Synthetic antigen ability to bind β1-adrenoreceptor autoantibodies

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

 

The invention relates to biochemistry and medicine, in particular to immunology, and serves to bind antibodies specific to β1-adrenergic receptors from plasma and human serum.

Cardiomyopathy is a group of diseases of the heart muscle of unknown etiology, among them the most threatening is dilated cardiomyopathy. Usually her suffer men of young and middle age. Dilated cardiomyopathy (DCMP) is one of the main causes of severe heart failure and the most frequent cause of heart transplantation. Despite advances in therapy DCMP mortality of patients suffering from the disease is very high. Within 10 years, 70% of such patients die. The pathogenesis of this disease is not fully understood, discusses the hypothesis of a chronic viral infection and genetic determinism, however, recent literature data indicate autoimmune nature of this severe disease [1, 2]. There is also literary evidence that the procedures of therapeutic apheresis leads to a significant improvement of the patients DCMP [3-5]. Autoantibodies detected in the serum of patients with DCMP are antibodies against its own antigens, such as myosin, actinolite translocator, sarcolemmal laminin, β1-adrenoreceptor the R, protein muscarinic M2-receptor and others [6]. Most antibodies belong to the IgG class of immunoglobulin with a molecular weight of about 150 kDa.

The level of autoantibodies to β1-adrenergic receptors in patients with DCMP varies with different authors, however, literature data clearly suggests 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 β1-adrenergic receptors leads to the development of DC in rabbits [8], therefore, the presence of antibodies of a given specificity plays a crucial role in the pathogenesis of this disease. In addition, it is shown that the presence of autoantibodies to β1-adrenergic receptors may be one of the factors in the development of tachyarrhythmias and acute heart failure [9, 10].

Therefore, determining the level of antibodies to β1-adrenergic receptors is extremely important for the possible diagnosis of dilated cardiomyopathy.

Known peptide used to determine the level of antibodies to β1-adrenergic receptors, which represent the area 197-222 [7, 11] 2nd loop β1-adrenergic receptors. The most detailed analysis of the ELISA using the peptide 2nd loop β1-adrenergic receptors is given in the article [7]. The level of the positive signal when breeding is esterwegen sera 1/40 in patients with DCMP was an average of 0.2 PU However, only in 13 patients out of 43 (31%) patients with DCMP was found statistically significant positive response (excess signal in ELISA in 2 and more times against a background of healthy donors with a negative response). In a population of healthy donors, the proportion of positive responses was 12% [7].

The main disadvantage of the used peptide is limited specificity, as this peptide can interact only antibodies to antigenic determinants of the 2nd loop β1-adrenergic receptors, presents the sequence of this peptide. While in patients with DCMP autoantibodies can be more as the 1-St and 2-nd loops, and complex two loops connected in a natural molecule β1-adrenergic receptors by a disulfide bond. Thus, the limited antigenic specificity of this peptide can, in turn, to explain the low sensitivity (the number of positive responses) in patients with DCMP.

The prototype of the proposed synthetic antigen is a combination of two individual peptide sequence 125-133 Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu (prototype I)corresponding to the 1st loop β1-adrenergic receptors and sequence 206-218 Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe (prototype II) of the 2nd loop β1-adrenergic receptors. Peptides immobilized on agarose matrix, Akti the new Enrichment, the resulting sorbent is the active ingredient column "Coraffin used to remove autoantibodies to β1-adrenergic receptors in patients with DCMP [3]. The said sorbent is a mechanical mixture of two sorbents with immobilized individual peptides that are specified in the patent [12].

A significant drawback of this sorbent is, first, that a mechanical mixture of the two sorbents with two individual peptides can bind antibodies specific for antigenic determinants located either on the 1st or 2nd extracellular loop β1-adrenergic receptors. Whereas in the natural molecule these two extracellular loops are connected by a disulfide bond and, therefore, form an additional antigenic determinant that can be formed of other autoantibodies specificity. Therefore, the affinity of such sorbent inevitably will not be sufficient to remove all of autoantibodies to β1-adrenergic receptors, which has been proved in their experiments the authors.

Based on the foregoing, the main task is to create a synthetic antigen which most closely mimic the antigenic properties of the natural antigen - β1-adrenergic receptors, which, consequently, would have a higher affinity.

Task d is highlighted by the synthesis of antigen, representing an individual chemical compound, in which the peptide sequence 125-133 and 206-218 β1-adrenergic receptors are connected by a disulfide bond, unlike a mixture (combination) of these peptides (prototype). The ability of such a synthetic antigen to bind autoantibodies to β1adrenoreceptor not obvious, since the inventive antigen - artificial design, non-linear epitope (in the case of lineinih epitope antigenicity can be predicted using a computer program for the analysis of amino acid sequences [13]), including 2 fragment β1-adrenergic receptors (125-133 and 206-218), far from each other and United in one molecule spontaneous oxidation of sulfhydryl groups of fragments 125-133 and 206-218 β1-adrenergic receptors using hydrogen peroxide [14].

