RU2356576C1 - SYNTHETIC ANTIGEN ABILITY TO BIND β1-ADRENORECEPTOR AUTOANTIBODIES - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to immunology. β₁-adrenoreceptor autoantibodies in human blood plasma/serum are bonded with a synthetic antigen containing nonapeptide (125-133) and tridecapeptide (208-218) of human β₁-adrenoreceptor sequences interbinded with disulfide bridge. The antigen is characterised with higher affinity as compared to applied 1st and 2nd loop β₁-adrenoreceptor sequences.

EFFECT: invention can be used for diagnostics and treatment of the patients suffering from dilated cardiomyopathy.

3 ex, 1 tbl, 1 dwg

Description

The invention relates to biochemistry and medicine, in particular to immunology, and serves to bind specific autoantibodies to a β ₁ -adrenoreceptor from human plasma and serum.

Cardiomyopathy is a group of diseases of the heart muscle of unknown etiology, among which the most threatening is dilated cardiomyopathy. As a rule, young and middle-aged men suffer from it. Dilated cardiomyopathy (DCMP) is one of the main causes of severe heart failure and the most common cause of heart transplant. Despite the success in the treatment of DCMP, the mortality rate of patients suffering from this disease is very high. Within 10 years, 70% of these patients die. The pathogenesis of this disease has not been fully studied - the hypotheses of chronic viral infection and genetic determination are considered, however, the latest literature data clearly indicate the autoimmune nature of this most serious disease [1, 2]. There is also literature evidence that therapeutic apheresis procedures lead to a significant improvement in the condition of patients with DCMP [3-5]. Autoantibodies found in the serum of patients with DCMP are antibodies against their own antigens, such as myosin, adenine nucleotide translocator, sarcolemal laminin, β ₁ -adrenergic receptor, muscarinic M2 receptor protein, etc. [6]. Most autoantibodies belong to the IgG class of immunoglobulins with a molecular weight of about 150 kDa.

The level of autoantibodies to β ₁ -adrenoreceptor in patients with DCMP varies among different authors, however, the literature data clearly indicate that these autoantibodies are most often found in patients with DCMP [7]. In animal models, it was shown that the introduction of a synthetic peptide corresponding to the sequence of the second loop of the β ₁ -adrenoreceptor leads to the development of DCMP in rabbits [8]; therefore, the presence of antibodies of this specificity plays a decisive role in the pathogenesis of this disease. In addition, it was shown that the presence of autoantibodies to the β ₁ -adrenoreceptor may be one of the factors in the development of tachyarrhythmias and acute heart failure [9, 10].

Therefore, the determination of the level of autoantibodies to the β ₁ -adrenoreceptor is extremely important for the possible diagnosis of dilated cardiomyopathy.

A known peptide is used to determine the level of autoantibodies to the β ₁ -adrenoreceptor, which is a portion 197-222 [7, 11] of the 2nd loop of the β ₁ -adrenoreceptor. The most detailed analysis of ELISA using the peptide of the 2nd loop of β ₁ -adrenoreceptor is given in [7]. The positive signal level in the dilution of the tested sera 1/40 in patients with DCMP was an average of 0.2 p.u. Moreover, only 13 patients out of 43 (31%) patients with DCMP showed a reliable positive response (exceeding the signal in ELISA by 2 or more times relative to the background of healthy donors with a negative response). The proportion of positive responses in the population of healthy donors was 12% [7].

The main disadvantage of the used peptide is limited specificity, since only autoantibodies to the antigenic determinant of the 2nd loop of the β ₁ -adrenoreceptor, represented by the sequence of this peptide, can interact with this peptide. Whereas in patients with DCMP, autoantibodies can be represented more broadly - both to the 1st and 2nd loops, and to the complex of two loops connected in the natural β ₁ -adrenoreceptor molecule by a disulfide bond. Thus, the limited antigenic specificity of this peptide can, in turn, explain the low sensitivity (the number of positive responses) in patients with DCMP.

