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CN112457410B - Antigen composition for detecting mycobacterium tuberculosis infection - Google Patents

Antigen composition for detecting mycobacterium tuberculosis infection Download PDF

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CN112457410B
CN112457410B CN202011221017.1A CN202011221017A CN112457410B CN 112457410 B CN112457410 B CN 112457410B CN 202011221017 A CN202011221017 A CN 202011221017A CN 112457410 B CN112457410 B CN 112457410B
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mycobacterium tuberculosis
antigen
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CN112457410A (en
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张文娅
张凌燕
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Maccura Biotechnology Co ltd
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    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

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Abstract

The invention relates to the field of medical detection, in particular to an antigen composition for detecting mycobacterium tuberculosis infection. The 15 matched specific combined polypeptides screened by the invention have higher sensitivity and stronger specificity of the result of the mycobacterium tuberculosis infection determination.

Description

Antigen composition for detecting mycobacterium tuberculosis infection
Technical Field
The invention relates to the field of medical detection, in particular to an antigen composition for detecting mycobacterium tuberculosis infection.
Background
Tuberculosis is an infectious disease caused by mycobacterium tuberculosis, and is one of ten causes of death worldwide. At present, the tuberculosis is mainly diagnosed according to clinical symptoms, chest X-ray films and bacteriological examination, and the traditional diagnosis methods are time-consuming and low in detection rate, so that the optimal time for early discovery and early treatment is delayed. In the early days, it was found that mycobacterium tuberculosis can stimulate T cells in vivo to generate immune response, and tuberculosis can be rapidly diagnosed by using purified antigen (PPD) of mycobacterium tuberculosis to prepare Tuberculin Skin Test (TST). However, as the intensive research on diagnostic methods shows, the tuberculin test has many disadvantages, for example, some antigen components of PPD, which is a pure protein derivative used in TST, are the same as those of BCG and nontuberculous mycobacteria in most environments, so that cross reaction easily occurs, TST has high false positive rate, and diagnostic specificity is low.
Currently, the main principle of the specific cellular immune response (i.e. gamma interferon release assay, IGRAs) of mycobacterium tuberculosis is adopted: the T lymphocytes (T cells) sensitized by the stimulation of the mycobacterium tuberculosis antigen can generate gamma-interferon when encountering the same antigen again, and the IGRAs judges whether the test subject has mycobacterium tuberculosis infection or not by detecting the gamma-interferon generated by the T cells in the sample under the stimulation of the mycobacterium tuberculosis specific antigen. Currently, there are two types of internationally mature IGRAs: firstly, the level of gamma-interferon released by sensitized T cells in whole blood after being stimulated by specific antigen of mycobacterium tuberculosis is detected; secondly, the number of effector T cells which can release gamma-interferon in peripheral blood mononuclear cells is measured under the stimulation of specific antigen of the mycobacterium tuberculosis. At present, reagents used for stimulation mainly comprise various combined antigens, such as commonly used antigen peptides such as ESAT-6, CFP-10 and the like, the detection sensitivity of a single antigen peptide cannot be met, and a plurality of antigen peptides are required to be jointly detected, so that the detection sensitivity is improved. In the process of preparing the antigen peptide, toxoid is easy to generate, so that a determination result generates false positive, thereby reducing the specificity of determination.
Disclosure of Invention
The invention combines the contradiction, obtains a combined polypeptide which has higher sensitivity and specificity and is used for detecting mycobacterium tuberculosis infection or tuberculosis and is needed as a stimulant.
The invention relates to an antigen composition, which comprises polypeptides with sequences shown as SEQ ID NO 1-15.
Alternatively, the antigenic composition described above, any 2 or more of the polypeptides shown in SEQ ID NO 1-15 are fused to form a fusion peptide.
The invention also provides nucleic acids encoding the antigenic compositions described above.
The invention also provides a vector containing the nucleic acid as described above.
The invention also provides a host cell comprising a nucleic acid as described above or a vector as described above.
The invention also provides a kit for detecting the infection of the mycobacterium tuberculosis, which contains the antigen composition.