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

Synthesis of peptides sequence 125-133 1st extracellular loop β1-adrenergic receptors (nonapeptide) and sequence 206-218 2nd loop β1-adrenergic receptors (tridecapeptide) was carried out according to a standard technique of synthesis on solid phase [15] using Fmoc* methodology on Wang resin. In R is the bot used derivatives of L-amino acids company Bachem (Switzerland), DIC, HOBT, TIBS company Fluka (Switzerland). For the synthesis of applied N is an organic, dichloromethane, piperidine, methanol and triperoxonane acid (Applied Biosystems, USA). DMF was purified by distillation over ninhydrin and barium oxide. To block functional groups of the side chains of amino acids was applied the following protection: tert-boutelou to carboxyl groups of aspartic and glutamic acids, the hydroxyl function of serine and tyrosine; tert-butyloxycarbonyl (Boc) - protection for ε-amino group of lysine; trailing (Trt) group carboxamide function of asparagine and Pmc for guanidino function of arginine. On the protection of the cysteine residues, see examples 1 and 2. The amino acid chain is increased by one amino acid, starting From the end, using the carbodiimide method with the addition of 1-hydroxybenzotriazole.

The synthesis was carried out on an automatic peptide synthesizer, Applied Biosystems model 431 And the standard program for a single condensation of Fmoc-amino acids. In each case came from 0.25 mmol Fmoc-aminoazotoluene (Bachem, Switzerland). For solid-phase synthesis used is a copolymer of styrene with 1% divinylbenzene with hydroxymethoxypethidine anchor group, with a particle size 200-400 mesh company Bachem (Switzerland).

Standard solid-phase synthesis Protocol includes the following stages:

Protocol for solid-phase synthesis

No.OperationReagentProcessing time
1Flushing5×NMP3 min
2The release of α-amino groups20% Pip/NMP10 min
3Flushing5×NMP3 min
4Activation1 mmol of Fmoc-amino acids + 1 mmol HOBt + 1 mmol of DIC in NMP20 min
5Condensation1 mmol of activated derivative Fmoc-amino acid in NMP90 min
6Flushing5×NMP3 min
List of abbreviations: Boc - tert-butyloxycarbonyl; Acm - acetamidomethyl; Asón - acetic acid; Buttert-butyl; DIC - N,N1-diisopropylcarbodiimide; DCM is dichloromethane; DMF IS N,N-dimethylformamide; DMSO - d6 - deuterated dimetilan XID; Fmoc - 9-fluorenylmethoxycarbonyl; HOBT is 1-hydroxybenzotriazole; NMP is N-organic; Pmc - 2,2,5,7,8-pentamethylchroman-6-sulfonyl; Pip - piperidine; TIBS - triisobutylene; TFA - triperoxonane acid; HPLC - high performance liquid chromatography; TPS - solid-phase synthesis of peptides

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

Synthesized peptides characterized data1H-NMR (1H-NMR spectra were taken on the spectrometer WH-500 Bruker 500MHz (Germany) in DMSO-d at 300 K, the concentration of the peptides was 2-3 m is/ml. Chemical shifts were measured relative to tetramethylsilane), mass spectrometry (mass spectra were recorded on the instrument PC-Kompact MALDI, Kratos, UK).

Example 1. The synthesis of the nonapeptide sequence 125-133 1st loop β1-adrenergic receptors H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-OH (predecessor (I)

Stage 1. Solid-phase synthesis of H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys(Acm)-Glu-Leu-OH (the predecessor of IAcm) was performed on the basis of 0.34 g Fmoc-Leu-polymer containing 0.25 mmol of the starting amino acid, in accordance with the above standard Protocol. Sulfhydryl group of the cysteine residue defended acetamidomethyl protection.

The final release and cleavage of the nonapeptide (IAcm) from the polymer were carried out in one stage by treatment of the corresponding nonparticipation a mixture of 10 ml of TFA and 0.5 ml of H2O for 2 hours and Then the polymer was filtered off, washed with 2×2 ml deblokiruyuschee mixture, the filtrate was evaporated and to the residue was added dry ether. The precipitate was filtered, washed with dichloromethane (3×3 ml), ether (3×5 ml), was dried in a vacuum desiccator. Obtained 0.25 g of crude product (IAcm), containing according to HPLC 94% of the target peptide.

Purification of the peptide was performed using preparative HPLC on a Beckman instrument (USA), using a column of diasorb-C16 T (25×250 mm), the particle size of the sorbent - 10 μm. As the eluents were used: buffer A - 0.1% aq is th solution of TFA and buffer B - 80% acetonitrile in water, the elution was performed with a gradient of 0.5% per minute buffer B to 100% buffer a, flow rate 10 ml/min, the Peptides were detected at a wavelength of 226 nm. The fraction containing the target product was combined and liofilizirovanny. In the end obtained 0.15 g (51% calculated on the starting amino acid attached to the polymeric carrier) trifenatate peptide (IAcm). The homogeneity of the product was determined using analytical HPLC, 98%. The amino acid composition according to1H-NMR: Glu 2, Ser 1, Gly 1, Leu 1, Phe 2, Hard 1, Cys 1.

