CN107121546A - Antigenic stimulus thing, kit and its application for detecting mycobacterium tuberculosis infection - Google Patents
Antigenic stimulus thing, kit and its application for detecting mycobacterium tuberculosis infection Download PDFInfo
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- CN107121546A CN107121546A CN201710119615.XA CN201710119615A CN107121546A CN 107121546 A CN107121546 A CN 107121546A CN 201710119615 A CN201710119615 A CN 201710119615A CN 107121546 A CN107121546 A CN 107121546A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
Abstract
The present invention provides a kind of antigenic stimulus thing for mycobacterium tuberculosis infection detection and the kit containing the antigenic stimulus thing, and the present invention also provides above-mentioned antigenic stimulus thing and applied in the reagent of detection mycobacterium tuberculosis infection.The antigenic stimulus thing includes at least one of the polypeptide as shown in sequence 1~11 in sequence table polypeptide or its analog, and these polypeptides are respectively derived from tuberculosis specific antigen polypeptide ESAT 6 and tuberculosis specific antigen peptide C FP 10.The periphery blood T lymphocyte that antigenic stimulus thing provided by the present invention is capable of effective stimulus tuberculosis infection patient produces IFN γ, so as to highly sensitive and high special diagnosis of tuberculosis infection, and is not influenceed by BCG inoculations or other underlying diseases.
Description
The application is application number 201410790793.1, applying date 2014.12.17, denomination of invention " for detecting tuberculosis point
The divisional application of antigenic stimulus thing, kit and its application of branch bacillus infection ".
Technical field
The invention belongs to biomedical inspection field, and in particular to a kind of antigen that detection is infected for mycobacterium tuberculosis
Stimulant and its application and the kit containing the antigenic stimulus thing.
Background technology
Tuberculosis is the infectious disease that a class as caused by mycobacterium tuberculosis seriously endangers human health.Although tuberculosis branch
Bacillus initially infects the respiratory system of human body, but it can diffuse to the almost all of organ of human body and cause corresponding disease
Occur.It is estimated that the population in the whole world about 1/3 has infected mycobacterium tuberculosis, and there are 8,000,000 emerging infectious diseases every year
Example, has 2,000,000 people to die from tuberculosis every year.The tuberculosis epidemic situation and mycobacterium tuberculosis infection conditions of China are all quite serious, are
One of 22, whole world tuberculosis high burden country, tuberculosis patient numerical digit occupies the second in the world.
Described mycobacterium tuberculosis infection, typically refers to the crowd with active tuberculosis or latent tuberculosis infects,
Can also be healthy contactee.Body has minority to develop into active tuberculosis after infection mycobacterium tuberculosis, shows corresponding
Clinical symptoms, such as heating, cough, expectoration, rabat are abnormal.Without corresponding clinical symptoms after most people infection, but machine
The mycobacterium tuberculosis that body can not clear all, as latent tuberculosis infects.Latent tuberculosis infects person is in hypoimmunity
When can develop into active tuberculosis.
Early diagnosis lungy is important for effectively controlling the propagation of progress lungy and mycobacterium tuberculosis to have
Meaning.The method of current diagnosis tuberculosis infection is limited, mainly includes tuberculine skin experiment (TST), Sputum smears, phlegm
Bacteria Culture, radioactivity X-ray, serology antibody antigen immune detection and PCR and nucleic acid hybridization etc..Mycobacterium tuberculosis
T cell gamma interferon (IFN-γ) release test (interferon-gamma release assay, IGRA) caused by infection
It is the new method that developed recently gets up, available for diagnostic activities tuberculosis and tuberculosis latent infection.U.S. FDA batch is obtained
Quasi- listing T-SPOT.TB kits (Oxford Immunotec Limited, Aingdon, United Kingdom) and
QuantiFERON-TB Gold kits are all based on IGRA principles, with the tuberculosis antigen specific protein of RD1 areas gene code
It is stimulus that -6KD Early insulin secretions targeting antigen (ESAT-6) and 10KD cultures, which filter albumen (CFP-10), in vain, by detecting periphery
The T lymphocytes of tuberculosis specificity release IFN-γ carry out diagnosis of tuberculosis infection in blood, and susceptibility and specificity are all higher.
There is more shortcoming and defect, tuberculine skin experiment in the conventional method of current diagnosis tuberculosis infection
(TST) (including pathogenic point of many mycobacterial species are contained in mycobacterium tuberculosis purified protein derivative (PPD) used in
Branch bacillus, environment mycobacteria and BCG) common to antigen molecule, therefore the poor specificity of PPD diagnosis of tuberculosis, it is impossible to accurate
Really distinguish PPD experimental result positive and be actually because BCG inoculations, the sensitization in contact environment after a variety of non-tuberculous mycobacterias also
It is that real mycobacterium tuberculosis infection is caused.Sputum smears method is although simple and easy to apply, but a big chunk tuberculosis patient phlegm
The inside might not have mycobacterium tuberculosis (so-called " phlegm is cloudy " tuberculosis).So, Sputum smears training approach has diagnosis positive
Rate is low, be easy to miss inspection, Sputum bacterial culture cycle length, culture success ratio low (only 80% or so) the shortcomings of.The nucleic acid of sputum specimen into
Go-on-go surveys the diagnosis predicament for not only facing " phlegm yin constipation core " but also program is complicated, false positive easily occur.Therefore, immunological technique
The important method just diagnosed into tuberculosis mycobacterial infections.
