 631-677). Depending on the organism, their molecular masses are between 15 and 40 kDa, whereby in most cells and tissues the small hsp's are synthesized even in the absence of stress and are present as cytosolic aggregates (heat shock granules) with a molecular mass of over 700 kDa (Arrigo et al., Mol. Cell Biol. 8: 5059-5071 (1988). Heat shock and chemical stress induce the synthesis of the small hsp's, which can then make up up to 1% of the total cell protein (Österreich et al., Biomed. Biochim. Acta 49 (1990), 219-226). In addition, the above-mentioned stress factors cause a change in the degree of phosphorylation of the proteins and in their cellular localization (Arrigo et al., Supra Zantema et al., J. Biol. Chem. 267 (1992), 12936-12941) . Despite their heterogeneity, the small hps's of the different organisms show matching hydrophobicity profiles and small regions of identical amino acid sequences (Lindquist & Craig, supra). In addition, clear sequence homologies with the also high molecular weight Associate-forming α-crystalline from vertebrates can be detected (Ingolia & Craig, Proc. Natl. Acad. Sei. USA 79 (1982), ₂ 360-2364). At the DNA level, sequence homologies of the small hsp's are described with one another.

In principle, all peptides or polypeptides that can be stabilized by heat shock proteins, i.e. essentially peptides, proteins, glycoproteins, lipoproteins and the like, and other substances which have a peptide content. In a preferred embodiment, recombinant or synthetically produced peptides or polypeptides are used. Such substances have a particular tendency to aggregate and adsorb and can be stabilized particularly advantageously by the process according to the invention.

In a particularly preferred embodiment of the invention, the hsp 25 from mouse is used, hsp 25 forms a 32mer (Mr = 800 kD) (Behlke et al., FEBS Letters, Vol. 288 No. 1.2 (1992), 119-122 ). The hsp 25 gene in the mouse is regulated by a heat shock promoter, so that the expression of the protein is induced by heat stress. The cloning and sequencing of a DNA sequence coding for hsp 25 and its expression in E. coli is described by Gaestel et al. in Eur. J. Bioche. 179 (1989), 209-213, J. Biol. Chem., Vol. 266., No. 22 (1991), pp. 14721-14724 and in DD-Al 273 071. Among other things, hsp 25 is particularly advantageous because, based on the 32mer, the protein-stabilizing effect can be measured on a molar basis even with a clear deficit. Furthermore, hsp 25 is extremely heat-resistant, and after incubation of the hsp alone at 100 ° C. for 15 minutes and subsequent cooling, its starting activity was found again, hsp 25 therefore appears to be a particularly advantageous candidate for the process according to the invention. Due to the possibility of recombinant production, it is also available in sufficient quantities. In a further embodiment of the invention, the small hsp is set in a molar ratio of preferably 0.0001: 1 to 1000: 1, particularly preferably from 0.01: 1 to 1000: 1 and most preferably from 0.1: 1 to 500: 1 based on the peptide or polypeptide to be stabilized. In this context, the concentration of hsp25 refers to a monomeric unit.

Another object of the present invention is the use of small heat shock proteins to stabilize peptides in aqueous solution. The term “peptide” denotes proteins comprising from 6 to 50 amino acids, preferably from 6 to 30 amino acids.

In a particular embodiment, the small hsp's are used to improve the storage stability of an aqueous solution which contains peptides and in particular for the long-term stabilization of a reagent for immunological determination methods which is in the form of an aqueous solution and contains immunologically reactive peptides. Usually, the instability of peptides in aqueous solution leads to a deterioration in the measurement results, especially when stored over a longer period of time. By adding small hsp's, on the other hand, the stability of the peptides is considerably improved, so that even after a longer period, e.g. after several weeks, no significant deterioration of measurement results, e.g. is observed in immunological determination methods.

Yet another object of the present invention is the use of small hsp's to improve the signal-to-noise ratio in a determination method which is carried out in the presence of at least one peptide and / or polypeptide in aqueous solution. The determination method is preferably an immunological determination method which is carried out using immunologically reactive peptides and / or polypeptides. Surprisingly, it has been found that in determination procedures by adding small hsp's, the determined blank values can be considerably reduced. Although the cause of this is not exactly known, it is assumed that the reduction in the blank values is based on the improved stabilization of the peptides and / or polypeptides present in the test solution. By reducing the blank values, the signal differentiation is improved and thus the measurement accuracy can be increased when using small hsp's according to the invention.