Optionally, the kit as described above, further comprising one or more of an anti-interferon-gamma antibody, a solid support, a reaction buffer, a wash solution, a positive control stimulus, and a negative control;
the gamma-interferon antibody is coupled with a signal substance.
Optionally, in the kit as described above, the interferon-gamma antibody is coated on the solid phase carrier.
Optionally, the kit as described above, wherein the solid support is selected from the group consisting of a test tube, an EP tube, a multiwell plate, a microplate well, and a microsphere.
Optionally, the kit as described above, wherein the signal substance is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme.
The invention also relates to application of the antigen composition in preparing a detection reagent or a kit for detecting specific T cell immune response caused by tuberculosis infection in vitro.
Optionally, the use as described above, the detection reagent or the kit is used for performing any one of the following methods:
enzyme-linked immunospot assay, enzyme-linked immunosorbent assay, immune colloidal gold assay, cytokine internal staining and T cell proliferation assay.
The matched specific combined polypeptide screened by the invention ensures that the sensitivity of the result of the infection determination of the mycobacterium tuberculosis is higher and the specificity is stronger.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention relates to an antigen composition, which comprises polypeptides with sequences shown as SEQ ID NO 1-15.
The invention relates to a combined polypeptide which is mainly prepared from the following proteins: early secreted antigen target (ESAT-6) and culture filtrate protein 10 (CFP 10), which are two recognized specific antigens in tuberculosis immunodiagnosis, and the two antigens have stronger immunological activity.
The inventors found that the combined use of ESAT-6 and CFP10 can exhibit higher sensitivity in the diagnosis of early tuberculosis, but in persistent infection, the secretion amount is gradually reduced, thereby reducing the sensitivity, so that the main dominant peptide fragments of the two antigens can be obtained as the composition of the mixed peptide according to the research. Rv2031c was shown to be expressed predominantly in the absence of oxygen in vitro from mycobacterium tuberculosis. Rv1174c is thought to be involved in reactivation of dormant Mycobacteria and to still cause growth of Mycobacterium tuberculosis after BCG ageing. Rv1985c is presumed to be a chromosome replication-initiating arrestin, a tuberculosis-specific protein capable of activating both cellular and humoral immunity. According to the invention, ESAT-6, CFP10 and the other 3 proteins, namely 5 proteins in total, are selected through experiments and are used as mixed polypeptides consisting of dominant peptide fragments, and after a biological sample of a mycobacterium tuberculosis infected person is stimulated, the gamma-interferon secreted by specific T cells generated by the mycobacterium tuberculosis infected person is measured, and the reaction is detected to show that the reaction is more sensitive and the specificity is higher.
An "antigenic portion" (which may or may not be soluble) of an antigen is a portion that is capable of reacting with serum obtained from a mycobacterium tuberculosis-infected individual (i.e., the absorption readings produced with serum from an infected individual are at least greater than three standard deviations of the absorption obtained with serum from an uninfected individual in the representative magnetic particle chemiluminescence assay described herein). A "Mycobacterium tuberculosis-infected individual" is a human that has been infected with Mycobacterium tuberculosis (e.g., has a intradermal skin test response to PPD of at least 0.5cm in diameter). Infected individuals may exhibit symptoms of tuberculosis, or may be asymptomatic for the disease.
The compositions and methods of the invention also include variants of the above polypeptides. As used herein, a "variant" is a polypeptide that differs from the native antigen only in conservative substitutions and/or modifications (such that the antigenic properties of the polypeptide are retained). Such variants can be generally identified by modifying one of the above-described polypeptide sequences using the representative methods described herein and evaluating the antigenic properties of the modified polypeptide.
A "conservative substitution" is a substitution in which one amino acid is substituted for another amino acid having similar properties, so that one skilled in the art of peptide chemistry can expect the secondary structure and hydrophilic properties of the polypeptide to be substantially unchanged. In general, the following group of amino acids represents conservative substitutions: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
Variants may also (or alternatively) be modified by, for example, amino acid deletions or additions with minimal effect on the antigenic properties, secondary structure and hydrophilic properties of the polypeptide. For example, the polypeptide may be linked to a signal (or leader) sequence at the N-terminus of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptides may also be linked to linkers and other sequences (e.g., poly-His) that facilitate synthesis, purification, and identification or enhancement of the polypeptide binding to a solid support. For example, the polypeptide may be linked to an immunoglobulin Fc region.