T-SPOT.TB kits and QuantiFERON-TB Gold kits complicated, expensive, cost are high,
Seriously constraining it is used for millions of active tuberculosis patients and hundreds of millions of tuberculosis latent infection crowd progress
Extensive tuberculosis examination and diagnosis.In addition, the Antigenic Peptide included in these kits it is extremely complex and also simultaneously non-principal in
The major histocompatibility antigen of compatriots carries out design, examination and the checking of Antigenic Peptide, so applicability needs with using effect
Further lifted.The A of Publication No. CN 102516356 Chinese patent application filters out 8-11 even from antigen Rv3615c
Continuous polypeptide fragment is tested (ELISPOT) diagnosis of tuberculosis as stimulus, using ELISpot and infected, but its sensitivity
Only 64%, the requirement of commercial kit is not reached much.The A of Publication No. CN 102297968 Chinese patent application is adopted
Antigenic stimulus thing is the combination of 9 polypeptides of the antigen of mycobacterium tuberculosis ESAT-6-6 and CFP-10 antigens, and uses
IFN-γ, TNF-α, the magnetic bead of five kinds of antibody parcels of IL-2, MIG and IP-10, while detecting five kinds of cells of cells and supernatant
The factor, this has resulted in its detection method and the complexity of interpretation of result, and needs to use flow cytometer, adds detection
Cost, limit it and promote the use of.The A of Publication No. CN 103604933 Chinese patent application be based on TNF-α cell because
Sub- ELISA detects the kit of active tuberculosis, and the antigenic stimulus thing that the kit is used is ESAT-6 and CFP-10 whole pieces
Albumen, the identification of effective epitope and T lymphocytic cell surfaces may be hindered yet with the space conformation of whole piece albumen
Molecule is combined, and it contains other epitopes may produce negative regulation, is influenceed the secretion of cell factor, is caused this
Kit is poor to the diagnosis effect of latent infection tuberculosis.
The content of the invention
It is an object of the invention to provide it is a kind of for mycobacterium tuberculosis infect detection antigenic stimulus thing and contain this
The kit of antigenic stimulus thing.Antigenic stimulus thing provided by the present invention is capable of the peripheral blood T of effective stimulus tuberculosis infection patient
Lymphocyte produces IFN-γ, so as to highly sensitive and high special diagnosis of tuberculosis infection, and is not inoculated with or it by BCG
The influence of his underlying disease.
The present invention is reaches its purpose, and the technical scheme of use is as follows：
First aspect present invention provides a kind of antigenic stimulus thing for being used to detect mycobacterium tuberculosis infection, and it includes such as sequence
At least one of polypeptide in list shown in sequence 1~11 polypeptide or its analog.Amino acid sequence is as shown in sequence 1~11
Polypeptide, sequence is as follows：
SEQ ID NO.1：AGIEAAASAIQGNVTSI
SEQ ID NO.2：YQGVQQKWDATATELNNALQNL
SEQ ID NO.3：RTISEAGQAMASTEGNVTGMFA
SEQ ID NO.4：MAEMKTDAATLAQEAGNF
SEQ ID NO.5：GAAGTAAQAAVVRFQEAA
SEQ ID NO.6：VVRFQEAANKQKQELDEI
SEQ ID NO.7：YQGVQQKWDATATELNNALQ
SEQ ID NO.8：ISEAGQAMASTEGNVTGMFA
SEQ ID NO.9：MAEMKTDAATLAQEA
SEQ ID NO.10：AAGTAAQAAVVRFQE
SEQ ID NO.11：VRFQEAANKQKQELD
Wherein, SEQ ID NO.1~3 and SEQ ID NO.7~8 derive from tuberculosis specific antigen polypeptide ESAT-6,
SEQ ID NO.4~6 and SEQ ID NO.9~11 derive from tuberculosis specific antigen peptide C FP-10.
It is preferred that, the antigenic stimulus thing is as the polypeptide shown in sequence in sequence table 1 and sequence 7~8 or its analog group
Into；Or, shown antigenic stimulus thing is made up of the polypeptide shown in sequence in sequence table 9~11 or its analog.
Highly preferred, the antigenic stimulus thing is made up of the polypeptide shown in sequence in sequence table 1~3 or its analog；
Or, the antigenic stimulus thing is made up of the polypeptide shown in sequence in sequence table 4~6 or its analog.Using this preferred side
Formula, its Detection results are optimal, and detection sensitivity is optimal, and 89% is up to the detection positive coincidence rate of the doubtful sample of tuberculosis infection,
Negative match-rate is up to 100%.Using preferred antigenic stimulus thing, the sample of a wide range of volunteer is tested, Ke Yiyou
Effect avoids interference of the pulmonary infection to result caused by other non-tuberculous mycobacterias, so as to effectively recognize mycobacterium tuberculosis sense
Dye, therefore the sensitivity with height and specificity.In addition, our kit positive coincidence rate (sensitivity and specificity) is bright
Aobvious to exceed existing patented technology, the sensitivity of some existing patented technologies is about 60% or so, and (such sensitivity is being faced
Hardly possible is normally used on bed).In addition, this patent kit can carry out tuberculosis examination to extensive unknown crowd, from embodiment 4
(Fig. 5, Fig. 6) as can be seen that examination 1 tubercular can be gone out from 37 unknown humans, exclude tumour, other pulmonary infections,
The interference of the diseases such as influenza, specificity and sensitivity with height, so as to be generally investigated suitable for large-scale crowd, the feature
It is particularly important for carrying out tubercular's examination in China, and it diagnoses sensitivity and is significantly better than existing patent with specificity
Technology.