The addition of small hsp according to the invention can in principle be used in all forms of determination methods, such as for stabilizing peptide or polypeptide antigens in direct or indirect detection methods for specific antibodies, for stabilizing analyte analogs in competitive immunoassays and for stabilizing calibration solutions ¬ gen. However, it is obvious to the person skilled in the art that the stabilization according to the invention of peptides or polypeptides can also be applied to other determination methods not explicitly described here.

A preferred embodiment of the invention comprises a method for the immunological determination of a specific antibody in a sample liquid, the sample liquid being incubated with at least one peptide or polypeptide antigen which is capable of binding with the antibody to be determined and detects the antibody by binding to the antigen, which is characterized in that the antigen is stabilized by adding one or more proteins from the family of the "small" heat shock proteins. Such a method is particularly preferably carried out as a heterogeneous immunoassay in the presence of a reactive solid phase.

An example of such a heterogeneous immunoassay is an indirect test format. The sample liquid containing the antibody to be determined is incubated with a stabilized antigen in the presence of a reactive solid phase is present in a form capable of binding to the solid phase, and a detection antibody directed against the antigen which bears a marking group. The qualitative and / or quantitative detection of the antibody to be determined can then be carried out by measuring the marking in the solid phase and / or in the liquid phase. The antigen capable of binding to the solid phase can be biotinylated and the solid phase can be coated, for example, with streptavidin or avidin.

Another preferred test format for the determination of antibodies is the double antigen bridge test. In this method, the sample liquid containing the antibody to be determined is incubated in the presence of a reactive solid phase with at least two antigens AGj and AG ₂ , the first antigen AGi being in a form capable of binding to the solid phase and the second antigen AG _{2 being} a labeling group wearing. The antibody to be determined is detected by determining the label in the solid phase and / or in the liquid phase, but preferably in the solid phase.

A further preferred embodiment of the invention is the stabilization of analyte analogs in a competitive immunoassay, in particular in a method for determining a peptide or polypeptide analyte in a sample liquid according to the principle of the competitive immunoassay, the sample liquid being counteracted with a the analyte-directed antibody and a peptide or polypeptide which competes with the analyte for the antibody and carries a labeling group, and detects the analyte by measuring the label, which is characterized in that the labeled peptide or polypeptide is added by adding a or several proteins from the family of small heat shock proteins stabilized.

Yet another embodiment of the invention relates to a method for determining reference values for the determination of a peptide or polypeptide analyte, with at least one a test solution which contains a known concentration of the analyte to be determined, incubated with an antibody directed against the analyte and, by quantifying the immune reaction between the antibody and the analyte in known concentration, forms at least one reference value, which is characterized in that the Antigen stabilized in the test solution by adding one or more proteins from the "small" heat shock protein family. The main advantage is that test solutions stabilized according to the invention improve the linearity of the test and, as described above, the durability or long-term stability of such test solutions can be increased considerably. In addition, it may be desirable to also add heat shock proteins to the sample liquid, in particular if the immunological determination method is not carried out directly after the sampling.

Yet another object of the present invention is a reagent kit for use in immunological test procedures, in which peptides or polypeptides are stabilized by adding one or more proteins from the family of small heat shock proteins. Such a reagent kit thus comprises in spatially separated form (a) at least one first reagent which contains a stabilized peptide or polypeptide, (b) at least one second reagent which contains a further immunologically reactive component, e.g. a detection antibody, and (c) optionally further auxiliary and / or detection reagents.

Auxiliary reagents are substances which are required, for example, to prepare the test solutions or to adjust their concentration, such as deionized water, buffers, etc. Detection reagents are substances with which an immune complex formed can be detected. A detection reagent preferably contains a substrate for a corresponding labeling group of the antigen or antibody. pers. Suitable methods or substances are known from the prior art.

A reagent kit according to the invention contains the reagents in a form which is dependent on the intended method to be used. Accordingly, reagents can be provided in which e.g. the stabilized peptide or polypeptide is in a form capable of binding to a reactive solid phase or carries a labeling group. Optionally, the reagent kit can also contain multiple reagents with different stabilized peptides or polypeptides, e.g. a reagent kit for a double antigen bridge test. Furthermore, the reagent kit can also contain one or more reagents which contain peptide or polypeptide antigens for establishing reference values, e.g. a calibration curve, in different known concentrations. The form in which the antigens are provided in the reagent kit according to the invention is not critical. Both solutions and lyophilisates can be used, which are then converted into a liquid form before first use.

