The subject of invention are peptides mimicking urease, methods of their manufactures, application as the agents for identification of diseases and the way of performance the test.

New informations indicated that autoimmune or atherosclerotic diseases are resulted by multifactorial interactions: genetical predispositions, environmental circumstances, but also chronic bacterial or viral infections. Mutlifactoriality of these diseases cause troubles in their diagnostics and prognostication. One of essential factor of the progress of of autoimmune diseases is molecular mimicry of pathogens; for instance in rheumathoidal arthritis significant similarity between human proteins and Proteus mirabilis hemolysines and ureases are observed. Also in atherosclerosis, which is sometimes regarded as autoimmune disease, similarity between human proteins and Hsp proteins from Helicobacter pylori and Chlamydia sp. is observed.

One of the common bacterial antigens are ureases. They are pathogenic factors in H. pylori and P mirabilis infections. But their role as an antigens for generation of antiurease antibodies - possible markers in autoimmune diseases - as rheumatoidal arthritis or atherosclerosis was not demonstrated yet.

In performed investigations fully chemically defined synthetic peptides - mimetics of conservative fragments of bacterial and plant ureases - were used. Their synthesis on cellulose carrier is one of the subjects on invention. These pepetides was useful for differentiation between populations of atherosclerotic patients and healthy persons. This observation is essential for application of urease mimicking peptides as the tools for detections of anti-urease antibodies - markers for autoimmune and atherosclerotic diseases.

According to invention the structure of peptides mimicking urease is presented with general formula:

where Rl indicates BK-61A: H ₂ N-Ser-Ile-Lys-Glu-Asp-Val- Gln-Phe-; R2 indicates BK-61B: H^-Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val- Gln-Phe-Ala-Asp-Ser-Arg-Ile-; R3 indicates BK-65A: H ₂ N-Gly-Arg-Arg-Asn-Leu-Lys-Trp- Met-Leu-Arg-; R4 indicates BK-65B: H ₂ N-Met-Leu-Met-Val-Cys-His-His-Leu-Asp-Pro-Ser- Ile-Pro-Glu-Asp- VaI-AIa-; R5 indicates BK-65C: H ₂ N-Met-Val-Met-Ile-Thr-His-His-Leu- Asn-Ala-Ser-Ile-Pro-Glu-Asp-Ile-Ala-; R6 indicates BK-65D: H ₂ N-Met-Leu-Met-Val-Cys- His-His-Leύ-Asn-Arg-Glu-Ile-Pro-Glu-Asp-Ile-Ala-.

Manufacturing of peptides mimicking urease according to invention is carried out in several steps consisting immobilization of 2,4-dichloro-6-methoxy-l,3,5-triazine on cellulose plate followed by treatment with solution of N-methylmorpholine in THF and removing of excess of NMM by washing with THF, treatment with solution of protected amino acid Ri-Re and conditioning of the plate for 4 h in the atmosphere saturated with DMF vapors, washing with dichloromethane, drying and treating with boiling toluene for 4h. Then, the plates were treated with acetic anhydride solution, washed with DMF and immersed in the piperidine solution in DMF for 15 min followed by thoroughly washing with DMF. The further modifications involving attachment of the next amino acids. It has been done by activation of all succeeding N-protected amino acids with triazine coupling reagent, followed by deprotection of Fmoc group by treatment with piperidine solution until all peptide sequence from Ri-R ₆ group have been assembled, hi the final step of the synthesis the plate was treated with trifluoroacetic acid in dichloromethane solution for 4 h in the presence of H2O and TIS, washed with DCM and ethanol and dried.

In another synthetic approach, the first amino acid was attached to cellulose support as above followed by attachment of protected peptides Ri-R ₆ prepared elsewhere and activated with triazine coupling reagent, treatment with trifluoroacetic acid in dichloromethane solution for 4 h in the presence of H2O and TIS, washing with DCM and ethanol and drying.