The prototype of the claimed synthetic antigen is a combination of two individual peptides of the sequence 125-133 Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu (prototype I), corresponding to the 1st loop of β ₁ -adrenoreceptor, and sequence 206-218 Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe (prototype II) from the 2nd loop of the β ₁ -adrenoreceptor. Peptides are immobilized on a BrCN activated agarose matrix, the resulting sorbent is the active ingredient in the Coraffin column used to remove autoantibodies to β ₁ -adrenoreceptor in patients with DCMP [3]. Mentioned sorbent is a mechanical mixture of two sorbents with immobilized individual peptides, as indicated in the patent [12].

A significant drawback of this sorbent is, firstly, that a mechanical mixture of two sorbents with two individual peptides allows the binding of autoantibodies specific to antigenic determinants located either only on the 1st or only on the 2nd extracellular loop of the β ₁ -adrenoreceptor . Whereas in a natural molecule, these two extracellular loops are connected by a disulfide bond and, therefore, form an additional antigenic determinant, to which autoantibodies of a different specificity can form. Consequently, the affinity of such a sorbent will inevitably be insufficient to remove all autoantibodies to the β ₁ -adrenoreceptor, which was proved in the authors own experiments.

Based on the foregoing, the urgent task is to create a synthetic antigen that closely repeats the antigenic properties of the natural antigen, the β ₁ -adrenoreceptor, which, therefore, would have a higher affinity.

The problem is solved by the synthesis of an antigen, which is an individual chemical compound in which the peptides of the sequence 125-133 and 206-218 β ₁ -adrenoreceptor are connected by a disulfide bond, in contrast to the mixture (combination) of these peptides (prototype). The ability of such a synthetic antigen to bind autoantibodies to β ₁ adrenergic receptor is not obvious, since the claimed antigen is an artificial construct that is not a linear epitope (in the case of linear epitopes, antigenicity can be predicted using computer programs for analysis of the amino acid sequence of a protein [13]), including 2 β fragments ₁ -adrenoceptor (125-133 and 206-218), widely separated from each other and connected in one molecule spontaneous oxidation of the sulfhydryl groups of fragments 125-133 and 206-218 β ₁ adrenoceptor with hydrogen peroxide [14].

The synthesis of the claimed synthetic antigen was carried out by oxidation of hydrogen peroxide with an equimolecular mixture of the precursors nonapeptide (125-133) and tridecapeptide (208-218).

The synthesis of peptides of the sequence 125-133 of the 1st extracellular loop of the β ₁ -adrenoreceptor (nonapeptide) and the sequence of 206-218 of the 2nd loop of the β ₁ -adrenoreceptor (tridecapeptide) was carried out using standard solid-phase synthesis technology [15] using Fmoc * - methodologies on Wang polymer. We used derivatives of L-amino acids from Bachem (Switzerland), DIC, HOBT, TIBS from Fluka (Switzerland). For the synthesis, N-methylpyrrolidone, dichloromethane, piperidine, methanol and trifluoroacetic acid were used (Applied Biosystems, USA). DMF was purified by distillation over ninhydrin and barium oxide. The following protections were used to block the functional groups of the amino acid side chains: tert-butyl for the carboxyl groups of aspartic and glutamic acids, the hydroxyl function of serine and tyrosine; tert-butyloxycarbonyl (Boc) - protection for the ε-amino group of lysine; trityl (Trt) - group for the carboxamide function of asparagine and Pmc - for the guanidine function of arginine. For the protection of cysteine residues, see examples 1 and 2. The amino acid chain was increased by one amino acid, starting from the C-terminus, using the carbodiimide method with the addition of 1-hydroxybenzotriazole.

The synthesis was carried out on an automated peptide synthesizer Applied Biosystems, model 431 A according to the standard program for a single condensation of Fmoc amino acids. In each case, 0.25 mmol of Fmoc aminoacylpolymer (Bachem, Switzerland) was proceeded. For solid phase synthesis, a styrene copolymer with 1% divinylbenzene with a hydroxymethylphenoxymethyl anchor group, with a particle size of 200-400 mesh from Bachem (Switzerland), was used.