The reagent kit according to the invention is suitable in a variety of immunological determination methods and the selection of the stabilized peptide or polypeptide is not critical. However, it has been found that the reagent kit according to the invention is used particularly advantageously when using synthetically or recombinantly produced peptides or polypeptides. In a preferred embodiment, the reagent kit is used to determine viral antigens or antibodies directed against viral antigens, and in a particularly preferred embodiment, peptides or polypeptides from the hepatitis C virus, for example from the areas NS 3 or NS-4 or from HIV, eg from the p24 range, stabilized. The present invention is illustrated by the following examples in conjunction with the accompanying figures. Show it:

Fig. 1 the stabilization of test solutions with known concentrations of troponin T by adding hsp25,

Fig. 2 shows the amino acid sequence of an HCV-NS3 polypeptide (a), a synthetic HCV-NS4 polypeptide (b) and a recombinant HIV-p24 polypeptide (c) and

Fig. 3 the improvement of the signal differentiation depending on the concentration of added hsp 25.

example 1

Stabilization of Troponin T test solutions

1.1 Preparation of the solutions a) Troponin T solution with 5 mol hsp25 (32-mer; 160 mol

Monomer) per 1 mol Troponin T: To 546 μl buffer (0.1 mol / 1 Tris, pH 8.0 / 100 mmol / 1 KCl / 2 mmol / 1 dithiothreitol (DTT) / 0.05% NaN ₃ ) become 100 μl Troponin T solution with a concentration of 0.43 mg / ml (in 5 mmol / 1 KP0 _< , pH 7.3 / 2.0 mol / 1 urea / 0.6 mol / 1 KCl / 2 mmol / 1 DTT / 0.05% NaN ₃ ) and 354 μl hsp25 solution with a concentration of 14.7 mg / ml (in 0.1 mmol / 1 Tris, pH 8.0 / 100 mmol / 1 KCl / 2 mmol / 1 DTT / 0.05% NaN ₃ ).

Recombinant hsp25 was produced as described in DE-A-42 39 969.6.

Conditions in the solution: Troponin T conc .: 0.043 μg / l hsp25 conc .: 5.204 mg / ml b) Control and comparison solutions:

Troponin T solution without hsp25:

The preparation was carried out as described under a). Instead of the hsp25 solution, only buffer is added.

Troponin T solutions with 5 mol or 10 ol bovine serum albumin (RSA) per 1 mol Troponin T: preparation:

100 μl of troponin T solution with a concentration are added to 800 or 700 μl of buffer (0.1 mol / 1 Tris, pH 8.0, 100 mmol / 1 KCl / 2 mmol / 1 DTT / 0.05% NaN ₃ ) of 0.43 mg / ml (in 5 mmol / 1 KPÖ _« , pH 7.3 / 2.0 mol / 1 urea / 0.6 mol / 1 KCl / 2 mmol / 1 DTT / 0.05% NaN ₃ ) and 100 or 200 μl RSA solution with a concentration of 4.2 mg / ml (in 0.1 mol / 1 Tris, pH 8.0 / 100 mmol / 1 KCl / 2 mmol / 1 DTT / 0.05% NaN ₃ ).

Conditions in the solution:

Troponin T conc .: 0.043 μg / ml

RSA conc .: 0.42 mg / ml or 0.84 mg / ml

The aim of this solution was to show that the stabilizing effect of hsp25 is not an unspecific protein effect.

hsp25 solution:

The preparation was carried out as described under a). Instead of

Troponin T solution was only used as a buffer.

This hsp25 solution was designed to visualize the effects of hsp25 on the Troponin T-ELISA test.

The solutions were all made on the same day and then stored under identical conditions for 5 days at room temperature. 1.2 Check the stability of Troponin T (TN-T) in the above solutions

The stability of troponin T in the solutions was determined by measuring the immunological activity of troponin T using the Troponin T ELISA from Boehringer Mannheim.

For this purpose, after 5 days of storage of the troponin T solution described under 1.1a), a series of dilutions of 0.65 - 12.5 ng / ml (0.65 / 1.8 / 4.8 and 12.5 ng / ml). The dilutions were then used in the ELISA test, as described in the package insert for the test.

The control and comparison solutions described under 1.1b) were diluted accordingly and also used in the ELISA test.

As a comparison benchmark, the corresponding series of dilutions of a troponin T sample stored at -80 ° C were carried out in the test.