In some embodiments, any 2 or more of the polypeptides set forth in SEQ ID NOS 1-15 are fused to form a fusion peptide.
The fusion peptides may be linked to each other by a linker peptide, which in some embodiments is a flexible linker peptide; in some embodiments, the amino acid sequence of the linking peptide is selected from one or more of Gly, ser, pro, ala, and Glu.
In some embodiments, the amino acid sequence of the linker peptide is selected from (GGGGS) n, (GGGS) n, (GGS) n, (GS) n, or (G) n, wherein n is selected from 1,2,3,4,5, or 6.
The invention also relates to nucleic acids of the antigenic compositions as described above.
The invention also relates to a vector containing a nucleic acid as described above.
The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection such that the genetic material element it carries is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or artificial chromosomes of P1 origin (PACs); bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.
The invention also relates to a host cell comprising a nucleic acid as described above, or a vector as described above.
According to a further aspect of the invention, the invention also relates to a kit for detecting mycobacterium tuberculosis infection containing the antigen composition as described above.
In some embodiments, the kit further comprises one or more of paired primary and secondary anti-interferon-gamma antibodies, a solid support, a reaction buffer, a wash solution, a positive control stimulus, and a negative control;
the primary antibody is conjugated to a signal substance.
In some embodiments, the positive control stimulus is a lectin.
In some embodiments, the anti-interferon-gamma antibody can be an anti-human or animal (e.g., mouse, rat, rabbit, sheep, and goat) interferon-gamma antibody.
In some embodiments, the second antibody is immobilized coated on the solid support.
In the context of the present invention, the term "coating" refers to both non-covalent association (e.g. adsorption) and covalent attachment (either by direct bonding of the antigen to a functional group on the support or by means of a cross-linking agent). An embodiment coated on magnetic particles is preferred. In such a case, the amount of the antibody coated per mg of the magnetic particle is preferably 1. Mu.g to 10. Mu.g, more preferably 5. Mu.g.
In some embodiments, the solid support is selected from a test tube, an EP tube, a multi-well plate, a microplate well, or a microsphere.
In the present invention, the term "microparticle" may be a sphere, a nearly sphere, a cube, a polyhedron, or an irregular shape. The diameter of the microspheres is preferably 10nm to 1mm, for example 100nm, 500nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400nm to 10 μm.
The fine particles are preferably magnetic fine particles, and the composition thereof contains a magnetic substance. The magnetic substance may be a metal (simple metal or alloy), a nonmetal, or a composite of a metal and a nonmetal. Metals such as iron, alnico, and the like; non-metals, e.g. ferrite non-metals (preferably Fe) 2 O 3 Or Fe 3 O 4 Magnetic nanoparticles); a composite of metal and non-metal such as neodymium iron boron rubber magnetic composite.
The multiwell plate is preferably an elisa plate, and may contain 8, 16, 32, 48, 64, 96 or more wells.
In some embodiments, the signal substance is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme.
The following non-limiting section lists these markers:
enzymes which produce a detectable signal, e.g.by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
Chromophores such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds (e.g. acridinium esters) and dyes.
Groups with electron density that can be detected by electron microscopy or by its electrical properties, such as conductivity, amperometry, voltage measurement and resistance.
A detectable group, such as one whose molecular size is sufficient to induce a detectable modification in its physical and/or chemical properties; such detection can be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic spectroscopy and tunneling).
Electron-dense substances, e.g. radioactive molecules (e.g. 32 P， 35 S or 125 I)。
According to a further aspect of the invention, the invention also relates to the use of the antigen composition as described above for the preparation of a detection reagent or kit for the in vitro detection of specific T cell immune responses caused by tuberculosis infection.
In some embodiments, the detection reagent or kit is used for detecting gamma-interferon by chemiluminescence, radioimmunoassay, enzyme-linked immunosorbent assay or colloidal gold-based techniques.
In another aspect, the present invention provides a method for diagnosing tuberculosis using the antigenic composition described above.