The standard solid phase synthesis protocol includes the following steps:

Solid Phase Synthesis Protocol

No. Operation Reagent Time of processing one Flushing 5 × NMP 3 min 2 The release of α-amino groups 20% Pip / NMP 10 min 3 Flushing 5 × NMP 3 min four Activation 1 mmol Fmoc amino acids + 1 mmol HOBt + 1 mmol DIC in NMP 20 minutes 5 Condensation 1 mmol activated derivative of Fmoc amino acids in NMP 90 min 6 Flushing 5 × NMP 3 min List of abbreviations: Boc - tert-butyloxycarbonyl; Acm - acetamidomethyl; AcOH - acetic acid; Bu ^t is tert-butyl; DIC — N, N ¹ -diisopropylcarbodiimide; DCM - dichloromethane; DMF — N, N-dimethylformamide; DMSO - d6 - deuterated dimethyl sulfoxide; Fmoc is 9-fluorenylmethoxycarbonyl; HOBT - 1-hydroxybenzotriazole; NMP - N-methylpyrrolidone; Pmc - 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Pip - piperidine; TIBS - triisobutylsilane; TFA - trifluoroacetic acid; HPLC - high performance liquid chromatography; TFS - solid-phase synthesis of peptides

The peptides were cleaved and released by trifluoroacetic acid with special additives that prevent side reactions. The sulfhydryl groups of cysteine residues were released by the action of mercury acetate. Linear nonapeptide and tridecapeptide were purified using preparative HPLC to 97-98% purity. Preparative HPLC was performed on a Beckman instrument (USA), peptides were detected at 226 nm. Peptides were eluted with a gradient of acetonitrile concentration in 0.1% TFA. For HPLC, acetonitrile from Technopharm (RF) was used. Analytical HPLC was performed on Ultrasphere ODS columns (Beckman, USA), (5 μm, 4.6 × 250 mm); on a chromatograph company Gilson (France). As eluents used buffer A - 0.1% TFA, pH 2.0, buffer B - 80% acetonitrile in buffer A, elution with a concentration gradient of buffer B in buffer A at a flow rate of 1 ml / min.

The synthesized peptides were characterized by ¹ H-NMR spectroscopy ( ¹ H-NMR spectra were recorded on a WH-500 Bruker 500 MHz spectrometer (Germany) in DMSO-d at 300 K, peptide concentration was 2-3 mg / ml. Chemical shifts were measured relative to tetramethylsilane), mass spectrometry (mass spectra were recorded on a PC-Kompact MALDI instrument, Kratos, England).

Example 1. Synthesis of the nonapeptide sequence 125-133 of the 1st loop of the β ₁ -adrenoreceptor H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-OH (precursor I)

Stage 1. Solid-phase synthesis of H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys (Acm) -Glu-Leu-OH (precursor I _Acm ) was carried out on the basis of 0.34 g of Fmoc-Leu-polymer containing 0.25 mmol starting amino acids, in accordance with the above standard protocol. The sulfhydryl group of the cysteine residue was protected with acetamidomethyl protection.

The final release and cleavage of the nonapeptide (I _Acm ) from the polymer was carried out in one step by treating the corresponding nonapeptidyl polymer with a mixture of 10 ml of TFA and 0.5 ml of H ₂ O for 2 hours. Then the polymer was filtered off, washed with 2 × 2 ml of the deblocking mixture, the filtrate was evaporated and dry ether was added to the residue. The precipitate was filtered off, washed with dichloromethane (3 × 3 ml), ether (3 × 5 ml), and dried in a vacuum desiccator. Received 0.25 g of crude product (I _Acm ) containing, according to HPLC, 94% of the target peptide.

The peptide was purified using preparative HPLC on a Beckman instrument (USA) using a 130T Diasorb-C16 column (25 × 250 mm), sorbent particle size 10 μm. As eluents, we used: buffer A — 0.1% aqueous TFA solution and buffer B — 80% acetonitrile in water, elution was performed with a gradient of 0.5% per minute of buffer B from 100% buffer A, and a flow rate of 10 ml / min. Peptides were detected at a wavelength of 226 nm. Fractions containing the desired product were combined and lyophilized. As a result, 0.15 g (51% based on the starting amino acid attached to the polymer carrier) of the peptide trifluoroacetate (I _Acm ) was _obtained . The homogeneity of the product, determined by analytical HPLC, is 98%. Amino acid composition according to ¹ H-NMR spectroscopy: Glu 2, Ser 1, Gly 1, Leu 1, Phe 2, Tug 1, Cys 1.

