Bioanalysis and peptides used in said bioanalysis

FIELD: chemistry.

SUBSTANCE: invention discloses peptides for detecting Mycobacterium tuberculosis, which consist of less than 18 amino acids and are capable of binding with the antibody against GRDIKVQFQSGGNNSPAV peptide (all sequences are given in a list of sequences). One of the peptides contains an NSPAX sequence, where X denotes methionine. More preferably, the peptides contain an NNSPAV sequence or have the type SGGNNSPAX, where X denotes methionine, leucine, alanine or valine. The invention describes a nucleotide sequence which codes the disclosed peptides, as well as an antibody or fragment thereof, capable of binding with said peptide. The invention discloses methods of detecting M.tuberculosis, involving ELISA substitutive analysis of a sample using a peptide capable of binding with the antibody against GRDIKVQFQSGGNNSPAV peptide or using the antidody.

EFFECT: use of the invention simplifies tests to determine anti-tuberculosis antibodies in a population owing to the artificially modified peptide sequences of T-cell epitope Ag85B M tuberculosis.

27 cl, 5 dwg

 

2420-152611RU/071

The BIOANALYSIS AND PEPTIDES USED IN IT

The technical field to which the invention relates

The invention relates to the peptides used in the analysis definitionMycobacterium tuberculosis, as well as to the analysis.

The level of technology

Tuberculosis is an infection caused by the bacteriumMycobacterium tuberculosis. Tuberculosis is a global problem in many countries, especially in the developing world and in many developing countries, the incidence of tuberculosis is increasing. Despite the fact that tuberculosis is a lung infection, it can also affect other parts of the body such as lymph nodes, skin and bones.

The diagnosis of tuberculosis, as well as provide treatment, is the primary tool against the spread of disease. The diagnosis can be made based on a combination of clinical signs, cultural examination, radiographs of the chest, histology tissue and fluid bronchial lavage, and using tuberculin skin test (also known as the standard of purified protein derivative or PPD skin test), such as the Mantoux test and test Ghiffa. The sample is based on immune response to proteinM. tuberculosisshowing the presence in the blood of the patient of antibodies againstM. tuberculosis.

For the rosedene samples must hold the patient's immunological provocation and then be evaluated to determine the test result. These stages make the sample time-consuming for use in population screening.

In the present invention, attempts were made to resolve some of these problems.

The invention

In a first General aspect, the invention relates to a peptide comprising less than 18 amino acids containing a sequence NSPAX, where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17), alanine (SEQ ID NO: 15) or valine (SEQ ID NO: 10), where the peptide is able to bind with the antibody directed against the peptide GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11). In the most preferred embodiments, the implementation of the peptide is associated with such an antibody.

Practically, such an antibody could be directed against the indicated peptide (SEQ ID NO: 11), supplemented With additional-amino acid at the C-terminal end, i.e. directed against GRDIKVQFQSGGNNSPAVC.

In the second aspect, therefore, the peptide contains up to 6 additional amino acids at the C and/or N-terminal end of the sequence NSPAX.

Preferably, therefore, the peptide according to the first or the second aspect contains the sequence NSPAX, and more preferably, the peptide contains the sequence NNSPAV (SEQ ID NO: 14).

In a more preferred aspect, the peptide contains the sequence SGGNNSPAX (SEQ ID NO: 26), where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17), alanine (SEQ ID NO: 15) or the'alene (SEQ ID NO: 10).

In any aspect of the invention it is preferable that the peptide differed from GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11).

Also in any aspect of the invention it is preferable that the peptide consisted of a sequence SGGNNSPAX (SEQ ID NO: 26), where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17), alanine (SEQ ID NO: 15) or valine (SEQ ID NO: 10).

In any peptide according to the invention it is preferable that X was a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17) or alanine (SEQ ID NO: 15). Particularly preferred peptides, in which X is a methionine (SEQ ID NO: 18) or alanine (SEQ ID NO: 15). Found that these replacements have the best stability Leu heat, which makes them suitable for use in hot climates.

The most preferred peptides according to any aspect of the invention additionally contain a label. The use of labeled peptide facilitates its use in analysis of the definition ofMycobacterium tuberculosis. Suitable labels include radioactive labels (e.g., by introducing a radioactive isotope or substitution of one or more atoms of the peptide them radioactive equivalent), that allows you to easily determine the presence or concentration of the peptide or complex containing the peptide, for example, using a scintillation counter or the like. You could also use other labels, so the e as mediated by the enzyme chromogenic label. Particularly preferred labels are fluorescent labels, including binding peptide with a fluorescent protein. Particularly suitable, however, is a fluorescent label, such as Alexa Fluor (e.g., Alexa Fluor 633).