Test format

The Troponin T-ELISA test is a 1-step sandwich assay. The sample containing troponin T is incubated in a streptavidin-coated tube with a biotinylated anti-TN-T antibody 1 and a peroxidase (POD) -labelled anti-TN-T antibody 2. An immunological complex (sandwich) is formed from the two antibodies and the troponin T, which is bound to the SA tube wall via the biotin. The complex is then detected after adding POD substrate (ABTS ^® ) by determining the POD activity.

1.3. Results

The results are shown in Fig. 1.

==> In the absence of hsp25 there is a significant decrease in immunological activity compared to fresh thawed troponin T solution (TN-T at -80 ° C).

==> No decrease in immunological activity is observed in the solution of TN-T with hsp25.

==> RSA has no stabilizing effect on troponin T. The stabilizing effect of hsp25 is based on an hsp25-specific effect and not on a pure protein effect.

==> The troponin T-ELISA test is not affected by the hsp25 concentration used.

Example 2

Stabilization of recombinant HCV and KIV antigens

The examples were created using the double antigen bridge test concept. In this method, the sample liquid is incubated with a biotinylated antigen that can bind to a streptavidin-coated solid phase and with an antigen that carries a labeling group. The immobilized immunological complex consisting of the antibody to be determined and two antigens is then detected. The labeling group in the examples shown was a ruthenium complex, and the detection was carried out via an electrochemiluminescence reaction (ECL counts).

The following antigens were used: a) HCV-NS3

The antigen corresponds to the helicase sequence of the hepatitis C virus (HCV) - non-structural protein 3 (NS3) and was produced recombinantly in E. coli according to DE-A-44 28 705.4. The amino acid sequence of the HCV-NS3 polypeptide is shown in Fig. 2a. b) HCV-NS4

The antigen corresponds to a partial sequence of the HCV-NS4 protein and was produced synthetically. The amino acid sequence of the synthetic HCV-NS4 peptide is shown in Fig. 2b (never = norleucine). c) HIV-p24

The antigen corresponds to a fusion antigen from the sequence of the core antigen p24 (Ghrayeb and Chang, DNA 5 (1986), 93-99) and further core antigen sequences pl7 and pl5 from HIV and was produced recombinantly in E. coli. The amino acid sequence of this recombinant polypeptide is shown in Fig. 2c.

The synthetic HCV-NS4 antigen was used both in biotinylated and in ruthenilated form with a concentration of 30 ng / ml in the reaction mixture.

The recombinant antigens HCV-NS3 and HIV-p24 were used both in biotinylated and in ruthenilated form with a concentration of 400 ng / ml in the reaction mixture.

The buffer composition in all experiments was as follows:

Hepes 50 mmol / 1, pH 7.4

Thesite 0.1%

MIT (N-methylisothiazolone HCl) 0.01%

CAA (2-chloroacetamide) 0.1%

Mixture of protease inhibitors

The hsp25 concentration based on hsp25 monomer varied in the range of 0/0, 1/1, 1/5, 1/10 mole antigen / mole hsp25.

The antigens were mixed with the hsp25-containing buffer immediately before carrying out the immunological reaction. The incubation time of sample antibodies and antigens was 20 minutes at 37 ° C. The results were as follows

Synthetic HCV / NS4 antigen

Sample buffer buffer with buffer buffer with buffer with buffer with RSA 0.5 X sp25 1/1 hsp25 1/5 large 1/1 large 1/5

Signal recovery after storage for 3 weeks at 35 ° C in X.

1 19 59 106 110 56 54 2 15 23 81 101 48 29

Adding hsp25 significantly improves signal recovery compared to the control (buffer alone) and comparison approaches (RSA and groEl). The superior effect of hsp25 over the large heat shock protein groEl is particularly remarkable.

b) Recombinant HCV / NS3 antigen

Control (without adding hsp25!

Signal signal differentiation

(ECL counts) (signal positive serum / signal negative serum)

Buffer 396000 neg. Serum 88000 pos. Serum 381000

Method according to the invention (with addition of hsp25)

Signal signal differentiation

(ECL counts (signal positive serum / signal negative serum)

Buffer 36000 neg. Serum 29000 pos. Serum 345000 12

By adding hsp25, a significant reduction in blank values is found for both buffer and negative serum. This results in a surprisingly high improvement in the signal-to-noise ratio or the signal differentiation.

c) Recombinant HIV / p24 antigen

Sample buffer with buffer with buffer with buffer with (positive) RSA 0.5 X hsp25 1/5 hsp25 1/10 hsp25 1/10 without antigen

Signat differentiation pos. Signal / neg. signal

1 13 15 16 1 2 17 19 23 1 3 18 21 22 1

Here, too, there is a significant improvement in the signal-to-noise ratio or signal differentiation if the antigen is stabilized with hsp25.