The method comprises the steps of stimulating immune cells (particularly T cells) in a biological sample by using the antigen composition, enabling the immune cells to release gamma-interferon, capturing through an anti-gamma-interferon antibody, and detecting the content of the gamma-interferon.
In some embodiments, the antigenic compositions have a working concentration of each of the polypeptides set forth in SEQ ID NOs 1-15 of 3 μ g/m to 7 μ g/mL, preferably 5 μ g/mL.
As used herein, a "biological sample" is any antibody-containing sample obtained from a patient. Preferably the sample is whole blood (preferably peripheral blood, more preferably purified peripheral blood mononuclear cells), serosal cavity fluid collection, ascites fluid and cerebrospinal fluid. The presence of such antibodies indicates early sensitization to mycobacterial antigens that may be indicative of tuberculosis.
Embodiments of the present invention will be described in detail with reference to examples.
Examples
1. The combination patterns of specific combination polypeptides of Mycobacterium tuberculosis, the polypeptide combinations used in examples 1-8 are shown in tables 1-8, and the results are shown in tables 9 and 10;
2. the combined peptide can be used for detecting tuberculosis infection and clinically detecting mycobacterium tuberculosis.
The detection kit comprises:
(1) Blood culture tube: specific combined polypeptide containing mycobacterium tuberculosis; phytohemagglutinin (PHA) solution positive control stimuli; a negative control;
(2) Immune magnetic particles: the anti-human or murine interferon-gamma monoclonal antibody is coated on the magnetic microparticles at a suitable coating ratio (preferably: 5. Mu.g/mg). Sealing redundant blank sites on the magnetic particles by biotin, and diluting the magnetic particles to working concentration by using a universal magnetic bead diluent after washing;
(3) Acridinium ester label: acridinium ester labeled anti-human or murine interferon-gamma monoclonal antibodies;
(4) A calibration product: an interferon-gamma antigen.
The test mode is as follows:
(1) Collecting: adopting venipuncture, and collecting a whole blood sample by using a heparin anticoagulant vacuum blood collection tube;
(2) Subpackaging: the collected whole blood sample is gently inverted and uniformly mixed for 3-5 times, and then the whole blood sample is subpackaged into a blank tube, a positive control tube and a combined polypeptide tube, wherein the average volume of each tube is 1mL;
(3) Culturing: after the culture tubes are turned and mixed evenly, the culture tubes are quickly placed at 37 ℃ for incubation for 18 +/-2 hours, and the culture tubes are kept closed and upright in the culture process;
(4) Centrifuging: centrifuging the cultured culture tube at 3000-5000 rpm for 10 min, and taking the upper plasma layer for chemiluminescence detection.
220 combined cases and 220 healthy volunteers were selected for the project, and 440 samples were selected in total.
The sample should meet the following requirements:
(1) The clinical manifestation of the case sample needs to be a diagnosed pulmonary tuberculosis patient, and the colony culture of the mycobacterium tuberculosis of the patient is positive;
(2) The amount of each sample is not less than 4mL, and each tube is ensured to have the same volume of 1mL;
(3) The samples should be left at room temperature prior to stimulation and should not be stimulated for more than 18 hours at the latest.