Preferably also, to any of the above peptides were selected form, i.e. essentially free of protein primary source (e.g., bacterial, fungal or mammalian proteins).

The above-described peptides used in the analysis definitionMycobacterium tuberculosis.

The invention relates to a nucleotide sequence that encodes any of the above peptides. This sequence is used in the biosynthesis of peptides of the present invention.

The invention relates to an antibody directed against SEQ ID NO: 11. Antibodies according to the invention can be polyclonal or monoclonal, or can be recombinant antibodies such as chimeric antibodies in which the constant region of the light and heavy chains of mouse replace sequences of human, or CDR-grafted antibody in which there are only complementary determining region, for example, mice. Antibodies according to the invention can also be antibodies person obtained, for example, by immunizing a transgenic animal capable of producing antibodies che is ovecka (see, for example, PCT application no WO 93/12227).

The invention relates to substitution ELISA (enzyme immunosorbent assay) to determine whetherMycobacterium tuberculosiscontaining the peptide according to any preceding paragraph. A suitable procedure to perform this analysis will be described below. Although it is preferred that such substitution analysis was additionally included antibody directed against SEQ ID NO: 11, or a fragment of such antibody having affinity in relation to SEQ ID NO: 11.

The invention relates to the analysis of ELISA to determine the presence ofMycobacterium tuberculosiscontaining antibody directed against SEQ ID NO: 11, or a fragment of such antibody having affinity in relation to SEQ ID NO: 11.

Additionally, the invention relates to substitution analysis to determine whetherMycobacterium tuberculosiscontaining the peptide according to the invention and the peptide corresponding to SEQ ID NO: 11.

Development test and description of the preferred embodiments

The test is based on the identified T-cell epitope Ag85BM. tuberculosis[Mustafa, A.S. et al, "Identification and HL Restriction of Nuturally Derived Thl-Cell Epitopes Secreted from the 1-Cell Epitopes from the SecretedMycobacterium tuberculosisAntigen 85B Recognised by Antigen-Specific Human CD4+T-Cell Lines", Infection and Immunity, 68(7), 3933-3940, 2000]presented an 18-dimensional peptide P3 (amino acids 19 to 36, denoted R) sequence GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11).

In one aspect the invention relates to the provision of competitive substitution ELISA (enzyme immunosorbent assay) - efficient ligand-replacement analysis. Antibodies againstM. tuberculosisin particular, directed to the epitope Ag85B, contact with the test plate. Peptides are able to bind with the antibodies, but are associated with antibodies, preferably labeled, for example, a fluorescent marker, with a lower affinity compared with antigenM. tuberculosis. If samples containing antigensM. tuberculosis(for example, the body itself), is brought into contact with a complex of the antibody-peptide, an antigen replaces the peptides that can be identified by the change in fluorescence. In another aspect of the invention suitable synthetic peptides receive, as described below:

To identify the target antigen with the aim of creating a library of analogues were synthesized 10 9-dimensional overlapping peptides, Charterhouse 18-dimensional P3 (i.e. 1-a, 2-a, 3-a etc. sequence SEQ ID NO: 11).

Below is the sequence 10 9-dimensional peptides using standard 1 letter amino acid code:

SEQ ID NO: 1: GRDIKVQFQ (1-a), denoted R-1

SEQ ID NO: 2: RDIKVQFQS (2-10aa), denoted P782-2

SEQ ID NO: 3: DIKVQFQSG (3-11aa), denoted P782-3

SEQ ID NO: 4: IKVQFQSGG (4-12aa), denoted P782-4

SEQ ID NO: 5: KVQFQSGGN (5-13aa), denoted P782-5

SEQ ID NO: 6: VQFQSGGNN (6-14aa), denoted P782-6

SEQ ID NO: 7: QFQSGGNNS (7-15aa), denoted P782-7

SEQ ID NO: 8: FQSGGNNSP (8-16aa), denoted P782-8

SEQ ID NO: 9: QSGNNSPA (9-17aa), denoted P782-9

SEQ ID NO: 10: SGGNNSPAV (10-18aa), denoted P782-10.

These peptides, along with 18-gauge P3 (SEQ ID NO: 11) were skanirovaniya against antisera (rabbit) against 18-dimensional P3 (SEQ ID NO: 11). When screening used the technique of competitive ELISA (enzyme immunosorbent assay), which was used to estimate the relative affinity of binding of 9-dimensional peptides with 18-dimensional peptide.