The analog, refers to that its characteristic is identical with the polypeptide, including it can equally be combined with antibody specificity,
This homeopeptide generally has at least 70% homology, preferably at least 90%, 95% homology.Its is unusual to come from
Replacement, insertion, missing and the modification of amino acid residue, can occur on the N-terminal in sequence, C-terminal or other any positions.
Polypeptide of the present invention can be synthesized by existing chemical synthesis, can also pass through those skilled in the art
The technique for gene engineering grasped is prepared, and a kind of representative method is that aforementioned polypeptides are made with solid-phase synthesis,
Its general principle is：The hydroxyl for the hydroxyl end amino acid that peptide chain will first be synthesized is same insoluble with the structure of covalent bond
Macromolecule resin is connected, and is then passed through using this amino acid combined on solid phase carrier as moiety and sloughs amino protecting group
And with excessive activated carboxyl component reaction, spreading peptide chain.Repeat (condensation → washing → deprotection → neutralization and washing → next
Wheel condensation) operation, the peptide chain length to be synthesized is reached, is finally cleaved peptide chain from resin, by the processing such as purifying,
Produce desired polypeptide.Wherein alpha-amido is referred to as BOC solid-phase synthesis with what BOC (tertbutyloxycarbonyl) was protected, and alpha-amido is used
The referred to as FMOC solid-phase synthesis of FMOC (9-fluorenylmethyloxycarbonyl) protections.
The antigenic stimulus thing that the present invention is provided can be applied to the detection of mycobacterium tuberculosis infection, its basic Cleaning Principle
It is：The antigenic stimulus thing provided by the present invention, stimulates the T cell of mycobacterium tuberculosis host, then detects T cell release
Cell factor, to determine whether T cell recognizes these polypeptides or the like, so that indirectly whether reflection host infected tuberculosis
Mycobacteria.
The antigenic stimulus thing that the present invention is provided can be answered in the reagent for being used for detecting mycobacterium tuberculosis infection is prepared
With.
The present invention also provides a kind of kit for being used to detect mycobacterium tuberculosis infection, and it is anti-comprising as described above
Primary stimuli thing, and capture antibody, detection antibody, the Streptavidin of horseradish peroxidase-labeled and 3- amino -9- ethyl clicks
Azoles (AEC) chromogenic substrate.
Further, the capture antibody is the monoclonal antibody of anti-human IFN-γ, and the detection antibody is biotin mark
The antibody of the anti-human IFN-γ of note.
Also include positive control stimulant and negative control reagent in kit, the antigen selected by positive control is to big absolutely
The T cell of most individuals can produce response, such as commercially available PHA (phytohemagglutin phytolectin), and negative control is added without antigenic component, selects
With culture medium or other buffer solutions.
Another aspect of the present invention provides a kind of method of detecting Mycobacterium tuberculosis infection in vitro, by previously described antigen
The T cell of stimulant and mycobacterium tuberculosis host are in contact, and by detecting the cell factor that T cell is secreted, determine that T cell is
The no identification antigenic stimulus thing, so that indirectly whether reflection host infected mycobacterium tuberculosis.
The host refers to people or other mammals, other mammals such as primate, ox, sheep, pig, small
The rodent such as mouse and rat.
Described T cell is generally in vivo by the antigen presensitization from mycobacterium tuberculosis, and these are by antigen sensibilization
T cell be typically found in the peripheral blood of host, also be present in bronchoalveolar lavage fluid, hydrothorax, cerebrospinal fluid, lymph node or its
He includes the tissue site of T cell.This T cell can be CD4+T cell or CD8+T cell.In the process, may be used
T cell is contacted in vitro or in vivo with peptide (antigenic stimulus thing), and can determine whether T cell recognizes in vitro or in vivo
Peptide.Generally, by determining whether state change or measure T cell of the T cell in the presence of peptide are combined with peptide, to determine that T is thin
Whether born of the same parents recognize the peptide.The state change of T cell can be that T cell starts secretory substance or secretion increase, and such as cell factor is special
It is not IFN-γ, IL-2 or TNF-α.It is preferred that determining the secretion of IFN-γ.Generally can be by making these materials and specific knot
The presence that agent combines and detects the conjugate and the complex of these materials is closed, to detect described material.Specificity knot
It is usually antibody, such as monoclonal antibody or polyclonal antibody to close agent, typically can buy or use standard from market
It is prepared by technology.Determine cell factor method be typically in field commonly use method, such as ELISA (EUSA),
ELISPOT (ELISpot experiment), Immunobloting (Western blot), intracellular cytokine dyeing, T cell
Proliferation experiment etc..