The essence of the invention is that membranes with peptides mimicking urease, depicted on the general formula presented below:

where Rl denote BK-61A: H ₂ N-Ser-Ile-Lys-Glu-Asp-Val- Gln-Phe-; R2 denote BK-61B: ftN-Cys-His-His-Leu-Asp-Lys-Ser-Ile-Lys-Glu-Asp-Val- Gln-Phe-Ala-Asp-Ser-Arg-Ile-; R3 denote BK-65A: H ₂ N-Gly-Arg-Arg-Asn-Leu-Lys-Trp- Met-Leu-Arg-; R4 denote BK-65B: H ₂ N-Met-Leu-Met-Val-Cys-His-His-Leu-Asp-Pro-Ser- Ile-Pro-Glu-Asp- VaI-AIa-; R5 denote BK-65C: H ₂ N-Met-Val-Met-Ile-Thr-His-His-Leu-Asn- Ala-Ser-Ile-Pro-Glu-Asp-Ile-Ala-; R6 denote BK-65D: H ₂ N-Met-Leu-Met-Val-Cys-His-His- Leu-Asn-Arg-Glu-Ile-Pro-Glu-Asp-Ile-Ala-, are applied in diagnostic tests for detection of markers characteristic for autoimmune diseases and atherosclerotic diseases.

Diagnostic tests were accomplished, according to invention, in ELISA plates by placing strips of the membranes with immobilized peptides into the wells, masking nonspecific bonding spaces with masking buffer, and then after removing excess of buffer incubating with human serum, washing the wells and treating with anti-human antibodies, washing and dying.

The invention apply to the membranes with immobilized peptides Ri-Re, which specifically reacts with antibodies of atherosclerotic patients.

The method of preparation and realization of the tests allow detections of antibodies, which could be markers of occurrence and progress of atherosclerotic and autoimmune diseases.

Examples presented below demonstrate the subject of invention.

Example 1. Synthesis of BK-61 A. Method 1.

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM (1.1 mL, 10 mmol) in THF (10 mL) and shaken for 30 h. Then the plate was washed with THF (3x10 mL) and treated with a solution of Fmoc-Phe-OH (1.937 g, 5 mmol) and NMM (0.55 mL, 5 mmol) in DCM (20 mL) for 4 h, washed with DCM (5x10 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. The Fmoc group was removed from the attached phenylalanine by treatment with a 25% solution of piperidine in DMF (20 mL) for 20 min followed by washing with DMF (3x10 mL) and DCM (2x10 mL).

The plate with the first amino acids immobilized was treated successively with next amino acids. In the first step it was treated with 1.222 g Fmoc-Gln(Trt)-OH, 0.27 g HOBt, 0.656 g DMTXNMMXBF ₄ ^" and 0.44 mL NMM in 6 mL DMF. Gentle shaken of the mixture was continued until coloration after treatment with Bromophenol Blue (BPB) was not more observed. Then, the plate was washed with DMF (3 x 6 mL), capped with 10 mL of 0.5 % solution of acetic anhydride in DMF, washed with DMF (3 x 10 mL), again treated with 25% solution of piperidine in DMF for 15 min. and washed with DMF (3 x 10 mL).

The following amino acids were attached to the cellulose consecutively:

1.222 g of Fmoc-Gln(Trt)-OH, 0.27 g HOBt, 0.656 g DMTXNMMXBF ₄ ^" and 4 rnmol NMM in 6 mL DMF;

0.678 g of Fmoc- VaI-OH, 0.27 g HOBt, 0.656 g DMTXNMMXBF ₄ ^" and 4 mmol NMM in 6 mL DMF;

0.822 g of Fmoc-Asp(OtBu)-OH, 0.27 g HOBt, 0.656 g DMTxNMMxBF ₄ ^" and 4 mmol NMM in 6 mL DMF;

0.850 g of Fmoc-Glu(OtBu)-OH, 0.27 g HOBt, 0.656 g DMTxNMMxBF ₄ ^" and 4 mmol NMM in 6 mL DMF;

0.936 g of Fmoc-Lys(Boc), 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM w 3 mL DMF;

0.706 g of Fmoc-Ile-OH, 0.27 g HOBt, 0.656 g DMTxNMMxBF ₄ ^" and 4 mmol NMM in 6 mL DMF;

0.766 g of Fmoc-Ser(tBu)-OH, 0.27 g HOBt, 0.656 g DMTxNMMxBF ₄ ^" and 4 mmol NMM in 6 mL DMF.