Briefly, the analysis included linking (cover) 18-dimensional P3 (R, SEQ ID NO: 11) with pads for ELISA, followed by incubation with different amounts of a competitive ligand (9-measures, SEQ ID NO: 1-10) in the presence of antisera (dilution 1:250). The level of binding of the antisera with 18-gauge P3 (R, SEQ ID NO: 11) was determined by processing anti-rabbit antibody goat, conjugated to peroxidase, followed by the addition of a chromogenic substrate ABTS (2,2-Azino-di-[3-ethylbenzthiazolinesulfonic]) and hydrogen peroxide, where after the appearance of green color was measured using counter dice as optical density (absorption). The development of staining is proportional to the level of binding of the original antisera to die.

To determine concentrations covering peptide and competitive peptides used for ELISA experiments, carried out the ELISA test, containing competitive ELISA 9-dimensional R-1 (GRDIKVQFQ) - SEQ ID NO: 1 is Rotel 18-dimensional R (GRDIKVQFQSGGNNSPAV) - SEQ ID NO: 11 (Mustafa, A.S. et al, above). This 9-Mer was chosen so that it is also overlap 18-dimensional P2 (YLQVPSPSMGRDIKVQFQ) - SEQ ID NO: 12. Based on these results, the authors showed that the concentration of peptides used were approached to conduct research using ELISA. Die for carrying out ELISA was covered with a 2 μm solution of 18-gauge P3 (R) - SEQ ID NO: 11 and used 500 μm solutions of competitive peptides, which are then subjected to four-fold serial dilution. The ELISA test results are presented in figure 1.

Then to determine the relative affinity of binding between ten 9-measures (with R-1 R-10) - SEQ ID NO: 1 through 10 - to 18-gauge P3 (R) - SEQ ID NO: 11 was carried out by mapping the epitope by ELISA. To obtain a quantitative assessment of the relative affinity of binding of the competing epitopes important selected range of concentration of peptide (linear part of the competition schedule). The results showed that the concentration range of 9-mers with R-1 R-9 (i.e. SEQ ID NO: 1 through 9) were observed associating with anticorodal in any significant amount; however, the 9-Mer R-10 (SGGNNSPAV) - SEQ ID NO: 10 is contacted anticorodal as much as 18-dimensional peptide P3 (SEQ ID NO: 11). This result is very important and he led the authors to think about the possible importance of the C-terminal residue of valine, because preds the corresponding 9-measure R-9 (QSGGNNSPA), SEQ ID NO: 9 there was no significant binding even though it overlaps the peptide R-10, SEQ ID NO: 10, 8 residues. The results of mapping of the epitope using ELISA is represented in figure 2(a) and 2(b). Then to test the importance of the C-terminal region epitope for binding to anticorodal identified the use of the ELISA of epitope-target. Were synthesized two 6-measure R-11 (GNNSPA) - SEQ ID NO: 13 P782-12 (NNSPAV) - SEQ ID NO: 14, which are truncated forms of the corresponding 9-mers, and they were subjected to competitive ELISA in accordance with the above, and also re-tested the 9-Mer R-10 (SEQ ID NO: 10), identified by epitope mapping by ELISA as significant epitope. The results of ELISA of epitope-target is shown in figure 3. The data clearly show reproducible results again indicate that:

9-Mer R-10 (SEQ ID NO: 10) is associated with anticorodal as strongly as P3 18-dimensional peptide R (SEQ ID NO: 11)

6-dimensional R-11 (SEQ ID NO: 13) is not associated with anticorodal and:

6-dimensional R-12 (NNSPAV) (SEQ ID NO: 14) is associated with anticorodal 2 times (1,99) is weaker than either the 9-dimensional R-10 (SEQ ID NO: 10)or 18-gauge R (SEQ ID NO: 11).

This result indicates the importance of the C-terminal region, which contains a valine residue. On the basis of this experiment it would be unwise to modify the N-limit the second region, to instead target the C-terminal valine residue to create some differences in the affinity of binding. Therefore, the authors decided to focus on the library, based on the 9-dimensional R-10 (SGGNNSPAV), SEQ ID NO: 10, and modify the C-terminal region, and synthesized peptides with sequence SGGNNSPA-X, where X represents a variable amino acid, including other hydrophobic and having the charge of amino acids.