Mass spectrum, m / z: 1165.5 [M + H] ⁺ , calculated 1164.7, for C ₅₄ H ₇₂ N ₁₀ О ₁₇ S ₁ .

Stage 2. Obtaining the nonapeptide H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-OH (precursor I)

0.05 g (0.043 mmol) of H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys (Acm) -Glu-Leu-OH (I _Acm ) was dissolved in 5 ml of 30% AcOH, 0.03 g (0.09 mmol) of acetate was added mercury in 1.5 ml of 30% AcOH, since the peptide is poorly soluble, the concentration of the solution was adjusted to 50% AcOH and stirred for 1.5 h at 20 ° С, then a stream of hydrogen sulfide was passed for 30 min. The precipitate was filtered off, washed with 2 × 5 ml of 30% AcOH. The filtrate was evaporated to a volume of ~ 2 ml and chromatographed on a column (25 × 250 mm) with Diasorb. Elution was performed with a gradient of buffer B (0.5% per minute, from 20 to 80%) in buffer A at a flow rate of 10 ml / min. The fractions corresponding to the target product were combined, evaporated, the residue was dissolved in water and lyophilized. Yield 0.038 g (80.0%). Mass spectrum (MALDI-MS): found m / z - 1095.2 ([M + H ⁺ ]), calculated 1094.2 for C ₅₁ H ₆₇ N ₉ O ₁₆ S.

Example 2. Synthesis of the tridecapeptide sequence 206-218 of the 2nd loop of the β ₁ -adrenoreceptor H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-OH (precursor II)

Stage 1. Solid-phase synthesis of H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys (Acm) -Asp-Phe-OH (precursor II _Acm ) was carried out on the basis of 0.37 g Fmoc A -Phe polymer containing 0.25 mmol of the starting amino acid, in accordance with the above standard protocol. The sulfhydryl groups of the Cys ⁴ and Cys ¹⁰ cysteine residues were protected with trityl protection, and Cys ^{11 with} acetamidomethyl.

The final release and cleavage of the tridecapeptide (II _Acm ) from the polymer was carried out in one step by treating the corresponding tridecapeptide peptide with a mixture of 10 ml of TFA, 0.25 ml of H ₂ O and 0.25 ml of TIBS for 2 hours. Then the polymer was filtered off, washed with 2 × 2 ml of the deprotection mixture the filtrate was evaporated and dry ether was added to the residue. The precipitate was filtered off, washed with dichloromethane (3 × 3 ml), ether (3 × 5 ml), and dried in a vacuum desiccator. Received 0.42 g of crude product (II _Acm ) containing, according to HPLC, 60% of the target peptide.

After that, the peptide was purified using preparative HPLC as in Example 1. As a result, 0.13 g (32% based on the starting amino acid attached to the polymer carrier) of trifluoroacetate peptide (II _Acm ) was _obtained . Product homogeneity determined by analytical HPLC was 98%. Amino acid composition of ¹ H-NMR spectroscopy: Asn 1, Asp 2, Ala 1, Phe 1, Tyr 1, Lys 1, Arg 2, Pro 1, Cys 3.

Mass spectrum, m / z: 1661.2 [M + H] ⁺ , calculated 1661.9 for C ₆₈ H ₁₀₄ N ₂₂ O ₂₁ S ₃ .

Stage 2. Obtaining the tridecapeptide H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-OH (precursor II)

The release of the Cys ¹¹ cysteine residue in the tridecapeptide H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys (Acm) -Asp-Phe-OH (II _Acm ) was carried out by the action of mercury acetate, as described in example 1.

The yield of tridecapeptide 0.027 g (90.0%). Mass spectrum (MALDI-MS): found m / z - 1591.9 ([M + H ⁺ ]), calculated 1590.8 for C ₆₅ H ₉₉ N ₂₁ O ₂₀ S ₃ .

