CN107121546B - For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection - Google Patents
For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection Download PDFInfo
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Abstract
The present invention provides a kind of antigenic stimulus object for mycobacterium tuberculosis infection detection and the kit containing the antigenic stimulus object, and the present invention also provides above-mentioned antigenic stimulus objects to be applied in the reagent of detection mycobacterium tuberculosis infection.The antigenic stimulus object includes that at least one of the polypeptide as shown in sequence 1~11 in sequence table polypeptide or its analog, 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 object provided by the present invention is capable of effective stimulus tuberculosis infection patient generates IFN-γ, so as to highly sensitive and high special diagnosis of tuberculosis infection, and is not influenced by BCG inoculation 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 object, kit and its application of branch bacillus infection ".
Technical field
The invention belongs to biomedical inspection fields, and in particular to a kind of antigen for mycobacterium tuberculosis infection detection
Stimulant and its application and kit containing the antigenic stimulus object.
Background technique
Tuberculosis is the infectious disease that one kind 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 lead to 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 of tuberculosis every year.The tuberculosis epidemic situation and mycobacterium tuberculosis infection conditions in China are all quite serious, are
One of 22 tuberculosis high burden countries in the whole world, tuberculosis patient numerical digit occupies the second in the world.
The mycobacterium tuberculosis infection, typically refers to the crowd with active tuberculosis or latent tuberculosis infects,
It is also possible to healthy contactee.For body after infecting mycobacterium tuberculosis, having a small number of development is active tuberculosis, is shown corresponding
Clinical symptoms, such as fever, cough, expectoration, rabat be 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 as active tuberculosis.
Early diagnosis lungy is for effectively controlling the propagation of progress lungy and mycobacterium tuberculosis with important
Meaning.The method of current diagnosis tuberculosis infection is limited, mainly includes tuberculine skin experiment (TST), sputum smear, 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 infecting
It is the new method that developed recently gets up, can be used for diagnostic activities tuberculosis and tuberculosis latent infection.U.S. FDA batch is obtained
Quasi- listing T-SPOT.TB kit (Oxford Immunotec Limited, Aingdon, United Kingdom) and
QuantiFERON-TB Gold kit is all based on IGRA principle, the tuberculosis antigen specific protein encoded with the area RD1 gene
White -6KD Early insulin secretion targeting antigen (ESAT-6) and 10KD culture filtration albumen (CFP-10) are stimulus, by detecting periphery
The T lymphocyte of tuberculosis specificity release IFN-γ carrys out diagnosis of tuberculosis infection in blood, and susceptibility and specificity are all higher.
There are more shortcoming and defect, tuberculine skin experiments for the conventional method of current diagnosis tuberculosis infection
(TST) containing there are many (including pathogenic point of mycobacterial species 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, Bu Nengzhun
Really distinguish the PPD experimental result positive and be actually because BCG inoculation, the sensitization in contact environment after a variety of non-tuberculous mycobacterias also
It is caused by real mycobacterium tuberculosis infection.Sputum smear method is although simple and easy, but a big chunk tuberculosis patient phlegm
The inside might not have mycobacterium tuberculosis (so-called " phlegm yin " tuberculosis).So sputum smear training approach has diagnosis positive
The disadvantages of rate is low, be easy to miss inspection, the Sputum bacterial culture period is long, culture success ratio is low (only 80% or so).The nucleic acid of sputum specimen at
Sorting surveys the diagnosis predicament for not only facing " phlegm yin constipation core " but also program is complicated, is easy to appear false positive.Therefore, immunological technique
The important method just diagnosed at tuberculosis mycobacterial infections.
T-SPOT.TB kit and QuantiFERON-TB Gold kit are complex in composition, expensive, cost is high,
It seriously constrains it and is used for millions of active tuberculosis patients and hundreds of millions of tuberculosis latent infection crowd progress
Extensive tuberculosis screening and diagnosis.In addition, the Antigenic Peptide for including in these kits it is extremely complex and and non-principal basis in
The major histocompatibility antigen of compatriots carries out design, screening and the verifying of Antigenic Peptide, so applicability and using effect need
Further promoted.The Chinese patent application of 102516356 A of Publication No. CN filters out 8-11 company from antigen Rv3615c
Continuous polypeptide fragment is infected as stimulus using elisa test (ELISPOT) diagnosis of tuberculosis, but its sensitivity
Only 64%, the requirement of commercial kit is much not achieved.The Chinese patent application of 102297968 A of Publication No. CN is adopted
Antigenic stimulus object is the combination of 9 polypeptides of -6 antigen of mycobacterium tuberculosis ESAT-6 and CFP-10 antigen, and uses
The magnetic bead that five kinds of IFN-γ, TNF-α, IL-2, MIG and IP-10 antibody wrap up, while detecting five kinds of cells of cells and supernatant
The factor, this has resulted in the complexity of its detection method and interpretation of result, and needs to increase detection using flow cytometer
Cost, limit its popularization and use.The Chinese patent application of 103604933 A of Publication No. CN be based on TNF-α cell because
The kit of sub- ELISA detection active tuberculosis, the antigenic stimulus object which uses is that ESAT-6 and CFP-10 are whole
Albumen, however since the space conformation of whole albumen may hinder the identification of effective epitope Yu T lymphocyte surface
Molecule combines, and it contains other epitopes may generate negative regulation, influences the secretion of cell factor, causes this
Kit is poor to the diagnosis effect of latent infection tuberculosis.