In one embodiment of the invention, T cell secretion of gamma-IFN is detected using ELISPOT methods.It is coated with advance
IFN-γ monoclonal antibody on solid phase carrier first combines to form compound with IFN-γ, the compound again with biotin labeling
The second IFN-γ antibody binding, then the Streptavidin of horseradish peroxidase-labeled and biotin specifically bind, most
Afterwards by the chromogenic reaction formation spot of enzyme and substrate, so as to reflect the T cell quantity being activated.T cell described in method
Can be in-vitro separation, it is of course also possible to be untreated either internal.In one embodiment, from periphery
Monocyte is separated in blood or other samples, it is that one kind can be by peptide submission that will include T cell and APC (antigen presenting cell), APC
To the cell of T cell.In one embodiment, peptide is directly added into itself in the experiment for containing T cell and APC.At this
In the experiment of sample, APC can be by peptide submission to T cell.When using being recognized by T cell without peptide by APC submissions, APC is not
It is required.
The time length that peptide is contacted with T cell can change according to for determining peptide knowledge method for distinguishing.It is representative
, 10 are added in each experiment5~107PMBC (PBMC), preferably 2.5 × 105~106.When peptide is direct
When adding in experiment, its concentration is 0.1~100 μ g/ml, preferably 1~10 μ g/ml.The typical time period that T cell is incubated together with peptide
It is 16~36 hours.
The antigenic stimulus thing that the present invention is provided and the kit containing this specific antigen stimulant, can be effectively in body
Outer detection mycobacterium tuberculosis infection, without being influenceed by inoculation BCG (BCG vaccine), with higher Sensitivity and Specificity,
It is not only suitable for the mycobacterium tuberculosis infection examination that mycobacterium tuberculosis infection clinical diagnosis is also applied for large-scale crowd.This hair
The kit of bright offer is designed according to Chinese's major histocompatibility complex, mycobacterium tuberculosis dominant antigen is verified in examination
Polypeptide, in CHINESE REGION, using effect is more preferable, with high specific and high sensitivity, of the invention in addition and foreign countries' kit phase
Than, it is cheap, more suitable for being promoted in China and developing country.
IFN-γ-ELISPOT the kits that the present invention is provided are in clinical practice, 55 mycobacterium tuberculosis height after testing
Spend suspected infection person's blood preparation, be diagnosed as mycobacterium tuberculosis infection patient 41, and through Sputum smears, Sputum bacterial culture,
Rabat and clinical symptoms are integrated into being diagnosed as patient 46 lungy, and its positive coincidence rate is up to 89.1%；With the present invention's
IFN-γ-ELISPOT kits are diagnosed as the patient 9 of non-tuberculosis, through Sputum smears, Sputum bacterial culture, rabat result and
Clinical symptoms diagnose all negative findingses, and its negative match-rate reaches 100%.The kit that the present invention is provided is in Diagnosis of Tuberculosis
In there is good clinical applicability, and specific good, sensitivity is high, and the coincidence rate with other diagnostic techniques is high.
Brief description of the drawings
Fig. 1：For the advantage polypeptide the result of IFN-γ-ELISPOT Diagnosis of Tuberculosis kits of the present invention：(A) this reagent
(spot number is IFN-γ-ELISPOT results after the stimulation cell of box tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3
123)；(B) this kit tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.4~6 stimulate the IFN-γ-ELISPOT after cell
As a result (spot number is 62)；(C) negative control (spot number is 0)；(D) positive (PHA) control (spot number is 801)；(E) it is not
The ground control of refinement born of the same parents.
Fig. 2：For the preferred polypeptide the result of IFN-γ-ELISPOT Diagnosis of Tuberculosis kits of the present invention：A is many in Fig. 2
The combined stimulation result (spot number is 160) of peptide antigen SEQID NO.1 and SEQID NO.7~8, B is this product this kit knot
Core specific polypeptide Antigenic Peptide SEQ IDNO.9~11 stimulate result after cell (spot number is 146), and C is negative control (spot
0) number is, and D be positive control (PHA) results of stimulation (spot number is 783), and E is to be not added with the ground control of cell (spot number is
0)。
Fig. 3：For this IFN-γ-ELISPOT kit diagnostic results of a tuberculosis suspected patient：(A) negative control (spot
Count as 1)；(B) the nonimmune advantage polypeptides of ESAT-6 stimulate the IFN-γ-ELISPOT results after cell (spot number is 5)；(C)
Tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3 stimulate the IFN-γ-ELISPOT results after cell (spot number is 22)；
(D) this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.4~6 stimulate the IFN-γ-ELISPOT result (spots after cell
Count as 19)；(E) PHA positive controls stimulate the IFN-γ-ELISPOT results after cell (spot number is 475).
Fig. 4：This IFN-γ-ELISPOT kits peptide sequence 1,7~11 for showing tuberculosis suspected patient is examined
Disconnected result.(A) is negative control (spot number is 1) in Fig. 4, and Fig. 4 (B) is that the nonimmune advantage polypeptides of ESAT-6 are stimulated after cell
IFN-γ-ELISPOT results (spot number is 4), Fig. 4 (C) is tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1 and SEQID
NO.7~8 stimulate the IFN-γ-ELISPOT results after cell (spot number is 38), and Fig. 4 (D) is that this product tuberculosis specificity is more
Peptide Antigenic Peptide SEQ ID NO.9~11 stimulate the IFN-γ-ELISPOT results after cell (spot number is 43), and Fig. 4 (E) is PHA
Positive control stimulates the IFN-γ-ELISPOT results after cell (spot number is 330).