Example 3. A synthetic antigen comprising a nonapeptide (125-133) and a tridecapeptide (208-218) of a β ₁ -adrenoreceptor sequence connected by a disulfide bond

0.007 g (0.006 mmol) of nonapeptide (site 125-133 β ₁ -adrenoreceptor) and tridecapeptide (208-218 β ₁ -adrenoreceptor) 0.009 g (0.006 mmol) was dissolved in 20 ml of 10% aqueous dioxane, 1 ml of 5% aqueous ammonia was added to pH 8.0. 0.1 ml of 3% aqueous hydrogen peroxide was introduced into the mixture and stirred for 10 min. The completeness of the oxidation of SH groups was monitored using Ellman's reagent (colorless solution). At the end of the reaction, the target product was chromatographed on a 25 × 600 mm column with Sephadex G-25 equilibrated with a 2% solution of acetic acid to remove low molecular weight impurities. Fractions containing the desired product were combined and lyophilized. Received 0.013 g of synthetic antigen (81%). Mass spectrum: 2679.8 ([M + H ⁺ ]), calculated 2680.9 for C ₁₁₆ H ₁₆₂ N ₃₀ O ₃₆ S ₄ .

Enzyme-linked immunosorbent assay of autoantibodies to β ₁ -adrenoreceptor on a synthetic antigen

Comparison of the specificity of the claimed synthetic antigen with the peptide described in the literature, which is the 2nd extracellular loop of the β ₁ -adrenoreceptor, was carried out under identical conditions for the production of indirect solid-phase ELISA. The methodology and conditions for the production of ELISA were similar to those described in the literature [7].

The following components were included in the reagent kit for the determination of autoantibodies: 96-well plate for immunoassay, Costar, Cat. No. 9018; synthetic antigen (see example 3) and peptide 197-222 of the 2nd extracellular loop of β ₁ -adrenoreceptor; 0.1 M Na-carbonate buffer; a solution for diluting samples, conjugates, a blocking solution and a solution for washing tablets - 0.01 M Na-phosphate buffer, pH 7.2, containing 8 g / L M NaCl, 0.1% Tween-20, 3% skimmed milk powder and bacteriostatic CG Caton 2.0 ml / L (FSBM-T); the solution for washing the tablets is 0.01 M Na-phosphate buffer pH 7.2, containing 0.15 M NaCl and 0.1% Tween-20 (FSB-T); conjugate - reagent for analysis: mouse monoclonal antibodies against human IgG labeled with biotin; streptavidin peroxidase conjugate; a solution of tetramethylbenzidine (TMB), 1 mmol / L in dimethylformamide; substrate buffer solution - sodium citrate 5.5-aqueous, 26 g / l; citric acid 6.92 g / l, sodium perborate 1.1 g / l and caton CG 2.0 ml / l; stop reagent - phosphoric acid 5%.

Analysis protocol

Before analysis, the components of the kit were kept at a temperature of + 20 ° С for 60 min. To immobilize the antigen on ELISA plates, peptide preparations were diluted to a concentration of 5 μg / ml in 0.1 M Na ₂ CO ₃ -NaHCO ₃ pH 9.6. 100 μl were introduced into the wells of the plates and incubated for 20 hours at + 20 ° C. To exclude nonspecific interactions, 100 μl of FSBM-T was added to the plate wells and incubated for 3 hours at + 20 ° С with shaking. To remove unbound antigens, the wells of the plate were washed 3 times with FSBM-T. Samples diluted in FSBM-T 20 and 100 times were pipetted into the corresponding wells of 100 μl, incubated at a temperature of + 4 ° C for 12 hours. Washed tablet FSBM-T at least 3 times. 100 μl of the working solution of the reagent for analysis in FSBM-T were added to the wells, incubated at a temperature of + 20 ° С for 60 min with shaking. The plate was washed with FSBM-T solution three times. 100 μl of the working solution of the conjugate in FSBM-T were added to the wells, the plate was incubated at a temperature of + 20 ° С for 30 min with shaking. Washed with a FSB-T solution four times, then 100 μl of a working solution of a freshly prepared substrate mixture (7 volumes of substrate buffer and 1 volume of TMB solution) were immediately added to the wells. The plate was closed with a lid and incubated at a temperature of + 20 ° С without stirring for 15 min, then 100 μl of stop reagent were added in the same sequence and by the same method as the substrate mixture was added. Within 5 minutes after adding the stop reagent, the absorbance was recorded in a plate at a wavelength of 450 nm.