Summary of the invention
The purpose of the present invention is to provide a kind of antigenic stimulus object for mycobacterium tuberculosis infection detection and contain this
The kit of antigenic stimulus object.Antigenic stimulus object provided by the present invention is capable of the peripheral blood T of effective stimulus tuberculosis infection patient
Lymphocyte generates IFN-γ, so as to highly sensitive and high special diagnosis of tuberculosis infection, and not by BCG be inoculated with or its
The influence of his underlying disease.
The present invention be reach its purpose, the technical solution adopted is as follows:
First aspect present invention provides a kind of antigenic stimulus object infected for detecting mycobacterium tuberculosis, and it includes such as sequences
At least one of polypeptide shown in sequence 1~11 polypeptide or its analog in list.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, NO.1~3 SEQ ID and NO.7~8 SEQ ID derive from tuberculosis specific antigen polypeptide ESAT-6,
NO.4~6 SEQ ID and NO.9~11 SEQ ID derive from tuberculosis specific antigen peptide C FP-10.
Preferably, antigenic stimulus object polypeptide as shown in sequence 1 in sequence table and sequence 7~8 or its analog group
At；Alternatively, shown antigenic stimulus object polypeptide shown in sequence 9~11 in sequence table or its analog form.
Highly preferred, antigenic stimulus object polypeptide shown in sequence 1~3 in sequence table or its analog form；
Alternatively, antigenic stimulus object polypeptide shown in sequence 4~6 in sequence table or its analog form.Using this preferred side
Formula, detection effect is best, and detection sensitivity is best, is up to 89% to the detection positive coincidence rate of the doubtful sample of tuberculosis infection,
Negative match-rate is up to 100%.Using preferred antigenic stimulus object, the sample of a wide range of volunteer is tested, Ke Yiyou
Effect avoids interference of the pulmonary infection caused by other non-tuberculous mycobacterias to result, to effectively identify 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 existing some patented technologies is about 60% or so, and (such sensitivity is being faced
Difficult normal use on bed).In addition, this patent kit can carry out tuberculosis screening to extensive unknown crowd, from embodiment 4
(Fig. 5, Fig. 6) as can be seen that screening 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 are generally investigated, the feature so as to be suitable for large-scale crowd
It is particularly important for carrying out tubercular's screening in China, and it diagnoses sensitivity and specificity is significantly better than existing patent
Technology.
The analog refers to that its characteristic is identical as the polypeptide, including it equally can with antibody specificity be combined,
This homeopeptide usually has at least 70% homology, preferably at least 90%, 95% homology.Its it is unusual 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,
The basic principle is that: it is first that the hydroxyl of the hydroxyl end amino acid for the peptide chain of being synthesized is same insoluble with the structure of covalent bond
Macromolecule resin is connected, and is then incorporated in the amino acid on solid phase carrier as moiety by sloughing amino protecting group using this
And with excessive activated carboxyl component reaction, spreading peptide chain.Repeat (condensation → washing → deprotection → neutralization and washing → next
Wheel condensation) operation, reach the peptide chain length to be synthesized, be finally cleaved peptide chain from resin, is handled by purifying etc.,
Up to desired polypeptide.Wherein alpha-amido is known as BOC solid-phase synthesis with what BOC (tertbutyloxycarbonyl) was protected, and alpha-amido is used
FMOC (9-fluorenylmethyloxycarbonyl) protection is known as FMOC solid-phase synthesis.
Antigenic stimulus object provided by the invention can be applied to the detection of mycobacterium tuberculosis infection, basic testing principle
Be: the antigenic stimulus object provided through the invention stimulates the T cell of mycobacterium tuberculosis host, then detects T cell release
Cell factor, to determine whether T cell identifies these polypeptides or the like, so that indirectly whether reflection host infected tuberculosis
Mycobacteria.