TABLE 1
Numbering Sequence of Quantity of
SEQ ID NO:1 EQQWNFAGIEAAASA 5μg/mL
SEQ ID NO:2 IQGNVTSIHSLLDEG 5μg/mL
SEQ ID NO:3 ELNNALQNLARTISE 5μg/mL
SEQ ID NO:4 ARTISEAGQAMASTE 5μg/mL
SEQ ID NO:5 QGQWRGAAGTAAQAA 5μg/mL
SEQ ID NO:6 QAAVVRFQEAANKQK 5μg/mL
SEQ ID NO:7 ISTNIRQAGVQYSRA 5μg/mL
SEQ ID NO:8 AEMKTDAATL 5μg/mL
SEQ ID NO:9 NIRQAGVQY 5μg/mL
SEQ ID NO:10 TAAQAAVVRF 5μg/mL
TABLE 2
Number of Sequence of Quantity of
SEQ ID NO:1 EQQWNFAGIEAAASA 5μg/mL
SEQ ID NO:2 IQGNVTSIHSLLDEG 5μg/mL
SEQ ID NO:3 ELNNALQNLARTISE 5μg/mL
SEQ ID NO:4 ARTISEAGQAMASTE 5μg/mL
SEQ ID NO:5 QGQWRGAAGTAAQAA 5μg/mL
SEQ ID NO:6 QAAVVRFQEAANKQK 5μg/mL
SEQ ID NO:7 ISTNIRQAGVQYSRA 5μg/mL
SEQ ID NO:8 AEMKTDAATL 5μg/mL
SEQ ID NO:9 NIRQAGVQY 5μg/mL
SEQ ID NO:10 TAAQAAVVRF 5μg/mL
SEQ ID NO:11 GRSEFAYGSFVRTVS 5μg/mL
SEQ ID NO:12 AYGSFVRTVSLPVGA 5μg/mL
SEQ ID NO:13 DEARRMWASAQNISG 5μg/mL
TABLE 3
Figure BDA0002762023750000081
Figure BDA0002762023750000091
TABLE 4
Number of Sequence of Quantity of
SEQ ID NO:1 EQQWNFAGIEAAASA 5μg/mL
SEQ ID NO:2 IQGNVTSIHSLLDEG 5μg/mL
SEQ ID NO:3 ELNNALQNLARTISE 5μg/mL
SEQ ID NO:4 ARTISEAGQAMASTE 5μg/mL
SEQ ID NO:5 QGQWRGAAGTAAQAA 5μg/mL
SEQ ID NO:6 QAAVVRFQEAANKQK 5μg/mL
SEQ ID NO:7 ISTNIRQAGVQYSRA 5μg/mL
SEQ ID NO:8 AEMKTDAATL 5μg/mL
SEQ ID NO:9 NIRQAGVQY 5μg/mL
SEQ ID NO:10 TAAQAAVVRF 5μg/mL
SEQ ID NO:11 GRSEFAYGSFVRTVS 5μg/mL
SEQ ID NO:12 AYGSFVRTVSLPVGA 5μg/mL
SEQ ID NO:13 DEARRMWASAQNISG 5μg/mL
SEQ ID NO:16 MATWFSAVFDGLGDV 5μg/mL
SEQ ID NO:17 LLDVRIEDQDHSARL 5μg/mL
SEQ ID NO:18 LREGVAMGAVTTERN 5μg/mL
SEQ ID NO:19 TTERNPVPGCRVHPL 5μg/mL
SEQ ID NO:20 RRAITRPTHFVPTTE 5μg/mL
SEQ ID NO:21 GFTAAARAGLGWGMF 5μg/mL
SEQ ID NO:22 GWGMFPEKLAASPLA 5μg/mL
TABLE 5
Figure BDA0002762023750000092
Figure BDA0002762023750000101
TABLE 6
Number of Sequence of Measurement of
SEQ ID NO:1 EQQWNFAGIEAAASA 5μg/mL
SEQ ID NO:2 IQGNVTSIHSLLDEG 5μg/mL
SEQ ID NO:3 ELNNALQNLARTISE 5μg/mL
SEQ ID NO:4 ARTISEAGQAMASTE 5μg/mL
SEQ ID NO:5 QGQWRGAAGTAAQAA 5μg/mL
SEQ ID NO:6 QAAVVRFQEAANKQK 5μg/mL
SEQ ID NO:7 ISTNIRQAGVQYSRA 5μg/mL
SEQ ID NO:8 AEMKTDAATL 5μg/mL
SEQ ID NO:9 NIRQAGVQY 5μg/mL
SEQ ID NO:10 TAAQAAVVRF 5μg/mL
SEQ ID NO:14 AVINTTCNYGQ 5μg/mL
SEQ ID NO:15 ASPVAQSYL 5μg/mL
TABLE 7
Figure BDA0002762023750000102
Figure BDA0002762023750000111
TABLE 8
Number of Sequence of Measurement of
SEQ ID NO:1 EQQWNFAGIEAAASA 5μg/mL
SEQ ID NO:2 IQGNVTSIHSLLDEG 5μg/mL
SEQ ID NO:3 ELNNALQNLARTISE 5μg/mL
SEQ ID NO:4 ARTISEAGQAMASTE 5μg/mL
SEQ ID NO:5 QGQWRGAAGTAAQAA 5μg/mL
SEQ ID NO:6 QAAVVRFQEAANKQK 5μg/mL
SEQ ID NO:7 ISTNIRQAGVQYSRA 5μg/mL
SEQ ID NO:8 AEMKTDAATL 5μg/mL
SEQ ID NO:9 NIRQAGVQY 5μg/mL
SEQ ID NO:10 TAAQAAVVRF 5μg/mL
SEQ ID NO:16 MATWFSAVFDGLGDV 5μg/mL
SEQ ID NO:17 LLDVRIEDQDHSARL 5μg/mL
SEQ ID NO:18 LREGVAMGAVTTERN 5μg/mL
SEQ ID NO:19 TTERNPVPGCRVHPL 5μg/mL
SEQ ID NO:20 RRAITRPTHFVPTTE 5μg/mL
SEQ ID NO:21 GFTAAARAGLGWGMF 5μg/mL
SEQ ID NO:22 GWGMFPEKLAASPLA 5μg/mL
TABLE 9
Figure BDA0002762023750000112