Mass spectrum, m/z: 1165.5 [M+H]+calculated 1164.7, for C54H72N10About17S1.

Stage 2. Getting nonapeptide H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys-Glu-Leu-OH (predecessor (I)

0.05 g (0.043 mmol) of H-Glu-Tyr-Gly-Ser-Phe-Phe-Cys(Acm)-Glu-Leu-OH (IAcm) was dissolved in 5 ml of 30% Asón, was added 0.03 g (0.09 mmol) of the acetate of mercury in 1.5 ml of 30% Asón, as the poorly soluble peptide drove the concentration of the solution up to 50% of the Asón and stirred 1.5 h at 20°C, then passed a current of hydrogen sulfide for 30 minutes the Precipitate was filtered off, washed with 2×5 ml of 30% Asón. The filtrate was evaporated to a volume of ~2 ml and was chromatographically on a column (25×250 mm) with Dearbom. Elution was performed with a gradient of buffer B (0.5% min, 20%to 80%) in the buffer And a flow rate of 10 ml/min fractions corresponding to the desired product were combined, evaporated, the residue rest rely in water and liofilizovane. Yield 0.038 g (80.0%). Mass spectrum (MALDI-MS): found m/z - 1095.2 ([M+H+]), calculated 1094.2 for C51H67N9O16S.

Example 2. Synthesis of tridecapeptide sequence 206-218 2nd loop β1-adrenergic receptors H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-OH (predecessor II)

Stage 1. Solid-phase synthesis of H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys(Acm)-Asp-Phe-OH (predecessor IIAcm) was performed on the basis of 0,37 g of Fmoc-Phe-polymer containing 0.25 mmol of the starting amino acid, in accordance with the above standard Protocol. Sulfhydryl groups of Cys residues4and Cys10cysteine was protected trailvoy protection and Cys11- acetamidomethyl.

The final release and removal tridecapeptide (IIAcm) from the polymer were carried out in one stage by treatment of the corresponding tridecapeptide a mixture of 10 ml of TFA, 0.25 ml of N2O and 0.25 ml of TIBS for 2 hours and Then the polymer was filtered off, washed with 2×2 ml deblokiruyuschee mixture, the filtrate was evaporated and to the residue was added dry ether. The precipitate was filtered, washed with dichloromethane (3×3 ml), ether (3×5 ml), was dried in a vacuum desiccator. Was obtained 0.42 g of crude product (IIAcm), containing according to HPLC 60% of the target peptide.

After that conducted clearing peptide using preparative HPLC as in example 1. Finally got 0.13 g (32% calculated on the starting s is nomikoto, attached to the polymeric carrier) trifenatate peptide (IIAcm). The homogeneity of the product was determined using analytical HPLC was 98%. The amino acid composition of1H-NMR: 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 C68H104N22O21S3.

Stage 2. Getting tridecapeptide H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys-Asp-Phe-OH (predecessor II)

Release cysteine residue Cys11in tridecapeptide H-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Cys(Acm)-Asp-Phe-OH (IIAcmwas conducted by action of the acetate of mercury, as described in example 1.

The output of tridecapeptide 0.027 g (90.0%). Mass spectrum (MALDI-MS): found m/z - 1591.9 ([M+H+]), calculated 1590.8 for C65H99N21O20S3.

Example 3. Synthetic antigen, comprising the nonapeptide (125-133) and tridecapeptide (208-218) sequence β1-adrenergic receptors, United by a disulfide bond

0.007 g (0.006 mmol) of nonapeptide (section 125-133 β1-adrenergic receptors) and tridecapeptide (208-218 β1-adrenergic receptors) 0.009 g (0.006 mmol) was dissolved in 20 ml of 10% aqueous dioxane was added 1 ml of 5% aqueous ammonia to a pH of 8.0. Made in a mixture of 0.1 ml of 3% aqueous hydrogen peroxide and stirred for 10 minutes complete oxidation of SH-groups was controlled using a reagent of Ellman (colourless solution). OK what nanii reaction, the target product was chromatographically on column 25×600 mm with Sephadex G-25, balanced 2% solution of acetic acid, to remove low molecular weight impurities. The fractions containing the desired product were combined and liofilizirovanny. Received 0.013 g of synthetic antigen (81%). Mass spectrum: 2679.8 ([M+H+]), calculated 2680.9 for C116H162N30About36S4.