Fig. 5：For using IFN-γ-ELISPOT kits of the present invention 37 volunteers are carried out with the result of tuberculosis examination.
Fig. 6：Shown by red block in Fig. 5 the tuberculosis positive is prompted for for wherein one IFN-γ-ELISPOT tuberculosis detection
Result：(A) negative control (spot number is 1)；(B) the nonimmune advantage polypeptides of ESAT-6 stimulate the IFN-γ after cell-
ELISPOT results (spot number is 6)；(C) tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3 stimulate the IFN- after cell
γ-ELISPOT results (spot number is 17)；(D) this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.4~6 stimulate thin
IFN-γ-ELISPOT results after born of the same parents (spot number is 9)；(E) PHA positive controls stimulate the IFN-γ-ELISPOT after cell
As a result (spot number is 466).
Embodiment
Technical scheme is described further below in conjunction with the accompanying drawings.Experimental method in following embodiments, such as
It is the conventional method of the art without specified otherwise.Experiment material used is city unless otherwise specified in embodiment
The % in routine biochemistry reagent, embodiment is sold, is weight/mass percentage composition unless otherwise specified.
Embodiment 1：The preparation of tuberculosis specific T-cells dominant antigen epitope polypeptide and IFN-γ-ELISPOT tuberculosis branches
The exploitation of bacillus infection diagnostic kit
T cell dominant antigen is designed and screens by bioinformatic analysis, and T cells examined in vitro immune response
Epitope, comprises the following steps：
(1) by bioinformatics technique and associated databases such as ProPred, MacVestor, Preotean etc., to RD1
The albumen ESAT-6 and CFP-10 of area's coding T cell antigen epitope are predicted, while analyzing it between different HLA partings
Identification relation.
(2) biological assays are combined, is designed in Overlap (overlap) method and preferably to go out 9 ESAT-6 more
Peptide and 11 CFP-10 polypeptides, each polypeptide include 9~22 amino acid.
(3) tuberculosis patient blood preparation, every part of 2~5ml are collected.Tuberculosis patient inclusion criteria is：There are cough, expectoration disease
Shape, rabat results abnormity, Sputum smears or Sputum bacterial culture are positive, and receive the tuberculosis therapy time less than three weeks, and HIV detections are cloudy
Property.
(4) with Ficoll lymphocyte separation mediums separating periphery blood monocytic cell (PBMC), PBMC through 5~10mlPBS or
After PRMI1640 is washed 2 times, being resuspended in the RPMI1640 culture mediums containing 10% hyclone (FBS, Life company), (Life is public
Department), and calculate cell quantity.
(5) ELISPOT methods detection T cell receives the IFN-γ discharged after stimulating, concretely comprises the following steps：In 96 with pvdf membrane
People's IFN-γ monoclonal antibody (ebioscience companies) is coated with orifice plate (Millipore companies) to stay overnight, RPMI1640+
2.5 × 10 are added per hole after 10%FBS closings5Individual cell and polypeptide, positive controls add phytohemagglutin phytolectin (PHA), negative
Control group adds culture medium or the solubilising reagent for dissolving polypeptide, and another set is not added with cell as ground control.Incubator
It is middle to be incubated 22 hours, the anti-human IFN-γ antibody of biotin labeling, the chain of horseradish peroxidase-labeled are sequentially added after board-washing
Mould Avidin, AEC (AEC) substrate colour developing, forms macroscopic punctation on pvdf membrane.As a result
Read on ELISPOT plate reading machines.Result judgement：Spot number >=10, and it is judged as sun more than 2 times of negative control hole spot numbers
Property result；Spot number ＜ 10, or less than 2 times negative control hole spot numbers are judged as negative findings.
(6) interpretation of result：According to frequency of the polypeptide to positive findings after tuberculosis patient stimulate the reaction, final screening obtains 3
Bar ESAT-6 dominant antigen epitope polypeptide SEQ ID NO.1~3 (its amino acid sequence can be found in sequence 1~3 in sequence table), 2
Bar ESAT-6 preferred antigens epitope polypeptide SEQ ID NO.7~8 (its amino acid sequence can be found in sequence 7~8 in sequence table), 3
Bar CFP-10 dominant antigen epitope polypeptide SEQ ID NO.4~6 (its amino acid sequence can be found in sequence 4~6 in sequence table), 3
Bar CFP-10 preferred antigens epitope polypeptide SEQ ID NO.9~11 (its amino acid sequence can be found in sequence 9~11 in sequence table).