Example 3

Concentration dependence of the effect of hsp25

The concentration dependence of the effect of hsp25 in stabilizing the HCV-NS3 antigen in a double antigen bridge test (carried out according to Example 2) is shown in Fig. 3.

From this it can be seen that the signal reduction in a negative sample can already be clearly seen with a molar ratio of the antigen to hsp25 of 1: 1. Even when large amounts of hsp25 are added, no significant signal reduction is found in positive samples. SEQUENCE LOG

(1) GENERAL INFORMATION: (i) LOGIN:

(A) NAME: Boehringer Mannheim

(B) STREET: Sandhofer Strasse 116

(C) LOCATION: Mannheim

(E) COUNTRY: Ger any

(F) POSTAL NUMBER: D-68298

(ii) DESCRIPTION OF THE INVENTION: Stabilization of peptides and polypeptides in immunological tests by adding small heat shock proteins. (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA) (vi) DATE OF THE URANE REGISTRATION

(A) REGISTRATION NUMBER: DE 195 02 386.2

(B) REGISTRATION DAY: JAN 26, 1995

(2) INFORMATION ON SEQ ID NO: 1: (i) SEQUENCE LABEL:

(A) LONG: 282 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Hepatitis C virus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Val Val Pro Gin Ser 1 5 10 15

Phe Gin Val Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr

20 25 30

Lys Val Pro Ala Ala Tyr Ala Ala Gin Gly Tyr Lys Val Leu Val Leu 35 40 45 Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys

50 55 60

Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr 65 70 75 80

Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp

85 90 95

Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys

100 105 110

His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp

115 120 125

Gin Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr

130 135 140

Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala 145 150 155 160

Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu

165 170 175

Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys

180 185 190

Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala

195 200 205

Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly

210 215 220

Asp Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly 225 230 235 240

Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gin Thr Val

245 250 255

Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr Ile Thr Leu Pro

260 265 270

Gin Asp Ala Val Ser Arg Thr Gin Arg Arg 275 280

(2) INFORMATION ABOUT SEQ ID NO: 2: (i) SEQUENCE LABEL:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Hepatitis C virus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Ser Gin His Leu Pro Tyr Ile Glu Gin Gly Never Never Leu Ala Glu Gin 1 5 10 15

Phe Lys Gin Gin Ala Leu Gly Leu Leu Gin Thr 20 25

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 328 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Human immunodeficiency virus type 1

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Thr Met Ile Thr Pro Ser Leu Ala Ala Gly Pro Asp Lys Gly Asn 1 5 10 15

Ser Ser Gin Val Ser Gin Asn Tyr Pro Ile Val Gin Asn Leu Gin Gly

20 25 30

Gin Met Val His Gin Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val

35 40 45

Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe

50 55 60

Ser Ala Leu Ser Glu Gly Ala Thr Pro Gin Asp Leu Asn Thr Met Leu 65 70 75 80

Asn Thr Val Gly Gly His Gin Ala Ala Met Gin Met Leu Lys Glu Thr

85 90 95

Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala

100 105 110

Gly Pro Ile Ala Pro Gly Gin Met Arg Glu Pro Arg Gly Ser Asp Ile

115 120 125

Ala Gly Thr Thr Ser Thr Leu Gin Glu Gin Ile Gly Trp Met Thr Asn

130 135 140

Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu 145 150 155 160 Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp

165 170 175

Ile Arg Gin Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe

180 185 190

Tyr Lys Thr Leu Arg Ala Glu Gin Ala Ser Gin Glu Val Lys Asn Trp

195 200 205

Met Thr Glu Thr Leu Leu Val Gin Asn Ala Asn Pro Asp Cys Lys Thr

210 215 220

Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr 225 230 235 240

Ala Cys Gin Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Ala

245 250 255

Glu Ala Met Ser Gin Val Thr Asn Ser Ala Thr Ile Met Met Gin Arg

260 265 270

Gly Asn Phe Arg Asn Gin Lys Lys Thr Val Lys Cys Phe Asn Cys Gly

275 280 285

Lys Glu Gly His Ile Ala Lys Asn Cys Arg Ala Pro Arg Lys Lys Gly

290 295 300

Cys Trp Lys Cys Gly Lys Glu Gly His Gin Met Lys Asp Cys Thr Glu 305 310 315 320

Arg Gin Ala Asn Phe Leu Gly Asn

325

Claims

PATENT CLAIMS

1. A method for determining an analyte in aqueous solution using immunologically reactive peptides or polypeptides and a stabilizer therefor, characterized in that the stabilizer comprises one or more proteins from the family of the "small" heat shock proteins (small hsp).