To create a library ELISA authors synthesized the following peptides:

SEQ ID NO 15: SGGNNSPAA denoted P783-1

SEQ ID NO 16: SGGNNSPAF denoted P783-2

SEQ ID NO 17: SGGNNSPAL denoted P783-3

SEQ ID NO 18: SGGNNSPAM denoted P783-4

SEQ ID NO 19: SGGNNSPAY denoted P783-5

SEQ ID NO 20: SGGNNSPAG denoted P783-6

SEQ ID NO 21: SGGNNSPAS denoted P783-7

SEQ ID NO 22: SGGNNSPAP denoted P783-8

SEQ ID NO 23: SGGNNSPAN denoted P783-9

SEQ ID NO 24: SGGNNSPAE denoted P783-10

SEQ ID NO 25: SGGNNSPAR denoted P783-11.

The authors have limited competitive concentrations of peptides used in the previous experiments, because it would be impossible to assess the relative affinity of the competitive binding peptides within a single study. The results of the competitive ELISA library (represented graphically in figure 4) show the range of the affinity of the binding relative to the 9-dimensional R-10 (SGGNNSPAV), SEQ ID NO: 10. Especially important is and epitopes are variants with alanine, leucine and methionine (SEQ ID NO: 15, 17 and 18), especially at concentrations epitopes 125 μm and 31.3 μm. Most preferred of these variants are variants with alanine and methionine, as they have high resistance to heat, which makes them particularly effective for use in hot climates.

The ELISA

A suitable method of analysis by ELISA of the present invention is as follows.

To determine the presence or absence ofMycobacterium tuberculosisthe patient sample is brought into contact with an antibody directed against SEQ ID NO: 11; determine the presence or absence of a complex formed between the peptide in the sample and the antibody; determine the amount of complex formed in a specified sample compared to the control, taken as the normal value, where increasing the amount of complex in the sample compared to a control indicates the presence ofMycobacterium tuberculosis. The main methods of implementation of the ELISA assays known to the person skilled in the art. Patient samples can be obtained in the form of liquids, such as blood, saliva, semen, and milk, or tissue samples such as biopsy material, properly obtained, for example, by homogenization. However, preferred are samples obtained from the breathing put the th. Especially preferred sputum or fluid obtained bronchial or pulmonary lavage. Most preferred, however, are samples obtained by inducing cough reflex in a patient by spraying aerosol and collection of the material obtained from the respiratory tract of the patient; these samples contain material derived from the trachea.

Substitution analysis ELISA

A suitable method for substitution analysis by ELISA of the present invention is as follows.

To determine the presence or absence ofMycobacterium tuberculosisget a composition comprising a peptide as described herein and defined in the claims, and the patient sample. The composition is brought into contact with an antibody directed against SEQ ID NO: 11; determine the presence or absence of a complex formed between the specified peptide and the antibody; and the amount of complex formed in the specified pattern, compared with the control, taken as the normal value, where the reduction of the complex in the sample compared to a control indicates the presence ofMycobacterium tuberculosis.

Professionals in this field know the major methods for substitution of ELISA assays. In addition, patient samples can be obtained in the form of body fluids such as blood is ü, saliva, semen, and milk, or tissue samples such as biopsy material obtained in a suitable manner, such as by homogenization. However, the preferred specimens obtained from the respiratory tract. Especially preferred sputum or fluid obtained bronchial or pulmonary lavage. Most preferred, however, are samples obtained by inducing cough reflex in a patient by spraying aerosol and collection of the material obtained from the respiratory tract of the patient; these samples contain material derived from the trachea.

1. Peptide for detection of Mycobacterium tuberculosis with less than 18 amino acids containing a sequence NSPAX, where X is a methionine (SEQ ID NO: 18), where the peptide has the ability to bind with the antibody against the peptide GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11).

2. The peptide according to claim 1, where the peptide contains up to 6 additional amino acids at the C - and/or N-end of the sequence NSPAX.

3. Peptide for detection of Mycobacterium tuberculosis with less than 18 amino acids containing a sequence NNSPAX, where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17) or Alani is (SEQ ID NO: 15), where the peptide has the ability to bind with the antibody against the peptide GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11).

4. The peptide according to claim 3, where the peptide contains the sequence SGGNNSPAX (SEQ ID NO: 26).

5. The peptide according to claim 4, where the peptide consists of a sequence SGGNNSPAX (SEQ ID NO: 26), where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17), alanine (SEQ ID NO: 15) or valine (SEQ ID NO: 10).

6. The peptide according to claim 5, where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17) or alanine (SEQ ID NO: 15).