Enzyme-linked immunosorbent assay of antibodies to β1-adrenergic receptors on synthetic antigen

Comparison of the specificity of the claimed synthetic antigen described in the literature by a peptide representing the 2nd extracellular loop β1-adrenergic receptors was performed in the same conditions plays an indirect solid-phase ELISA. Methods and conditions of production ELISA were similar to those described in the literature [7].

Part of a set of reagents for the detection of autoantibodies consisted of the following components: 96-well plate for immunoassay, the firm "Costar"cat. No. 9018; synthetic antigen (see example 3) and peptide 197-222 2nd extracellular loop β1-adrenergic receptors; 0.1 M Na-carbonate buffer; sample diluent, conjugates, the blocking solution and the solution for washing tablets - 0,01M Na-phosphate buffer pH of 7.2, containing 8 g/l M NaCl, 0.1% tween-20, 3% non-fat dry milk and bacteriostatic Katon CG 2.0 ml/l (PSBM-T); the solution for washing tablets - 0.01 M Na-phosphate buffer pH of 7.2, containing 0.15 M NaCl and 0.1% tween-20 (the SAT-T); conjugate - reagent for analysis: mouse monoclonal antibodies against human IgG labeled with Biotin; conjugate streptavidin-peroxidase; solution of tetramethylbenzidine (TMB), 1 mm/l in dimethylformamide; substrate buffer solution - sodium citrate 5.5-water, 26 g/l; citric acid 6,92 g/l, sodium perborate 1.1 g/l and Katon CG 2.0 ml/l; stop-reagent phosphoric acid 5%.

Protocol analysis

Prior to testing the components of the kit kept at a temperature of +20°C for 60 minutes For immobilization of the antigen on the pads for ELISA drugs peptides were diluted to a concentration of 5 μg/ml in 0.1 M Na2CO3-NaHCO3the pH of 9.6. Made in the wells of tablets, 100 μl, and incubated for 20 hours at 20°C. To eliminate nonspecific interactions in wells contributed 100 ál FSBM-T and incubated for 3 hours at +20°C with shaking. To remove unbound antigen, the wells were washed 3 times FSBM, Samples, diluted in FSBM-T 20 and 100 times, made a pipette into the respective wells 100 ál, incubated at a temperature of +4°C for 12 hours. Washed tablet FSBM-T is not less than 3 times. Contributed to the wells 100 ál of working solution reagent for analysis in FSBM-T, incubated at a temperature of +20°C for 60 min with shaking. Washed tablet solution FSBM-T three times. Contributed to the wells 100 ál of working what about the solution of the conjugate in FSBM-T, incubated the plate at a temperature of +20°C for 30 min with shaking. Washed with a solution of the FSB-T four times, then immediately brought into the wells 100 ál of working solution of freshly prepared substrate mixture (7 volumes substrate buffer and 1 volume of solution TMB). Covered the plate with a lid and incubated at a temperature of +20°C without stirring for 15 min, then brought to 100 µl of stop solution in the same sequence and the same method, which was made of the substrate mixture. Within 5 min after adding the stop reagent was detected optical density in the plate at a wavelength of 450 nm.

The binding definition of the sorbent with immobilized synthetic antigen and characterization of specificity

Immobilization of the inventive synthetic antigen and associate with them a combination of peptide sequences 125-133 and 206-218 molecule β1-adrenergic receptors on the prototype brachionichthyidae Sepharose 4 FF conducted in accordance with the standard method of immobilization of protein ligands on agarose matrix.

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

The concentration immobiliza the aqueous ligand was assessed by the difference in the number of peptide used for the reaction of immobilization and remaining in solution after incubation with the matrix. The concentration of peptide in solution was measured by optical absorption at 280 nm. After immobilization, the sorbent was washed with 10 volumes of distilled water. Then to the resulting sorbent (2 ml suspension containing 1 ml of settled gel) was added to 0.5 ml of 1M ethanolamine solution was acidified with HCl to pH 8. 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 sorbents with immobilized declare synthetic antigen and the prototype was performed affinity chromatography of blood plasma of patients DCMP patients and healthy donor. The ratio of the volume of the sorbent to the plasma volume was 2:1, the incubation time is 1 hour. After incubation, the sorbents were washed in 50-fold volume of buffer a mixture of 0.01 M KH2PO4-NaOH pH 7.0, bound peroxidase protein was suirable 20-fold volume of buffer a mixture of 0.2m glycine-HCl pH 2,5.

Results

The sensitivity and specificity of detection of autoantibodies to β1-adrenergic receptors

Comparison of the specificity of the claimed synthetic antigen described in the literature peptide site 197-222 2nd extracellular loop β1-adrenergic receptors was performed in the experience with uni is zirovanii conditions. Conditions setting ELISA corresponded to the conditions of the ELISA described for the prototype [7]. Determination of autoantibodies was performed in the group of patients with DCMP (group 1, n=9), in a mixed group of patients with reduced ejection fraction of the left ventricle (as with DC, and tachyarrhythmias) (group 2, n=16) and in the group of healthy donors (group 3, n=24).