After completion of amino acid coupling the plate was treated 4 h with 20 mL 50% TFA solution in DCM with 3% v/v H ₂ O and 2% v/v TIS added, then washed with DCM (3 x 20 mL), EtOH (3 x 20 mL) and dried in vacuum desiccator. The plate was used for diagnostic tests without further modifications.

Example 2. Synthesis of BK-61A. Method 2

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM (1.1 mL, 10 mmol ) in THF (10 mL) and shaken for 30 h. Then the plate was washed with THF (3x10 mL) and treated with a solution of Fmoc-Phe-OH (1.937 g, 5 mmol ) and NMM (0.55 mL, 5 mmol ) in DCM (20 mL) for 4 h, washed with DCM (5x10 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. The Fmoc group was removed from the attached phenylalanine by treatment with a 25% solution of piperidine in DMF (20 mL) for 20 min followed by washing with DMF (3><10 mL) and DCM (2x10 mL). The plate was reacted with Fmoc-Ser(tBu)-Ile-Lys(Boc)-Glu(OtBu)-Asp(OtBu)-Val-Gln(Trt)- COOH (m/z = 1549 MALDI) (spitted from chlorotrityl resin) activated with 0.328 g DMTXNMMXBF ₄ until no reaction with BPB solution was observed. Then, the plate was washed with DMF (3 x 3 mL). Protecting groups were removed by immersing the plate for 4 h in 10 mL of the mixture of 50 % TFA solution in DCM and 3% v/v H ₂ O and 2% v/v TIS. The plate was washed successively with DCM (3 x 10 mL) and EtOH (3 x 10 mL), dried in vacuum dessiccator and directly used in diagnostic tests.

BK-61 A prepared by method 1 and method 2 were equally useful and gave analogous results in all tests.

Example 3. Synthesis of BK-61B-method 1

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM in THF (5 mL) and shaken for 3O h. Then the plate was washed with THF (3x5 mL) and treated with a solution of 0.883 g Fmoc-Ile-OH and NMM (0.55 mL, 2.5 mmol ) in DCM (5 mL) for 4 h, washed with DCM (5x5 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. The Fmoc group was removed from the attached isoleucine by treatment with a 25% solution of piperidine in DMF (10 mL) for 20 min followed by washing with DMF (3x5 mL) and DCM (2x5 mL).

The plate with the first amino acids immobilized was treated successively with next amino acids. In the first step it was treated with 0.649 g Fmoc-Arg(Pbf)-OH 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 0.22 mL NMM in 3 mL DMF. Gentle shaken of the mixture was continuead until coloration after treatment with Bromophenol Blue (BPB) was not more observed. Then, the plate was washed with DMF (3 x 6 mL), capped with 5 mL of 0.5 % solution of acetic anhydride in DMF, washed with DMF (3 x 10 mL), again treated with 25% solution of piperidine in DMF for 15 min. and washed with DMF (3 x 10 mL).

The following amino acids were attached to the cellulose consecutively:

0.383 g Fmoc-Ser(tBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.411 g Fmoc-Asp(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF; 0.311 g Fmoc-Ala-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.487 g Fmoc-Phe-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.611 g Fmoc-Gln(Trt)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.339 g Fmoc-Val-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF/ and 2 mmol NMM in 3 mL DMF;

0.411 g Fmoc-Asρ(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.425 g Fmoc-Glu(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.468 g Fmoc-Lys(Boc)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Ile-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.383 g Fmoc-Ser(tBu)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.468 g FmOc-LyS(BoC)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmoL NMM in 3 mL DMF;

0.411 g Fmoc-Asp(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF/ and 2 mmol NMM in 3 mL DMF;

0.620 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.620 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.586 g Fmoc-Cys(Trt)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF.