Determination of sorbent binding to immobilized synthetic antigen and specificity characterization

The claimed synthetic antigen and the corresponding combination of peptides of the sequence 125-133 and 206-218 of the β ₁ -adrenoreceptor molecule of the prototype for the bromine-activated Sepharose 4 FF were immobilized according to the standard method of immobilizing protein ligands on an agarose matrix.

The peptides were dissolved to a concentration of 0.3-2.5 mg / ml and immobilized on an agarose matrix, for which solutions of the peptides in 0.2 M borate buffer with a pH of 8.0 were incubated for 10 hours with a pre-activated bromine cyan agarose matrix.

The concentration of the immobilized ligand was estimated by the difference in the amount of peptide used for the immobilization reaction and remaining in the solution after incubation with the matrix. The concentration of the peptide in the solution was measured by optical absorption at 280 nm. At the end of the immobilization, the sorbent was washed with 10 volumes of distilled water. Then, 0.5 ml of a 1 M solution of ethanolamine acidified with HCl to pH 8 was added to the resulting sorbent (2 ml of suspension containing 1 ml of settled gel). The mixture was stirred at 20 ° C for 30 minutes, then the sorbent was washed with 20 volumes of water.

To determine the amount of adsorbed antibodies on a sorbent with the claimed synthetic antigen and prototype immobilized, affinity chromatography of the blood plasma of patients with DCMP and a healthy donor was performed. The ratio of the sorbent volume to the plasma volume was 2: 1, the incubation time was 1 hour. After incubation, the sorbents were washed with a 50-fold volume of a buffer mixture of 0.01 M KH ₂ PO ₄ -NaOH with a pH of 7.0, the bound protein was eluted with a 20-fold volume of a buffer mixture of 0.2 M glycine-HCl pH 2.5.

results

The sensitivity and specificity of detection of autoantibodies to β ₁ -adrenoreceptor

A comparison of the specificity of the claimed synthetic antigen with the peptide of the 197-222 portion of the 2nd extracellular loop of the β ₁ -adrenoreceptor described in the literature was carried out in the experiment with standardized conditions. The conditions for the production of ELISA corresponded to the ELISA conditions described for the prototype [7]. Autoantibodies were determined in the group of patients with DCMP (group 1, n = 9), in the mixed group of patients with a reduced ejection fraction of the left ventricle (both with DCMP and tachyarrhythmias) (group 2, n = 16) and in the group of healthy donors ( group 3, n = 24).

The results of ELISA sensitivity for determining autoantibodies to β ₁ -adrenoreceptor in plasma / serum samples of patients of the studied groups and healthy donors are shown in the table.

Table. Comparison of the average signal in ELISA between different groups Sample The patients Healthy donors group 1, n = 6 group 2, n = 16 group 3, n = 24 The inventive synthetic antigen 0.32 ± 0.12 * 0.36 ± 0.08 ** 0.22 ± 0.04 peptide 197-222 of the β ₁ -adrenoreceptor sequence 1.1 ± 0.9 1.2 ± 0.7 0.8 ± 0.2

The data are presented as mean ± deviation for confidence probability by Student's criterion, the level of statistical significance of the results ** p <0.005, * p <0.1.

An acceptable level of statistical significance (p <0.05) between the group of healthy donors and groups of patients in the authors' own experiments was observed only for ELISA using a synthetic antigen, indicating a greater specificity of the interaction of the claimed synthetic antigen with autoantibodies to β ₁ -adrenoreceptor than peptide 197-222.

According to published data, the detection rate of autoantibodies to β ₁ -adrenoreceptor in patients with DCMP on peptide 197-222 of the β ₁ -adrenoreceptor sequence is 13 of 42 positive responses, which is 30%

[7]. In our own experiments using the inventive synthetic antigen, the detection rate of autoantibodies to β ₁ -adrenoreceptor in patients with DCMP was 44% (4 positive responses out of 9 patients with DCMP), while on peptide 197-122 of the β ₁ -adrenoreceptor sequence - 33% (3 positive response from 9 patients with DCMP) (data not shown). The signal considered to be a positive response was two times the value of the average background level. The data obtained indicate a greater sensitivity of the method for detecting autoantibodies to β ₁ -adrenoreceptor when using the inventive synthetic antigen.