Results the sensitivity of the ELISA for the detection of autoantibodies to β1-adrenergic receptors in samples of plasma/serum of patients studied groups and healthy donors are shown in the table.

Table.
A comparison of the average signal in the ELISA between different groups
SamplePatientsHealthy donors
group 1, n=6group 2, n=16group 3, n=24
The inventive synthetic antigen0,32±0,12*0,36±0,08**0,22±0,04
peptide 197-222 sequence β1-adrenergic receptors1,1±0,91,2±0,7 0,8±0,2

Data are presented as mean ± deviation confidence level by student's criterion, the level of statistical significance **p<0,005, *p<0,1.

An acceptable level of statistical significance (p<0,05) between the group of healthy donors and patients in their experiments, the authors observed only for ELISA using synthetic antigen, indicating that greater specificity of the interaction of the inventive synthetic antigen with autoantibodies to β1-adrenergic receptors than peptide 197-222.

According to literature data, the frequency of detection of autoantibodies to β1-adrenoreceptor in patients with DCMP at the peptide 197-222 sequence β1-adrenergic receptors are 13 positive responses from 42 that is 30%

[7]. In our own experiments using the inventive synthetic antigen frequency of detection of autoantibodies to β1-adrenergic receptors in patients with DCMP was 44% (4 positive response from 9 patients DCMP), whereas the peptide 197-122 sequence β1-adrenergic receptors - 33% (3 positive response from 9 patients DCMP) (data not shown). A positive response was considered signal, two times greater than the average background level. The data obtained indicate more sensitive the ti method of detecting autoantibodies to β 1-adrenergic receptors the use of the claimed synthetic antigen.

Binding of autoantibodies to β1-adrenergic receptors on the sorbents

It should be noted that in the literature there are no data to compare the sorption characteristics of the inventive synthetic antigen with the prototype. Therefore, the sorption properties of the sorbent, containing a synthetic antigen, and the sorbent with the prototype was tested by the method of affinity chromatography from plasma of human blood in the same conditions in vitro experiments. This used the blood plasma DCMP patients (patient 1 and 2), patients with tachyarrhythmia (patient 3) and a healthy donor. The amount of bound peroxidase with the tested sorbents of immunoglobulin G when carrying out affinity chromatography individual plasmas of patients with DCMP and plasma of a healthy donor is shown in the drawing.

In the drawing a Number of related autoantibodies to β1-adrenergic receptors from the plasma of patients and plasma of healthy donors on sorbents with immobilized prototype and declare a synthetic antigen.

According to the results of own researches of the authors of the average number of adsorbed antibodies for sorbents with immobilized declare a synthetic antigen (the amount of IgG in the eluate with 1 ml sorbent) in the experiments, three samples of blood plasma of patients with DCMP sostav the lo 227±35 μg, while the prototype 70±65 µg (p<0,05).

Thus, the claimed synthetic antigen allows significantly higher sensitivity and affinity to bind antibodies from plasma/serum of patients with presence of autoantibodies to β1-adrenergic receptors compared with the prototype.

Literature

1. San Martin M.A. et al. Dilated cardiomyopathy and autoimmunity: an overview of current knowledge and perspectives. // Rev Esp Cardiol. - 2002. - V.55 (5) P.514-524.

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

3. Rönspeck W. et al. Peptide based adsorbers for therapeutic immunoadsorption. // Ther Apher Dial. - 2003. -. V.7 (1). - P.91-97.

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 GA and other Apheresis immunoglobulin - a new approach to the treatment of severe forms of dilated cardiomyopathy. // Cardiology. - 2002 - No. 6 - P.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 dosage cardiac hypertrophy in rabbits. // J Card Fail. - 1999. - V.5 (3). - P.246-254.

9. Zhang L et. al. Autoantibodies are 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 arrhythmis and sinus node dysfunction. // Circulation. 2001; 103 (13): 1765-71.

11. Iwata M. Et al, Autoimmunity Against the increasing interest among Second Loop of β1-Adrenergic Receptors Dosage of β-Adrenergic Receptor Desensitization and Myocardial Hypertrophy In Vivo. // Circulation Research. - 2001 - V.88. - P.578-586.

12. Rönspeck W. Peptides for combating the autoantibodies are that are responsible for dilatative cardiomyopathy (DCM). // EP 1214350.

13. Hopp N., Woods, K., Prediction of antigenic determinants from amino acid sequences. // Proc. Natl. Acad. Sci. USA. 1981. V.72. 72. P.3824-3828.

14. Mwizerwa, Astromega, Amy, Avicularia, Crouse, Mavoungou, Idisplay. The use of hydrogen peroxide for circuit disulfide bridges in peptides. // Bioorganic chemistry, 2004, vol 30, No. 2, s-125.