In all coupling steps, with every succeeding N-protected amino acids, the progress of synthesis was monitored by detection of free amino group using colored reaction with BPB, until the negative color test was established. After completion of coupling cellulose plate was washed with DMF (3 x 3 mL), treated with 5 mL 0.5% solution of acetic anhydride in DMF, again washed with DMF (3 x 5 mL), treated with 25% solution of piperidine in DMF for 15 min. and washed with DMF (3 x 5 mL). Side chains protecting groups was removed by 4 h treatment of the plate with the mixture of 50 % TFA solution in DCM with 3% v/v H ₂ O and 2% v/v TIS followed by washing with DCM (3 x 10 mL) and EtOH (3 x 10 mL) and drying in vacuum dessiccator. The plate prepared according to this procedure was used directly in diagnostic tests.

Example 4. Synthesis of BK-61B. Method 2

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM in THF (5 mL) and shaken for 30 h. Then the plate was washed with THF (3x5 mL) and treated with a solution of 0.883 g Fmoc-Ile-OH and NMM (0.55 mL, 2.5 mmol ) in DCM (10 mL) for 4 h, washed with DCM (5x5 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. Then, the cellulose plate was treated with 5 mL 0.5% solution of acetic anhydride in DMF and again washed with DMF (3 x 5 mL). The Fmoc group was removed from the attached isoleucine by treatment with a 25% solution of piperidine in DMF (10 mL) for 20 min followed by washing with DMF (3x5 mL) and DCM (2x5 mL).

FmocCys(Trt)-His(Trt)-His(Trt)-Leu-Asp(OtBu)-Lys(Boc)-Ser (tBu)-Ile-Lys(Boc)- Glu(OtBu)-Asp(OtBu)-Val-Gln(Trt)-Phe-Ala-Asp(OtBu)-Ser(tBu)- Arg(Pbf)-COOH (m/z = 4104 MALDI), prepared under standard conditions of solid phase synthesis on chlorotrityl resin, was activated with DMTXNMMXBF ₄ ^" and coupled to cellulose plate with isoleucine immobilized as described above. The progress of coupling was monitored by detection of free amino group using colored reaction with BPB, until the negative color test was established. After completion of coupling cellulose plate was washed with DMF (3 x 3 mL), treated with 5 mL 0.5% solution of acetic anhydride in DMF, again washed with DMF (3 x 5 mL), treated with 25% solution of piperydine in DMF for 15 min. and washed with DMF (3 x 5 mL). Side chains protecting group were removed by 4 h treatment of the plate with the mixture of 50 % TFA solution in DCM with 3% v/v H ₂ O and 2% v/v TIS followed by washing with DCM (3 x 10 mL) and EtOH (3 x 10 mL) and drying in vacuum dessiccator. The plate prepared according to this procedure was used directly in diagnostic tests.

Example 5. Synthesis of BK-65A. Method 1

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM in THF (5 mL) and shaken for 30 h. Then the plate was washed with THF (3x5 mL) and treated with a solution of 1.622 g Fmoc-Arg(Pbf)-OH and NMM (0.55 mL, 2.5 mmol ) in DCM (5 mL) for 4 h, washed with DCM (5x5 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. Then, the cellulose plate was treated with 5 mL 0.5% solution of acetic anhydride in DMF and again washed with DMF (3 x 5 mL). The Fmoc group was removed from the attached Fmoc-Arg(Pbf)-OH by treatment with a 25% solution of piperidine in DMF (10 mL) for 20 min followed by washing with DMF (3x5 mL) and DCM (2x5 mL).

The plate with the first amino acids immobilized was treated successively with next amino acids. In the first step it was treated with 0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 0.22 mL NMM in 3 mL DMF. Gentle shaken of the mixture was continuead until coloration after treatment with Bromophenol Blue (BPB) was not more observed. Then, the plate was washed with DMF (3 x 6 mL), capped with 5 mL of 0.5 % solution of acetic anhydride in DMF, washed with DMF (3 x 10 mL), again treated with 25% solution of piperidine in DMF for 15 min. and washed with DMF (3 x 10 mL).