The binding of autoantibodies to β ₁ -adrenoreceptor on sorbents

It should be noted that there is no data in the literature that allows us to compare the claimed synthetic antigen with the prototype according to the sorption characteristics. Therefore, the sorption properties of the sorbent containing the synthetic antigen and the sorbent with the prototype were tested by affinity chromatography with human blood plasma under the same conditions in vitro experiments. For this, the blood plasma of patients with DCMP (patient 1 and 2), a patient with tachyarrhythmia (patient 3), and a healthy donor were used. The number of class G immunoglobulins bound to the tested sorbents during affinity chromatography of individual plasmas of patients with DCMP and healthy donor plasma is shown in the drawing.

In the drawing - the Number of associated autoantibodies to β ₁ -adrenoreceptor from the plasma of patients and the plasma of healthy donors on sorbents with an immobilized prototype and the claimed synthetic antigen.

According to the authors' own research, the average number of adsorbed antibodies for a sorbent with the claimed synthetic antigen immobilized (the amount of IgG in the eluate with 1 ml of sorbent) in experiments with three blood plasma samples of patients with DCMP was 227 ± 35 μg, whereas in the prototype 70 ± 65 μg (p <0.05).

Thus, the claimed synthetic antigen allows with significantly greater sensitivity and affinity to bind autoantibodies from plasma / serum of patients with the presence of autoantibodies to β ₁ -adrenoreceptor compared to the prototype.

Literature

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2. Fu M. et al. Is cardiomyopathy an autoimmune disease? // 2002.- V.51 (4). - P.208-212.

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4. Staudt A. et al. Immunohistological changes in dilated cardiomyopathy induced by immunoadsorption therapy and subsequent immunoglobulin substitution. // Circulation. - 2001 .-- V.103 (22). - P.2681-2686.

5. Konovalov G.A. et al. Apheresis of immunoglobulins - a new approach to the treatment of severe forms of dilated cardiomyopathy. // Cardiology. - 2002 - No. 6 - S.123-127

6. Matoba Y. et al. Therapeutic Left Ventricular Assist Device and Apheresis on Dilated Cardiomyopathy. // Artificial Organs. - 2004 .-- V.28 (2). - P.171-181.

7. Magnusson Y. et al. Autoimmunity in idiopathic dilated cardiomyopathy. Characterization of antibodies against the beta 1-adrenoceptor with positive chronotropic effect. // Circulation. - 1994 .-- V.89 (6). - P.2760-2767.

8. Matsui S. et al. Active immunization of combined betal-adrenoceptor and M2-muscarinic receptor peptides induces cardiac hypertrophy in rabbits. // J Card Fail. - 1999 .-- V.5 (3). - P.246-254.

9. Zhang L et. al. Autoantibodies against the myocardial betal-adrenergic and M2-muscarinic receptors in patients with congestive heart failure Chin Med J (Engl). 2002; 115 (8): 1127-31.

10. Chiale PA, et. al. Differential profile and biochemical effects of antiautonomic membrane receptor antibodies in ventricular arrhythmias and sinus node dysfunction. // Circulation. 2001; 103 (13): 1765-71.

11. Iwata M. Et al, Autoimmunity Against the Second Extracellular Loop of β ₁ -Adrenergic Receptors Induces β-Adrenergic Receptor Desensitization and Myocardial Hypertrophy In Vivo. // Circulation Research. - 2001 - V.88. - P.578-586.

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14. M.B. Sidorova, A. S. Molokoedov, A. A. Azmuko, E. V. Kudryavtseva, E. Krause, M. V. Ovchinnikov, J. D. Bespalova. The use of hydrogen peroxide for the closure of disulfide bridges in peptides. // Bioorganic chemistry, 2004, volume 30, No. 2, pp. 115-125.

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Claims (1)

A synthetic antigen for binding of autoantibodies to a β ₁ -adrenoreceptor in human blood plasma, comprising a nonapeptide (125-133) and a tridecapeptide (208-218) of a human β ₁ -adrenoreceptor sequence, characterized in that the claimed antigen is an individual chemical compound in which both peptides are linked by a disulfide bond.

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