7. The peptide according to any one of p-6, where X is a methionine (SEQ ID NO: 18) or alanine (SEQ ID NO: 15).

8. The peptide according to any one of claims 1 to 6, optionally containing the label.

9. The nucleotide sequence encoding a peptide according to any one of claims 1 to 8.

10. Antibody against the peptide with the amino acid sequence presented in SEQ ID NO: 11, or a fragment having affinity binding in relation to SEQ ID NO: 11, used for detection of Mycobacterium tuberculosis.

11. The method of determining the presence of Mycobacterium tuberculosis in the sample, where the method includes substitution ELISA specified pattern, where the procedure specified analysis includes the use of a peptide comprising less than 18 amino acids containing a sequence NSPAX, where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17), alanine (SEQ ID NO: 15) or valine (SEQ ID NO: 10), where the peptide has the ability to bind the antibody against the peptide GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11).

12. The method according to claim 11, where the peptide contains up to 6 additional amino acids at the C - and/or N-end of the sequence NSPAX.

13. The method according to claim 11 or 12, where the peptide contains the sequence NNSPAX.

14. The method according to item 13, where the peptide contains the sequence NNSPAV (SEQ ID NO: 14).

15. The method according to claim 11 or 12, where the peptide contains the sequence SGGNNSPAX (SEQ ID NO: 26), where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17), alanine (SEQ ID NO: 15) or valine (SEQ ID NO: 10).

16. The method according to claim 11 or 12, where the peptide is not GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11).

17. The method according to claim 11, where the peptide consists of a sequence SGGNNSPAX (SEQ ID NO: 26), where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17), alanine (SEQ ID NO: 15) or valine (SEQ ID NO: 10).

18. The method according to any of § § 11, 12 or 17, where X is a methionine (SEQ ID NO: 18), leucine (SEQ ID NO: 17) or alanine (SEQ ID NO: 15).

19. The method according to p, where X is a methionine (SEQ ID NO: 18) or alanine (SEQ ID NO: 15).

20. The method according to claim 11 or 12, where the specified method further comprises using the antibody of claim 10.

21. The method according to claim 11 or 12, where the method additionally includes the use of peptide GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11).

22. The method according to claim 11 or 12, comprising the following stages:
(i) a composition containing a specified peptide and the sample of the patient;
(ii) bringing into contact the specified composition with an antibody against GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11);
iii) determining the presence or absence of the complex, formed between the specified peptide and the antibody; and
(iv) comparing the amount of complex formed in the specified pattern with normal controls, where increasing the amount of complex in the sample compared to a control indicates the presence of Mycobacterium tuberculosis.

23. The method according to item 22, where the patient sample contains a sample of the trachea.

24. The method of determining the presence of Mycobacterium tuberculosis in the sample, where the method includes substitution ELISA specified pattern and where the procedure specified analysis includes the use of the antibody of claim 10.

25. The method according to paragraph 24, which includes stages:
(i) bringing into contact of the patient sample with the antibody against GRDIKVQFQSGGNNSPAV (SEQ ID NO: 11);
(ii) determining the presence or absence of a complex formed between the specified peptide and the antibody; and
(iii) comparing the amount of complex formed in the specified pattern with normal controls, where increasing the amount of complex in the sample compared to a control indicates the presence of Mycobacterium tuberculosis.

26. The method according A.25, where the patient sample contains a sample obtained from the lungs.

27. The method according to p. 25 or 26, where the antibody is observed enzyme.



 

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42 cl, 29 dwg, 18 tbl, 22 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention is aimed at compound in accordance with formula (R2R3)-B1-A1-c(A2-A3-A4-A5-A6-A7-A8-A9)-A10-A11-A12-A13-B2-B3-R1, which act as ligands for one or several melanocortin receptors, their pharmaceutically acceptable salts.

EFFECT: elaboration of method of peptide application and obtaining pharmaceutical compositions, which include said peptides.

132 cl, 4 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to application of peptide of formula (I): ((X)1(Y)m)n where peptide contains 3 to 200 amino acids and where 1, m and n represent integers within 0 to 10; X and Y which can be identical or different, represent cationic amino acids chosen from arginine and lysine in preparing drugs for treating fungal infection.

EFFECT: ensured applicability of peptide for preparing a drug for treating fungal infection.

26 cl, 53 dwg, 2 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: there are described polypeptides eliciting an immune response on H.influenzae. There is offered an antibody selectively binding specified polypeptides. The invention also concerns an immunogenic composition containing the described polypeptides.

EFFECT: invention can be used in medicine for treating or preventing a disease or an infection caused by Hinfluenzae, such as otitis media.

9 cl, 3 tbl

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