According to the results in the table above, the sensitivity of the reagent can be increased to some extent by adding the combined polypeptide to the specific example 1, but the change is not large; within a certain range, the results of the combination of the polypeptides 1 to 15 in the specific example 3 are best, and the specificity is not obviously changed while the sensitivity is improved. And the sensitivity is not obviously improved and the specificity is reduced by adding the new combined polypeptide. The results show that the antigen polypeptide library has higher reactivity in tuberculosis patients, and the specificity is also highest compared with the results of healthy volunteers. Therefore, the combined polypeptide is used for detecting gamma-interferon and can produce effective diagnosis results for tuberculosis patients.
As is clear from the results of examples 1 to 8, 10 polypeptides in ESAT-6 and CFP-6 had a large influence on the measurement results, so that the gradient of the polypeptide addition amount was attempted in two ways
Example 9: the adding amount of 1-15 peptides is 5 mug/mL.
Example 10: the adding amount of 1-15 peptides is 7 mug/mL.
Example 11: the adding amount of 1-10 peptides is 7 mug/mL; the adding amount of 11-15 peptides is 5 mug/mL.
Watch 10
Figure BDA0002762023750000121
According to the results in the table, the adding amount of the polypeptide is adjusted on the basis of the results in example 7, and the results in example 9 show that the best results are obtained, and the best effect can be achieved by combining the sensitivity and the specificity when the amounts of the first 1 to 15 strips are all 5 mug/mL; therefore, the detection of interferon-gamma using the preferred combined polypeptide added amount can produce effective diagnosis results for tuberculosis patients.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Mike Bio-Ltd
<120> antigen composition for detection of Mycobacterium tuberculosis infection
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> artificial sequence
<400> 1
Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala
1 5 10 15
<210> 2
<211> 15
<212> PRT
<213> artificial sequence
<400> 2
Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
1 5 10 15
<210> 3
<211> 15
<212> PRT
<213> artificial sequence
<400> 3
Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu
1 5 10 15
<210> 4
<211> 15
<212> PRT
<213> artificial sequence
<400> 4
Ala Arg Thr Ile Ser Glu Ala Gly Gln Ala Met Ala Ser Thr Glu
1 5 10 15
<210> 5
<211> 15
<212> PRT
<213> artificial sequence
<400> 5
Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr Ala Ala Gln Ala Ala
1 5 10 15
<210> 6
<211> 15
<212> PRT
<213> artificial sequence
<400> 6
Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys Gln Lys
1 5 10 15
<210> 7
<211> 15
<212> PRT
<213> artificial sequence
<400> 7
Ile Ser Thr Asn Ile Arg Gln Ala Gly Val Gln Tyr Ser Arg Ala
1 5 10 15
<210> 8
<211> 10
<212> PRT
<213> artificial sequence
<400> 8
Ala Glu Met Lys Thr Asp Ala Ala Thr Leu
1 5 10
<210> 9
<211> 9
<212> PRT
<213> artificial sequence
<400> 9
Asn Ile Arg Gln Ala Gly Val Gln Tyr
1 5
<210> 10
<211> 10
<212> PRT
<213> artificial sequence
<400> 10
Thr Ala Ala Gln Ala Ala Val Val Arg Phe
1 5 10
<210> 11
<211> 15
<212> PRT
<213> artificial sequence
<400> 11
Gly Arg Ser Glu Phe Ala Tyr Gly Ser Phe Val Arg Thr Val Ser