Antigenic stimulus object provided by the invention can be answered in reagent of the preparation for detecting mycobacterium tuberculosis infection
With.
The present invention also provides a kind of for detecting the kit of mycobacterium tuberculosis infection, and it includes as described above to resist
Primary stimuli object, and the Streptavidin and 3- amino -9- ethyl click of capture antibody, detection antibody, horseradish peroxidase-labeled
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.
It further include positive control stimulant and negative control reagent in kit, antigen selected by positive control is to big absolutely
The T cell of most individuals can generate response, and such as commercially available PHA (phytohemagglutin phytolectin), negative control is added without antigenic component, select
With culture medium or other buffers.
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 the cell factor of detection T cell secretion, determine that T cell is
The no identification antigenic stimulus object, 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 rodents such as mouse and rat.
The T cell is usually in vivo by the antigen presensitization from mycobacterium tuberculosis, 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 is also possible to CD8+T cell.In the method, may be used
It contacts T cell with peptide (antigenic stimulus object) in vitro or in vivo, and can determine whether T cell identifies in vitro or in vivo
Peptide.In general, by measuring state change or measurement T cell of the T cell in the presence of peptide whether in conjunction with peptide, to determine that T is thin
Whether born of the same parents identify the peptide.The state change of T cell can be T cell and start 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 the secretion of measurement IFN-γ.It usually can be by making these substances and specific knot
The presence that agent combined and detected the conjugate and the complex of these substances is closed, to detect the substance.Specificity knot
Closing agent is usually antibody, for example perhaps standard generally can be bought or use to polyclonal antibody from market to monoclonal antibody
Technology preparation.Measure cell factor method be usually common method in field, as ELISA (enzyme-linked immunosorbent assay),
ELISPOT (elisa test), 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 method.Coating in advance
IFN-γ monoclonal antibody on solid phase carrier first forms compound in conjunction with IFN-γ, the compound again with biotin labeling
The second IFN-γ antibody combine, then horseradish peroxidase-labeled Streptavidin Yu biotin specifically bind, most
Spot is formed by the chromogenic reaction of enzyme and substrate afterwards, to reflect the T cell quantity being activated.T cell described in method
It can be in-vitro separation, it is of course also possible to be untreated either intracorporal.In one embodiment, from periphery
Separate monocyte in blood or other samples, will include T cell and APC (antigen presenting cell), APC be it is a kind of can be by peptide submission
To the cell of T cell.In one embodiment, peptide itself is directly added into the experiment for containing T cell and APC.At this
In the test of sample, APC can be by peptide submission to T cell.When using by T cell identification without peptide by APC submission, APC is not
It is required.
The length of time that peptide is contacted with T cell can change according to for measuring peptide knowledge method for distinguishing.It is representative
, 10 are added in each experiment5~107Peripheral blood mononuclear cells (PBMC), preferably 2.5 × 105~106.When peptide is direct
When being added in experiment, concentration is 0.1~100 μ g/ml, preferably 1~10 μ g/ml.The typical time period that T cell and peptide are incubated with
It is 16~36 hours.
Antigenic stimulus object provided by the invention and kit containing this specific antigen stimulant, can be effectively in body
Outer detection mycobacterium tuberculosis infection, without being influenced by inoculation BCG (BCG vaccine), sensibility and specificity with higher,
It is not only suitable for the mycobacterium tuberculosis infection screening 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 screening
Polypeptide, in CHINESE REGION, using effect is more preferable, has high specific and highly sensitive, furthermore of the invention and foreign countries' kit phase
Than, it is cheap, more suitable for being promoted in China and developing country.
IFN-γ-ELISPOT kit provided by the invention in clinical application, through detect 55 mycobacterium tuberculosis height
Spend suspected infection person blood preparation, be diagnosed as mycobacterium tuberculosis infection patient 41, and through sputum smear, Sputum bacterial culture,
Rabat and clinical symptoms, which are integrated into, is diagnosed as patient 46 lungy, and positive coincidence rate is up to 89.1%；With of the invention
IFN-γ-ELISPOT kit is diagnosed as patient 9 of non-tuberculosis, through sputum smear, Sputum bacterial culture, rabat result and
Clinical symptoms diagnose all negative findings, and negative match-rate reaches 100%.Kit provided by the invention is in Diagnosis of Tuberculosis
In there is good clinical applicability, and specificity is good, sensitivity is high, high with the coincidence rates of other diagnostic techniques.