2. The method according to claim 1, characterized in that the hsp in a molar ratio of 0.0001: 1 to 1000: 1 and preferably from 0.01: 1 to 1000: 1 based on hsp monomer: peptide or polypeptide used.

3. The method according to claim 1 or 2, characterized in that one uses immunologically reactive polypeptides or peptides which are produced recombinantly or synthetically.

4. The method according to any one of the preceding claims, characterized in that the hsp 25 from the mouse and / or recombinant hsp 25 is used.

5. Use of one or more proteins from the family of small heat shock proteins to stabilize peptides in aqueous solution.

6. Use according to claim 5 to improve the storage stability of an aqueous solution which contains peptides.

7. Use of one or more proteins from the family of small heat shock proteins to improve the signal-to-noise ratio in a determination method. drive, which is carried out in the presence of at least one peptide and / or polypeptide in aqueous solution.

8. A method for the immunological determination of a specific antibody in a sample liquid, the sample liquid being incubated with at least one peptide or polypeptide antigen which is capable of binding to the antibody to be determined and the antibody being detected by binding to the antigen. characterized in that the antigen by adding one or more

Proteins from the family of "small" heat shock proteins stabilized.

9. The method according to claim 8, characterized in that one carries out the incubation and / or the detection in the presence of a reactive solid phase.

10. The method according to claim 9, characterized in that the sample liquid in the presence of a solid phase with an antigen, which is present in a form capable of binding to the solid phase, and with a detection antibody directed against the antigen, which carries a labeling group, and incubated detect the antibody to be determined by measuring the label in the solid phase and / or in the liquid phase.

11. The method according to claim 9, characterized in that the sample liquid is incubated with at least two antigens AG _j and AG ₂ in the presence of a solid phase, the first antigen AG _{X being} in a form which is bindable to the solid phase and the second antigen AG - carries a marker group and the antibody to be determined by measuring the marking in the solid phase and / or in the liquid phase.

12. A method for determining a peptide or polypeptide analyte in a sample liquid according to the principle of competitive immunoassay, wherein the sample liquid with an antibody directed against the analyte and a peptide or polypeptide that competes with the analyte for the antibody and carries a labeling group , incubated and the analyte detected by measuring the label, characterized in that the labeled peptide or polypeptide is stabilized by adding one or more proteins from the family of small heat shock proteins.

13. A method for determining reference values for the determination of a peptide or polypeptide analyte, wherein at least one test solution containing a known concentration of the analyte to be determined is incubated with an antibody directed against the analyte and by quantifying the immune reaction between Antibody and the analyte present in known concentration forms at least one reference value, characterized in that the antigen in the test solution is stabilized by adding one or more proteins from the "small" heat shock protein family.

14. Reagent kit for use in immunological test procedures, comprising in spatially separated form

(a) at least one first reagent which contains an immunologically reactive peptide or polypeptide,

(b) at least one second reagent which contains a further immunologically reactive component and

(c) optionally further auxiliary and / or detection reagents, characterized in that at least one first reagent containing an immunologically reactive peptide or polypeptide is stabilized by the addition of one or more proteins from the family of the "small" heat shock proteins.

15. Reagent kit according to claim 14, characterized in that the peptide or polypeptide is present in a form capable of binding to a reactive solid phase.

16. Reagent kit according to claim 14 or 15, characterized in that the peptide or polypeptide carries a labeling group.

17. Reagent kit according to one of claims 14 to 16, characterized in that the peptide or polypeptide is produced recombinantly or synthetically.

18. Reagent kit according to one of claims 14 to 17, characterized in that the reagents are present as a solution or lyophilisate.

19. Use of a reagent kit according to one of claims 14 to 18 for the determination of viral antigens or of antibodies directed against viral antigens.

20. Use according to claim 19, characterized in that one uses a peptide or polypeptide from HCV or HIV.