Fig. 1 is a typical IFN-γ-ELISPOT result of the examination of polypeptide 1~6.Wherein Fig. 1 (A) is that this kit tuberculosis is special
Property the combined stimulation cell of polypeptide antigen peptide SEQ ID NO.1~3 after IFN-γ-ELISPOT results (spot number is 123), Fig. 1
(B) it is the IFN-γ-ELISPOT after the combined stimulation cell of this kit tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.4~6
As a result (spot number be 62), Fig. 1 (C) be negative control (spot number is 0), and Fig. 1 (D) be that the positive (PHA) compares that (spot number is
801), Fig. 1 (E) is the ground control for being not added with cell.Fig. 2 for sequence 1,7~11 polypeptide examination a typical IFN-γ-
ELISPOT results.Fig. 2A is that (spot number is the combined stimulation result of polypeptide antigen peptide SEQ ID NO.1 and SEQID NO.7~8
160), Fig. 2 B are to tie after the combined stimulation cell of this product this kit tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.9~11
Really (spot number is 146), Fig. 2 C are negative control (spot number is 0), and Fig. 2 D are positive control (PHA) results of stimulation (spot number
For 783), Fig. 2 E are the ground control for being not added with cell (spot number is 0).
Embodiment 2：IFN-γ-ELISPOT mycobacterium tuberculosis the infection diagnostic reagent kits that the present invention is developed, comprising：
(1), antigenic stimulus thing, by the amino acid sequence that is filtered out in embodiment 1 as shown in sequence 1-11 in sequence table
Polypeptide in one or two kinds of above polypeptide combination, or the analog of these polypeptides；
(2) antibody, is captured, is the monoclonal antibody of anti-human IFN-γ, is purchased from ebioscience companies；
(3) antibody, is detected, is the antibody of the anti-human IFN-γ of biotin labeling；
(4), the Streptavidin of horseradish peroxidase-labeled；
(5), AEC (AEC) chromogenic substrate；
(6), positive control stimulant, is phytohemagglutin phytolectin (PHA)；
(7), negative control thing, is culture medium (such as RPMI1640+10%FBS) or the solubilising reagent for dissolving polypeptide
(such as DMSO).
Embodiment 3：IFN-γ-ELISPOT the kits of the present invention are applied to mycobacterium tuberculosis and infect suspected patient
Clinical diagnosis
Purpose：IFN-γ-ELISPOT mycobacterium tuberculosis the infection diagnostic reagent kits of the present invention are examined in Diagnosis of Tuberculosis
Clinical applicability, specificity, sensitivity and the coincidence rate with other diagnostic techniques.
(1) 55 mycobacterium tuberculosis height suspected infection person's blood preparations, every part of 2~5ml are collected.
(2) with Ficoll lymphocyte separation mediums separating periphery blood monocytic cell (PBMC), PBMC through 5~10mlPBS or
After PRMI1640 is washed 2 times, the RPMI1640 culture mediums containing 10% hyclone (FBS) are resuspended in, and calculate cell quantity.
(3) ELISPOT methods detection T cell receives the IFN-γ discharged after stimulating, concretely comprises the following steps：In 96 with pvdf membrane
People's IFN-γ monoclonal antibody is coated with orifice plate to stay overnight, and 2.5 × 10 are added per hole after RPMI1640+10%FBS closings5Individual cell
And (or preferred polypeptide combination is SEQ ID NO.1 polypeptides to the polypeptide of polypeptide SEQ ID1~3 and the polypeptide of SEQ ID NO.7~8
Combination) or polypeptide SEQ ID4~6 (or combination of preferred polypeptide SEQ ID NO.9~11), positive controls addition plant blood
Solidifying element (PHA), negative control group adds 10%FBS/1640 culture mediums or dissolves the solubilising reagent DMSO of polypeptide, and another set is not
Refinement born of the same parents are used as ground control.It is incubated 22 hours in incubator, the anti-human IFN-γ that biotin labeling is sequentially added after board-washing resists
Body, the Streptavidin of horseradish peroxidase-labeled, the colour developing of AEC (AEC) substrate, the shape on pvdf membrane
Into macroscopic punctation.As a result read on ELISPOT plate reading machines.Result judgement：Spot number >=10, and more than 2
Times negative control hole spot number is judged as positive findings；Spot number ＜ 10, or less than 2 times negative control hole spot numbers are judged as
Negative findings.
(4) interpretation of result：In 55 doubtful mycobacterium tuberculosis infected patients, through Sputum smears, Sputum bacterial culture, rabat with
And clinical symptoms are integrated into being diagnosed as patient 46 lungy, knot is diagnosed as with the IFN-γ-ELISPOT kits of the present invention
The patient of core mycobacterial infections 41, therefore positive coincidence rate is 89.1%；Diagnosed with this IFN-γ-ELISPOT kits
For the patient 9 of non-tuberculosis, tied through all feminine genders of Sputum smears, Sputum bacterial culture, rabat result and clinical symptoms diagnosis
Really, therefore negative match-rate reaches 100%.Fig. 3 show this IFN-γ-ELISPOT kits of a tuberculosis suspected patient
(the antigenic stimulus thing that its used kit of the testing result contains is polypeptide SEQ ID NO.1~3 or polypeptide SEQ to diagnostic result
ID NO.4~6).Fig. 3 (A) is negative control (spot number is 1), and Fig. 3 (B) is that the nonimmune advantage polypeptides of ESAT-6 stimulate cell
IFN-γ-ELISPOT results (spot number is 5) afterwards, Fig. 3 (C) is tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3
IFN-γ-ELISPOT results (spot number is 22) after combined stimulation cell, Fig. 3 (D) is that this product tuberculosis specific polypeptide resists
IFN-γ-ELISPOT results (spot number is 19) after the combined stimulation cell of former peptide SEQ ID NO.4~6, Fig. 3 (E) is PHA
Positive control stimulates the IFN-γ-ELISPOT results after cell (spot number is 475).Fig. 4 show a tuberculosis suspected patient
The diagnostic results of preferred IFN-γ-ELISPOT kits (the antigenic stimulus thing that its used kit of the testing result contains is
Polypeptide SEQ ID NO.1 are combined with SEQ ID NO.7~8 or polypeptide SEQ ID NO.9~11 are combined).Fig. 4 (A)
For negative control (spot number is 1), Fig. 4 (B) is that the nonimmune advantage polypeptides of ESAT-6 stimulate the IFN-γ-ELISPOT after cell
As a result (spot number be 4), Fig. 4 (C) is the combined stimulation cell of tuberculosis specific polypeptide SEQ ID NO.1 and SEQ ID NO.7~8
IFN-γ-ELISPOT results (spot number is 38) afterwards, Fig. 4 (D) is this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID
IFN-γ-ELISPOT results (spot number is 43) after the combined stimulation cell of NO.9~11, Fig. 4 (E) is PHA positive controls thorn
Swash the IFN-γ-ELISPOT results after cell (spot number is 330).