1 5 10 15
<210> 12
<211> 15
<212> PRT
<213> artificial sequence
<400> 12
Ala Tyr Gly Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala
1 5 10 15
<210> 13
<211> 15
<212> PRT
<213> artificial sequence
<400> 13
Asp Glu Ala Arg Arg Met Trp Ala Ser Ala Gln Asn Ile Ser Gly
1 5 10 15
<210> 14
<211> 11
<212> PRT
<213> artificial sequence
<400> 14
Ala Val Ile Asn Thr Thr Cys Asn Tyr Gly Gln
1 5 10
<210> 15
<211> 9
<212> PRT
<213> artificial sequence
<400> 15
Ala Ser Pro Val Ala Gln Ser Tyr Leu
1 5
<210> 16
<211> 15
<212> PRT
<213> artificial sequence
<400> 16
Met Ala Thr Trp Phe Ser Ala Val Phe Asp Gly Leu Gly Asp Val
1 5 10 15
<210> 17
<211> 15
<212> PRT
<213> artificial sequence
<400> 17
Leu Leu Asp Val Arg Ile Glu Asp Gln Asp His Ser Ala Arg Leu
1 5 10 15
<210> 18
<211> 15
<212> PRT
<213> artificial sequence
<400> 18
Leu Arg Glu Gly Val Ala Met Gly Ala Val Thr Thr Glu Arg Asn
1 5 10 15
<210> 19
<211> 15
<212> PRT
<213> artificial sequence
<400> 19
Thr Thr Glu Arg Asn Pro Val Pro Gly Cys Arg Val His Pro Leu
1 5 10 15
<210> 20
<211> 15
<212> PRT
<213> artificial sequence
<400> 20
Arg Arg Ala Ile Thr Arg Pro Thr His Phe Val Pro Thr Thr Glu
1 5 10 15
<210> 21
<211> 15
<212> PRT
<213> artificial sequence
<400> 21
Gly Phe Thr Ala Ala Ala Arg Ala Gly Leu Gly Trp Gly Met Phe
1 5 10 15
<210> 22
<211> 15
<212> PRT
<213> artificial sequence
<400> 22
Gly Trp Gly Met Phe Pro Glu Lys Leu Ala Ala Ser Pro Leu Ala
1 5 10 15

Claims (12)

1. An antigen composition, which comprises a polypeptide with a sequence shown as SEQ ID NO 1-15.
2. A nucleic acid encoding the antigenic composition of claim 1.
3. A vector comprising the nucleic acid of claim 2.
4. A host cell comprising the nucleic acid of claim 2 or the vector of claim 3.
5. A kit for detecting infection with Mycobacterium tuberculosis comprising the antigen composition of claim 1.
6. The kit of claim 5, further comprising one or more of paired primary and secondary anti-interferon gamma antibodies, a solid support, a reaction buffer, a wash solution, an interferon gamma antigen standard, a positive control stimulus, and a negative control;
the primary antibody is conjugated to a signal substance.
7. The kit of claim 6, wherein the second antibody is immobilized coated on the solid support; the solid phase carrier is selected from test tubes, EP tubes, multi-well plates, micro-reaction plate concave holes or microspheres.
8. The kit of claim 7, wherein the solid support is a magnetic microparticle.
9. The kit according to claim 7, wherein the signal substance is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme.
10. The kit according to claim 9, wherein the signal substance is an acridinium ester.
11. Use of the antigen composition of claim 1 for the preparation of a detection reagent or kit for the in vitro detection of specific T cell immune responses caused by tuberculosis infection.
12. The use of claim 11, wherein the detection reagent or the kit is used for detecting gamma-interferon by chemiluminescence, radioimmunoassay, enzyme-linked immunosorbent assay or colloidal gold-based technique.
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