The following amino acids were attached to the cellulose consecutively:

0.371 g Fmoc-Met-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.527 g Fmoc-Trp(Boc)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ and 2 mmol NMM in 3 mL DMF;

0.468 g Fmoc-Lys(Boc)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.597 g Fmoc-Asn(Trt)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF/ and 2 mmol NMM in 3 mL DMF;

0.649 g Fmoc-Arg(Pbf)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.649 g Fmoc-Arg(Pbf)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.297 g Fmoc-Gly-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF.

In all coupling steps, with every succeeding N-protected amino acids, the progress of synthesis was monitored by detection of free amino group using colored reaction with BPB, until the negative color test was established. After completion of coupling cellulose plate was washed with DMF (3 x 3 mL), treated with 5 mL 0.5% solution of acetic anhydride in DMF, again washed with DMF (3 x 5 mL), treated with 25% solution of piperydine in DMF for 15 min. and washed with DMF (3 x 5 mL). Side chains protecting groups was removed by 4 h treatment of the plate with the mixture of 50 % TFA solution in DCM with 3% v/v H ₂ O and 2% v/v TIS followed by washing with DCM (3 x 10 mL) and EtOH (3 x 10 mL) and drying in vacuum dessiccator. The plate prepared according to this procedure was used directly in diagnostic tests.

Example 6. Synthesis of BK-65B. Method 1

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM in THF (5 mL) and shaken for 30 h. Then the plate was washed with THF (3x5 mL) and treated with a solution of 0.777 g Fmoc-Ala-OH and NMM (0.55 mL, 2.5 mmol ) in DCM (5 mL) for 4 h, washed with DCM (5x5 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. Then, the cellulose plate was treated with 5 mL 0.5% solution of acetic anhydride in DMF and again washed with DMF (3 x 5 mL). The Fmoc group was removed from the attached Fmoc-Ala-OH by treatment with a 25% solution of piperidine in DMF (10 mL) for 20 min followed by washing with DMF (3x5 mL) and DCM (2x5 mL).

The plate with the first amino acids immobilized was treated successively with next amino acids. In the first step it was treated with 0.339 g Fmoc- VaI-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^' and 0.22 mL NMM in 3 mL DMF. Gentle shaken of the mixture was continuead until coloration after treatment with Bromophenol Blue (BPB) was not more observed. Then, the plate was washed with DMF (3 x 6 mL), capped with 5 mL of 0.5 % solution of acetic anhydride in DMF, washed with DMF (3 x 10 mL), treated with 25% solution of piperidine in DMF for 15 min. and washed again with DMF (3 x 10 mL).

The following amino acids were attached to the cellulose consecutively:

0.411 g Fmoc- ASp(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.425 g Fmoc-Glu(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.337 g Fmoc-Pro-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Ile-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.383 g Fmoc-Ser(tBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF; 0.337 g Fmoc-Pro-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^' and 2 mmol NMM in 3 mL DMF;

0.411 g Fmoc- Asp(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF/ and 2 mmol NMM in 3 mL DMF;

0.619 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.619 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.586 g Fmoc-Cys(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.339 g Fmoc-Val-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.371 g Fmoc-Met-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.371 g Fmoc-Met-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

In all coupling steps, with every succeeding N-protected amino acids, the progress of synthesis was monitored by detection of free amino group using colored reaction with BPB, until the negative color test was established. After completion of coupling cellulose plate was washed with DMF (3 x 3 mL), treated with 5 mL 0.5% solution of acetic anhydride in DMF, again washed with DMF (3 x 5 mL), treated with 25% solution of piperidine in DMF for 15 min. and washed with DMF (3 x 5 mL). Side chains protecting groups was removed by 4 h treatment of the plate with the mixture of 50 % TFA solution in DCM with 3% v/v H ₂ O and 2% v/v TIS followed by washing with DCM (3 x 10 mL) and EtOH (3 x 10 mL) and drying in vacuum dessiccator. The plate prepared according to this procedure was used directly in diagnostic tests.