Embodiment 4：Applicability of the IFN-γ-ELISPOT kits of the present invention in unknown crowd's tuberculosis examination
Purpose：IFN-γ-ELISPOT mycobacterium tuberculosis infection diagnostic reagent kit of the present invention is examined (used in the embodiment
Kit, combination or polypeptide SEQ ID NO.4~6 of its antigenic stimulus thing contained for polypeptide SEQ ID NO.1~3
Combination) applicability in large-scale crowd tuberculosis examination, specificity and sensitivity.
(1) 37 negative volunteer blood's samples of HIV detections, every part of 2~3ml are collected.These inoculated BCG epidemic diseases per capita
Seedling.
(2) with Ficoll lymphocyte separation mediums separating periphery blood monocytic cell (PBMC), PBMC through 5~10mlPBS or
After PRMI1640 is washed 2 times, the RPMI1640 culture mediums containing 10% hyclone (FBS) are resuspended in, and calculate cell quantity.
(3) ELISPOT methods detection T cell receives the IFN-γ discharged after stimulating, concretely comprises the following steps：In 96 with pvdf membrane
People's IFN-γ monoclonal antibody is coated with orifice plate to stay overnight, and 2.5 × 10 are added per hole after RPMI1640+10%FBS closings5Individual cell
And the polypeptide of sequence 1~3 or the polypeptide of sequence 4~6, positive controls addition phytohemagglutin phytolectin (PHA), negative control group, which is added, cultivates
Base or the solubilising reagent for dissolving polypeptide, another set are not added with cell as ground control.It is incubated 22 hours in incubator, after board-washing
Sequentially add the anti-human IFN-γ antibody of biotin labeling, the Streptavidin of horseradish peroxidase-labeled, 3- amino -9- second
Base carbazole (AEC) substrate is developed the color, and macroscopic punctation is formed on pvdf membrane.As a result read on ELISPOT plate reading machines
Take.Result judgement：Spot number >=10, and it is judged as positive findings more than 2 times of negative control hole spot numbers；Spot number ＜ 10
It is individual, or less than 2 times negative control hole spot numbers are judged as negative findings.
(4) interpretation of result：Fig. 5 is the tuberculosis detection part knot that this IFN-γ-ELISPOT kits are applied to unknown crowd
Really, shown result (the visible figure that the positive is prompted for for wherein one IFN-γ-ELISPOT tuberculosis detection of red block in Fig. 5
6), wherein Fig. 6 (A) is negative control (spot number is 1), and Fig. 6 (B) is that the nonimmune advantage polypeptides of ESAT-6 are stimulated after cell
IFN-γ-ELISPOT results (spot number is 6), Fig. 6 (C) is that tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3 are combined
IFN-γ-ELISPOT the results after cell (spot number is 17) are stimulated, Fig. 6 (D) is this product tuberculosis specific polypeptide Antigenic Peptide
IFN-γ-ELISPOT results (spot number is 9) after the combined stimulation cell of SEQ ID NO.4~6, Fig. 6 (E) is that the PHA positives are right
According to stimulating the IFN-γ-ELISPOT results (spot number be 466) after cell, the result point out volunteer's IFN-γ-
The detection of ELISPOT tuberculosis is positive, also illustrates have 1 to detect positive in IFN-γ-ELISPOT tuberculosis in 37 volunteer's samples,
The volunteer is by further detection afterwards, it was demonstrated that be active tuberculosis mycobacterial infections, illustrate it is of the invention develop IFN-γ-
ELISPOT kits are applicable to the generaI investigation of large-scale crowd tuberculosis and not by BCG vaccine inoculation or other underlying diseases etc.
Influence.In addition, 7~11 couples of similar unknown crowds of preferred polypeptide sequence have also been carried out with large-scale tuberculosis examination experiment, hair
Existing preferred polypeptide sequence 7~11, which has, is similar to specificity and spirit that 1~6 pair of large-scale crowd of peptide sequence carries out tuberculosis examination
Quick property, will not be repeated here.