15. Barany G., Merrifild R.B. Solid phase synthesis. // The Peptide. Analysis, Synthesis, Biology. V2. Special Methods in Peptide Synthesis. Part. A. / Ed. .Gross, J.Meienhofer. - New York; Academic Press, 1980, P.3-254.

Synthetic antigen for binding of autoantibodies to β1-adrenergic receptors in the plasma of human blood, including the nonapeptide (125-133) and tridecapeptide (208-218) sequence β1-adrenergic receptors person, characterized in that the inventive antigen is an individual chemical compound in which both peptides linked by a disulfide bond.



 

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3 ex

FIELD: medicine.

SUBSTANCE: invention refers to virology, namely, to methods of immunosorbent production. Method of immunosorbent production virus-specific antibody binding is offered. Method includes inorganic sorbent-carrier incubation with virus-containing liquid. Ultradisperse oxygen-containing graphite is used for inorganic sorbent-carrier as foamed particles of the stratified graphite, containing oxygen in amount 12-14 mass % per 73-76 mass % of carbon. Specific surface of graphite is 1500-2000 m3/g, particle size is 25-50 mcm. Graphite is pre-boiled in distilled water. Produced suspension of sorbent-carrier at graphite content not less than 2 mass % is incubated with virus-containing liquid at temperature 5-35°C. Virus-containing liquid contains, e.g. influenza virus.

EFFECT: produced immunosorbent for virus-specific antibody binding has high sorptive power, enables to bind completely virus-specific antibodies of immune serum.

2 cl, 5 tbl

FIELD: medicine; veterinary science.

SUBSTANCE: produced plasma after it is separated from blood and refined from trypanosome deposition is processed with 20-25% polyethylene glycol solution taken in proportion equal to blood plasma volume. Then produced mixture is kept at room temperature for 12-15 min, recentrifuged at 6000 revolutions/min for 15-20 min, after that supernatant is removed, and produced deposition is used as trypanosome exoantigen for serological reaction. At that its activity should be 95-97%.

EFFECT: timely finding of sick animals and possibility to take emergency measures for invasion elimination.

2 tbl

FIELD: medicine; gastroenterology.

SUBSTANCE: cell lysate is received by double destruction of bacterial cells: first, treatment with 1% sodium desoxycholate solution at a rate of 0.5 ml solution to 0.2 ml suspension with concentration (5-7) × 1010 microbes/ml, with following stirring at magnetic agitator in thermostat at 40°С for 2 hours, second, by performing 5 per 45 sec cycles of ultrasound disintegration, after which, the obtained suspension is precipitated by centrifuging at 8000 rpm for 40 min, and the obtained cell lysate is used as a sensitin, which is immobilised at polymer carrier with the following lyophilisation. In sensitin, the protein concentration 1.5-2 mg/m with wide immunoglobulin spectrum is received. As sensitin carrier, the globular polymeric particles with diameter 1.5 mcm and containing 1.3 mmol/g aldehyde groups can be used, and the lysate-produced immobilisation of micro spheres is performed by use of 0.1 mol carbonate buffer with pH 9.2. Interlocking of free aldehyde groups is performed by adding 2.0 ml 0.5% gelatose on 0.9% sodium chloride to suspension with carrier and soluble antigen, and leave to stay at constant stirring at the agitator at 20°С fro 120 min.

EFFECT: method provides the quantitative determination of antibodies to HPylory and efficient application for controlling the eradicative therapy.

2 tbl, 2 ex, 4 cl

FIELD: medicine, veterinary.

SUBSTANCE: specific antibodies to virus of goose enteritis are determined by IEA where virus received following injection of epizootic strain "P-75" of enteritis virus, is used as an antigen, and macro porous glass with diameter not less than 700 is used for purification of virus-containing material, the goose Ig G specific immunoperoxidase conjugate is used as an anti-specific agent, and the IEA results are calculated by use of the formula: titer = antiIg[2.02(lg S/P)+3.51], where S is for optical density of tested serum; P is for optical density of the positive control.

EFFECT: method reduces the test time and economical costs.

2 tbl, 2 ex

FIELD: medical engineering.