Example 7. Synthesis of BK-65C. Method 1

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM in THF (5 mL) and shaken for 30 h. Then the plate was washed with THF (3x5 mL) and treated with a solution of 0.777 g Fmoc- AIa-OH and NMM (0.55 mL, 2.5 mmol ) in DCM (5 mL) for 4 h, washed with DCM (5x5 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. Then, the cellulose plate was treated with 5 mL 0.5% solution of acetic anhydride in DMF and again washed with DMF (3 x 5 mL). The Fmoc group was removed from the attached Fmoc- AIa-OH by treatment with a 25% solution of piperidine in DMF (10 mL) for 20 min followed by washing with DMF (3x5 mL) and DCM (2x5 mL).

The plate with the first amino acids immobilized was treated successively with next amino acids. In the first step it was treated with 0.339 g Fmoc- VaI-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 0.22 mL NMM in 3 mL DMF. Gentle shaken of the mixture was continued until coloration after treatment with Bromophenol Blue (BPB) was not more observed. Then, the plate was washed with DMF (3 x 6 mL), capped with 5 mL of 0.5 % solution of acetic anhydride in DMF, washed with DMF (3 x 10 mL), treated with 25% solution of piperidine in DMF for 15 min. and washed again with DMF (3 x 10 mL).

The following amino acids were attached to the cellulose consecutively:

0.411 g Fmoc-Asp(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.425 g Fmoc-Glu(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF/ and 2 mmol NMM in 3 mL DMF;

0.337 g Fmoc-Pro-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^' and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Ile-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.383 g Fmoc-Ser(tBu)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF/ and 2 mmol NMM in 3 mL DMF;

0.311 g Fmoc-Ala-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.597 g Fmoc-Asn(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.619 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.619 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 2 mmol NMM in 3 mL DMF; 0.397 g Fmoc-Thr(tBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Ile-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF/ and 2 mmol NMM in 3 mL DMF;

0.371 g Fmoc-Met-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.339 g Fmoc- VaI-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.371 g Fmoc-Met-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

In all coupling steps, with every succeeding N-protected amino acids, the progress of synthesis was monitored by detection of free amino group using colored reaction with BPB, until the negative color test was established. After completion of coupling cellulose plate was washed with DMF (3 x 3 mL), treated with 5 mL 0.5% solution of acetic anhydride in DMF, again washed with DMF (3 x 5 mL), treated with 25% solution of piperidine in DMF for 15 min. and washed with DMF (3 x 5 mL). Side chains protecting groups was removed by 4 h treatment of the plate with the mixture of 50 % TFA solution in DCM with 3% v/v H ₂ O and 2% v/v TIS followed by washing with DCM (3 x 10 mL) and EtOH (3 x 10 mL) and drying in vacuum dessiccator. The plate prepared according to this procedure was used directly in diagnostic tests.

Example 8. Synthesis of BK-65D. Method 1

A cellulose plate with immobilized DCMT was treated with a 1 M solution of NMM in THF (5 mL) and shaken for 3O h. Then the plate was washed with THF (3><5 mL) and treated with a solution of 0.777 g Fmoc-Ala-OH and NMM (0.55 mL, 2.5 mmol ) in DCM (5 mL) for 4 h, washed with DCM (5x5 mL), and soaked to remove excess solvent. The plate was immersed in dry boiling toluene for 4 h and then soaked and dried thoroughly in a vacuum desiccator. Then, the cellulose plate was treated with 5 mL 0.5% solution of acetic anhydride in DMF and again washed with DMF (3 x 5 mL). The Fmoc group was removed from the attached Fmoc-Ala-OH by treatment with a 25% solution of piperidine in DMF (10 mL) for 20 min followed by washing with DMF (3x5 mL) and DCM (2x5 mL).

The plate with the first amino acids immobilized was treated successively with next amino acids. In the first step it was treated with 0.353 g Fmoc-Ile-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ ^" and 0.22 mL NMM in 3 mL DMF. Gentle shaken of the mixture was continued until coloration after treatment with Bromophenol Blue (BPB) was not more observed. Then, the plate was washed with DMF (3 x 6 mL), capped with 5 mL of 0.5 % solution of acetic anhydride in DMF, washed with DMF (3 x 10 mL), treated with 25% solution of piperidine in DMF for 15 min. and washed again with DMF (3 x 10 mL).