The above described is only a preferred embodiment of the present invention, any formal limitation not is done to the present invention, therefore
All contents without departing from technical solution of the present invention, the technical spirit according to the present invention any is simply repaiied to made for any of the above embodiments
Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.
Claims (8)
1. a kind of antigenic stimulus thing for being used to detect mycobacterium tuberculosis infection, it is characterised in that the antigenic stimulus thing is by sequence
Sequence 1 and polypeptide or its analog composition shown in sequence 7~8 in list.
2. the antigenic stimulus thing described in claim 1 is being prepared for detecting the application in the reagent that mycobacterium tuberculosis infects.
3. a kind of kit for being used to detect mycobacterium tuberculosis infection, it is characterised in that it is comprising as claimed in claim 1
Antigenic stimulus thing, and capture antibody, detection antibody, the Streptavidin of horseradish peroxidase-labeled and 3- amino -9- ethyls
Carbazole chromogenic substrate.
4. kit according to claim 3, it is characterised in that the capture antibody resists for the monoclonal of anti-human IFN-γ
Body, the detection antibody is the antibody of the anti-human IFN-γ of biotin labeling.
5. a kind of method of detecting Mycobacterium tuberculosis infection in vitro, it is characterised in that by the antigenic stimulus described in claim 1
The T cell of thing and mycobacterium tuberculosis host are in contact, and by detecting the cell factor that T cell is secreted, determine whether T cell is known
Not described antigenic stimulus thing.
6. method according to claim 5, it is characterised in that the host refers to people or other mammals.
7. method according to claim 5, it is characterised in that the cell factor is IFN-γ, IL-2 or TNF-α, institute
State peripheral blood, bronchoalveolar lavage fluid, hydrothorax, cerebrospinal fluid, lymph node or other groups for including T cell that T cell derives from host
Knit position.
8. method according to claim 5, it is characterised in that adopt and be secreted into the following method to T cell cell factor
Row detection：ELISA, ELISPOT, Western blot, intracellular cytokine dyeing, T cell proliferation experiment.
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| CN201410790793.1A CN104597239B (en) | 2014-12-17 | 2014-12-17 | Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant |
| CN201710119615.XA CN107121546B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
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Publications (2)
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| CN201410790793.1A Active CN104597239B (en) | 2014-12-17 | 2014-12-17 | Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant |
| CN201710119273.1A Pending CN107102137A (en) | 2014-12-17 | 2014-12-17 | Antigenic stimulus thing, kit and its application for detecting mycobacterium tuberculosis infection |
| CN201710119615.XA Expired - Fee Related CN107121546B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
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| CN201410790793.1A Active CN104597239B (en) | 2014-12-17 | 2014-12-17 | Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant |
| CN201710119273.1A Pending CN107102137A (en) | 2014-12-17 | 2014-12-17 | Antigenic stimulus thing, kit and its application for detecting mycobacterium tuberculosis infection |
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| CN105527446B (en) * | 2016-01-11 | 2017-12-08 | 扬州大学 | A kind of ELISA kit of polypeptide antigen detection mycobacterium tuberculosis var bovis antibody |
| CN105884872B (en) * | 2016-05-18 | 2019-11-29 | 首都医科大学附属北京胸科医院 | The antigen polypeptide and application thereof of LppZ antibody identification |
| CN105954521B (en) * | 2016-07-08 | 2017-04-12 | 广州华弘生物科技有限公司 | MTB (Mycobacterium Tuberculosis) infection diagnosis kit |
| CN106353501A (en) * | 2016-08-19 | 2017-01-25 | 南通表源生物技术有限公司 | Tuberculosis T cell detection kit and detection method thereof |
| CN108254553A (en) * | 2016-12-29 | 2018-07-06 | 广东希格生物科技有限公司 | For the albumen Rv1791 of specific detection mycobacterium tuberculosis infection |
| CN110865187A (en) * | 2019-10-16 | 2020-03-06 | 华中科技大学同济医学院附属同济医院 | Method for calculating average spot area of ESAT-6 antigen hole based on enzyme-linked immunosorbent assay kit |
| CN114859062B (en) * | 2022-05-08 | 2023-07-07 | 吉林大学 | Chemiluminescent immunoassay method based on double-enzyme amplification system |
| CN115078732A (en) * | 2022-06-09 | 2022-09-20 | 广州迪澳医疗科技有限公司 | Specific stimulating antigen of mycobacterium tuberculosis, kit and application thereof |
| CN116102629B (en) * | 2022-09-03 | 2023-09-08 | 武汉中纪生物科技有限公司 | Mycobacterium tuberculosis T cell epitope polypeptide and application thereof |
| CN117304284B (en) * | 2023-08-07 | 2024-03-19 | 梅州市人民医院(梅州市医学科学院) | Mycobacterium tuberculosis specific T cell HLA-A 0201 restriction epitope peptide and application thereof |
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| Publication number | Publication date |
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| CN107064498A (en) | 2017-08-18 |
| CN107102137A (en) | 2017-08-29 |
| CN107064498B (en) | 2019-03-22 |
| CN104597239B (en) | 2017-04-26 |
| CN107121546B (en) | 2019-03-22 |
| WO2016095273A1 (en) | 2016-06-23 |
| CN104597239A (en) | 2015-05-06 |
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