SUBSTANCE: device has substrate having polymeric working layer on it, produced from copolymer based on methacrylic acid derivatives with biological macromolecules (probes) immobilized thereon. The substrate is manufactured from activated or not activated glass, metal or polymer material. The working layer has macroporous monolithic copolymer glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass with affine biological probes immobilized thereon. Probe-copolymer proportion is 2-10 mg/g of copolymer, for protein, 1-20 mg/g of copolymer for peptide and for oligonucleotide, nucleic acid - 0.5-3 mg/g of copolymer, pore radius of 0.4-1.5 mcm, it has thickness of 50-700 microns and is manufactured as continuous or discrete microcellular layer. The method for manufacturing biochip involves preparing substrate, producing working layer by monomer copolymerization on methacrylic acid derivatives base, immobilizing biological macromolecules - probes on forming copolymer, washing, drying the received biochip. Radical copolymerization of glycidyl methacrylate and ethylene glycol methacrylate taken in (50:70)-(50:30) proportions by mass is carried out for producing working layer with photo-or thermal initiation in poregenic solvent medium being applied. Proportion of the sum of monomer volumes to solvent volume being equal to 6:9, initiator concentration in reactionary medium being equal to 0.2-1.0% by weight, given reaction mixture is placed on substrate as continuous or discrete layer. Macroporous monolithic continuous or discrete microcellular layer is formed as a result of copolymerization on the substrate. Then, covalent immobilization of biological macromolecules is carried out in the layer pores or their direct synthesis on formed copolymer with its native or modified epoxy groups being used. Biological affine probe is produced. The probe is introduced into copolymer in quantity of 2-10 mg/g of copolymer for fiber, for peptide - 1-20 mg/g of copolymer and for oligonucleotide or nucleic acid - 0.5-3 mg/g of copolymer.

EFFECT: manufacturing reusable biochip with predetermined controllable and reproduced quality.

17 cl

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology. Description is given of a single-strand T-cell receptor (scTCR), containing an α segment, formed by a sequence of a variable region in a TCR chain, joined with the N end of the extracellular sequence with constant region in the TCR chain, a β segment, formed by a sequence of the variable region of the α TCR chain, joined with the N end of the extracellular sequence with constant region of the β TCR chain, and a linker sequence, joining the C end of the α segment with the N end of the β segment, or vice versa. Extracellular sequences of constant regions of α and β segments are joined by a disulphide bond. Extracellular sequences of constant regions can correspond to constant regions of α and β chains of native TCR, cut-off at their C ends such that, cysteic residues, which form the inter-chain native disulphide bond of the TCR, are excluded, or extracellular sequences of constant regions which are in the α and β segments, can correspond to constant regions of α and β chains of native TCR, in which cysteic residues, which form the native inter-chain disulphide bond, are replaced by another amino acid residue, or there is no uncoupled cysteic residue, which is in the β chain of the native TCR. This invention makes available a new class of alpha/beta analogues of scTCR, in which there is a disulphide bond between residues of a single amino acid, contributing to stability of the bond between the alpha and beta regions of the molecule.

EFFECT: such TCR are suitable for screening or for therapeutic purposes.

3 cl, 14 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: proposed here is an isolated cyclic peptide, with amino acid sequence Cys-Ile-Xaa-Ser-Cys (SEQ ID NO:7); where Xaa is an amino acid residue, chosen from a group comprising Asp, Asn, Glu and Gin, and containing a disulphide bond between two Cys residues, which can be used as a selective antagonist of R-cadherin of mammals.

EFFECT: invented selective peptide-antagonists of R-cadherin can be used for inhibiting targeting of hematopoietic stem cells (HSC) on a developing vascular tree, for inhibiting cytoadherence caused by R-cadherin and inhibiting retina angiogenesis.

8 cl, 12 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biotechnology. Description is given of a phage particle, exposing on its surface, T-cell receptor (TCR), which is a human scTCR. The exposed dTCR polypeptide pair or exposed scTCR polypeptide contains a series of α and β chains of extra-cellular constant lg of the native TCR domain. A disulphide bond joins amino acid residues of the given series of chains of the constant lg domain, where the given disulphide bond is between cysteic residues. Presented is a library of polypeptide pairs of mutated TCR or scTCR polypeptides, exposed on the described phage particle. Nucleic acid, which encodes the described phage particle, is also presented. A method of identifying TCRs is invented. The invention can be used for making various TCR libraries, for identification of high-affinity TCRs.

EFFECT: possibility of making various TCR libraries for identification of high-affinity TCRs.

17 cl, 25 ex, 63 dwg, 2 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: variants of the combined protein which contain the extracellular domain of a human receptor of a hormone of growth and the domain which includes alarm sequence for joining glycosylphosphatidylynozyte (GPI) anchors are offered.

EFFECT: effective medical product for acromegalia and gigantism treatment.

8 cl, 16 dwg

FIELD: genetic engineering.

SUBSTANCE: invention refers to polypeptides GPCR Drosophila melanogaster (DmGPCR) and polynucleotides detecting and coding such polypeptides. Construction of expressing vectors based on specified polynucleotides and production of containing host cells enables to apply DmGPCR as insecticides. Besides, invention refers to methods of DmGPCR-bindnig, method of DmGPCR expression and activity modulator identification, method of insect population control using DmGPCR-antibody, DmGPCR antisense polynucleotides, DmGPCR binding partner or modulator.

EFFECT: new application of polypeptides.

35 cl, 7 tbl, 10 ex

FIELD: technological processes.