The following amino acids were attached to the cellulose consecutively:

0.411 g Fmoc-Asp(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.425 g Fmoc-Glu(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.337 g Fmoc-Pro-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Ile-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.425 g Fmoc-Glu(OtBu)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.645 g Fmoc-Arg(Pbf)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.597 g Fmoc-Asn(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.619 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.619 g Fmoc-His(Trt)-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.586 g Fmoc-Cys(Trt)-OH, 0.135 g HOBt, 0.328 g DMTxNMMxBF ₄ ^" and 2 mmol NMM in 3 mL DMF;

0.339 g Fmoc-Val-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.371 g Fmoc-Met-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.353 g Fmoc-Leu-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF;

0.371 g Fmoc-Met-OH, 0.135 g HOBt, 0.328 g DMTXNMMXBF ₄ - and 2 mmol NMM in 3 mL DMF; In all coupling steps, with every succeeding N-protected amino acids, the progress of synthesis was monitored by detection of free amino group using colored reaction with BPB, until the negative color test was established. After completion of coupling cellulose plate was washed with DMF (3 x 3 mL), treated with 5 mL 0.5% solution of acetic anhydride in DMF, again washed with DMF (3 x 5 mL), treated with 25% solution of piperydine in DMF for 15 min. and washed with DMF (3 x 5 mL). Side chains protecting groups was removed by 4 h treatment of the plate with the mixture of 50 % TFA solution in DCM with 3% v/v H ₂ O and 2% v/v TIS followed by washing with DCM (3 x 10 mL) and EtOH (3 x 10 mL) and drying in vacuum dessiccator. The plate prepared according to this procedure was used directly in diagnostic tests.

BIOLOGICAL INVESTIGATION

Example 9. Procedure of immunological test.

Microtitrated plate wells were filled up with 300 μl of blocking buffer (3% bovine serum albumine in buffer TBS pH 7.45) and whole plate was incubated in ambient temperature during 2 hours with shaking. Than to each well small pieces (ca. 5 x 5 mm) of cellulose plate with immobilized synthetic peptide mimicking urease were added and blocked nonspecifical binding sites overnigth with shaking. After remowing of blocking bufifer the pieces of cellulose were incubated with 200 μl of human serum, diluted 1:1000 with 1% BSA in buffer TBS pH 7.45, during 2 hours with shaking. Then cellulose were washed two times with 300 μl of buffer TBS pH 7.45 supplemented by 0.4% of Tween80 and once with TBS buffer only. Than 200 μl of anti-human antibodies coniugated with horseradish peroxidase, diluted 1:1000 with 1% BSA in buffer TBS pH 7.45 was added to each well, followed by 1 hour incubation, in ambient temperature, with shaking. After next washing (as described previously) colour reaction was developed with addition of 300 μl of substrate buffer (solution 3 mg/ml of 4-chloro-l-naphtol in methanol, diluted 1:5 in buffer TBS pH 7.45 and supplemented with 0,5 μl of 30% H ₂ O ₂ per each mililiter) to each well and incubation durig 20 minutes in dark. Reaction was stopped by removing of substate buffer followed by two times of washing with 300 μl of distilled water, and cellulose was leaved in dark to dry.

Level of the human antibody binding to each peptide mimicking urease was determined by scanning of cellulose pieces and processing of gray level using ImageJ programme.

The results:

At Fig. the reactions of human antibodies, from voluntary blood donors (white squares) and atherosclerotic patients (black triangles) sera with synthetic peptide mimicking urease, are presented. Tab.l contains the statistical analysis of the results presented in Fig. Synthetic peptides mimicking urease are as follows: BK-65 A- 10-aminoacid side fragment of H. pylori urease BK-65 B - flap region characteristic for i.a. Proteus sp. urease BK-65 C - flap region characteristic for i.a. Staphylococcus sp. urease BK-65 D — flap region characteristic for Canavalia ensiformis urease BK-61 A- 8-aminoacid flap region fragment of H. pylori urease BK-61B - 19-aminoacid flap region fragment of H. pylori urease

Table 1. Statistical analysis of the reactions of human antibodies, from voluntary blood donors and atherosclerotic patients sera with synthetic peptide mimicking urease.