SUBSTANCE: method suggests protein of adipocyte plasma membrane, method of its preparation and complex based on this protein. Protein has molecular mass of 115 kilodaltons and has the ability to start-up tyr-phosphorylation of insulin-receptor proteins substrate in adipocyte. Method of protein preparation provides for adipocytes preparation out of rat, mouse or human tissues and plasma membranes extraction out of them. Then plenty of domains are isolated with high content of cholesterol hcDIG, which are treated with solution trypsin/NaCl. Centrifugation is done and protein fraction SDS-polyacrylamide gel is segregated with electrophoresis. Prepared protein fraction in amount of 115 kilodaltons is eluated from this gel. Complex constitutes activated protein and is formed during its combination with one of compounds from group: YCN-PIG, YMN-PIG, YCN or lcGcel.

EFFECT: protein in its activated form allows regulating glucose utilization bypassing insulin signal chain.

7 cl, 20 dwg, 1 tbl

FIELD: technological processes.

SUBSTANCE: this invention is related to biotechnology, to be more precise, to preparation of proteins out of milk, and may be used for prevention or treatment disorders related to metabolism in bones and immune function. Osteoprotegrin is prepared out of human or cow milk and has glycolysis profile that produces polypeptide with molecular mass of approximately 80, 130 and 200 kilodaltons. Prepared protein is used in structure of food substance and pharmaceutical composition for prevention or treatment of disorders related with bone remodeling, and/or immune disorders. Also prepared protein is added to make fodder.

EFFECT: invention allows simple and efficient preparation of stable biologically active form of protein osteoprotegrin out of natural sources.

20 cl, 7 dwg, 1 ex

FIELD: medicine, biotechnology, pharmaceutical industry.

SUBSTANCE: method involves increasing part of the most active conformation of a glycosylated recombinant protein secreted by mammalian cell by its contact with a reagent for coupled oxidation-reduction. The proposed promotion method of the most active conformation of protein is used in a method for preparing a glycosylated recombinant protein in its the most active conformation. Configuration isomer of protein prepared by the indicated method for preparing a glycosylated recombinant protein in it's the most active conformation used in a method for preparing the protein composition for its administration to user and/or a patient or for consumption by user and/or patient. Using of the proposed invention provides enhancing activity of glycosylated protein prepared by a method of recombinant DNAs using a mammalian cell.

EFFECT: improved preparing method, valuable properties of protein.

27 cl, 9 dwg, 2 tbl, 3 ex

FIELD: biotechnology, immunology, biochemistry, medicine.

SUBSTANCE: invention proposes peptide concatemer inducing production of antibodies against apolipoprotein B-100 that inhibit lipase effect and inhibit binding LDL with LDL receptors. This concatemer consists of amino acid sequence of peptide repeating four times. Amino acid sequence is given in the invention description. Also, invention describes a concatemer-base vaccine used in treatment and prophylaxis of obesity and a method for preparing concatemer in E. coli cells using a vector. Invention discloses a polynucleotide encoding concatemer and expressing vector comprising the indicated polynucleotide. Using the invention provides inhibition of obesity.

EFFECT: valuable medicinal properties of concatemer and vaccine.

7 cl, 16 dwg, 1 tbl, 6 ex

FIELD: immunology, biotechnology.

SUBSTANCE: invention relates to variants of nucleic acid construct (NK-construct) encoding of MUC1 antigen based on seven full repeated VNTR-units. Variants include NK-constructs selected from group containing MUC1 based on seven full repeated VNTR-units, MUC1 based on seven full repeated VNTR-units without signal sequence, MUC1 based on seven full repeated VNTR-units without signal sequence, transmembrane and cytoplasm domains, full MUC1 based on seven full repeated VNTR-units without transmembrane and cytoplasm domains, as well as mutants of abovementioned variants, wherein at least one VNTR is mutated to reduce of glycosylation potential. Disclosed are NK-constructs additionally containing epitopes selected from group: FLSFHISNL, NLTISDVSV or NSSLEDPSTDYYQELQRDISE. Also described are variants of expressing plasmide carrying NK-construct represented as DNA, protein having anti-tumor activity, encoded with NK-construct and pharmaceutical composition with anti-tumor activity based on said protein, NK-construct or plasmide. Application of NK-construct and protein for producing of drug for treatment or prevention of MUC-1 expressing tumors; method for therapy by using NK-construct, protein, or plasmide also are disclosed.

EFFECT: NK-constructs with increased anti-tumor activity.

20 cl, 25 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, namely to immunodiagnostics, and can be used in life-time serodiagnostics dirofilariasis in animals and human. The process involves producing the purified somatic antigen Dirofilaria repens by sequential mechanical and ultrasonic homogenisation combined with protein extraction in aqueous saccharose solution 0.25 M, followed by settling the supernatant fluid in cooled acetone and dissolving the vacuum-dried deposition in potassium-phosphate buffer.

EFFECT: method involves producing the high-specific and high-sensitive somatic antigen.

3 ex, 3 tbl, 1 dwg

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