Detailed description of the invention
Fig. 1: for the advantage polypeptide verification result of IFN-γ-ELISPOT Diagnosis of Tuberculosis kit of the present invention: (A) this reagent
(spot number is IFN-γ-ELISPOT result after box tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3 stimulation cell
123)；(B) this kit tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.4~6 stimulates 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)；It (E) is not
The ground control of refinement born of the same parents.
Fig. 2: for the preferred polypeptide verification result of IFN-γ-ELISPOT Diagnosis of Tuberculosis kit of the present invention: A is more in Fig. 2
Peptide antigen SEQID NO.1 and the combined stimulation of SEQID NO.7~8 result (spot number is 160), B is this kit of this product knot
Core specific polypeptide Antigenic Peptide SEQ IDNO.9~11 stimulates result (spot number is 146) after cell, and C is negative control (spot
For number for 0), D be positive control (PHA) stimulus result (spot number is 783), and E is the ground control of cell to be not added (spot number is
0)。
Fig. 3: as an example of tuberculosis suspected patient this IFN-γ-ELISPOT kit diagnostic result: (A) negative control (spot
1) points is；(B) IFN-γ-ELISPOT result after the nonimmune advantage polypeptide stimulation cell of ESAT-6 (spot number is 5)；(C)
Tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3 stimulates the IFN-γ-ELISPOT result after cell (spot number is 22)；
(D) this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.4~6 stimulates the IFN-γ-ELISPOT result (spot after cell
19) points is；(E) IFN-γ-ELISPOT result after PHA positive control stimulation cell (spot number is 475).
Fig. 4: this IFN-γ-ELISPOT kit polypeptide sequence 1,7~11 of tuberculosis suspected patient examines as an example of shown
Disconnected result.(A) is negative control (spot number is 1) in Fig. 4, and Fig. 4 (B) is after the nonimmune advantage polypeptide of ESAT-6 stimulates cell
IFN-γ-ELISPOT result (spot number is 4), Fig. 4 (C) is tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1 and SEQID
NO.7~8 stimulates the IFN-γ-ELISPOT result (spot number is 38) after cell, and Fig. 4 (D) is that this product tuberculosis specificity is more
Peptide Antigenic Peptide SEQ ID NO.9~11 stimulates the IFN-γ-ELISPOT result (spot number is 43) after cell, and Fig. 4 (E) is PHA
Positive control stimulates the IFN-γ-ELISPOT result after cell (spot number is 330).
Fig. 5: for the result that using IFN-γ-ELISPOT kit of the present invention 37 volunteers are carried out with tuberculosis screening.
Fig. 6: to prompt for the tuberculosis positive shown by red block in Fig. 5 for the detection of wherein an example IFN-γ-ELISPOT tuberculosis
Result: (A) negative control (spot number be 1)；(B) IFN-γ-after the nonimmune advantage polypeptide stimulation cell of ESAT-6
ELISPOT result (spot number is 6)；(C) tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3 stimulates the IFN- after cell
γ-ELISPOT result (spot number is 17)；(D) this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.4~6 stimulation is thin
IFN-γ-ELISPOT result after born of the same parents (spot number is 9)；(E) IFN-γ-ELISPOT after PHA positive control stimulation cell
As a result (spot number is 466).
Specific embodiment
Technical scheme is described further with reference to the accompanying drawing.Experimental method in following embodiments, such as
It is the conventional method of the art without specified otherwise.Experimental material used in embodiment is city unless otherwise specified
Conventional biochemical reagent is sold, the % in embodiment is unless otherwise specified mass percentage.
Embodiment 1: the preparation of tuberculosis specific T-cells dominant antigen epitope polypeptide and IFN-γ-ELISPOT tuberculosis branch
The exploitation of bacillus infection diagnostic kit
T cell dominant antigen is designed and screened by bioinformatic analysis and T cells examined in vitro immune response
Epitope, comprising the following steps:
(1) by bioinformatics technique and associated databases such as ProPred, MacVestor, Preotean etc., to RD1
The T cell antigen epitope of the albumen ESAT-6 and CFP-10 of area's coding are predicted, while analyzing it between different HLA partings
Identification relationship.
(2) biological assays are combined, designed in Overlap (overlap) method and preferably go out 9 ESAT-6 is 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 are as follows: have cough, expectoration disease
Shape, rabat results abnormity, sputum smear or Sputum bacterial culture are positive, and receive the tuberculosis therapy time less than three weeks, and HIV detection is negative
Property.
(4) use Ficoll lymphocyte separation medium separating periphery blood monocytic cell (PBMC), PBMC through 5~10mlPBS or
After PRMI1640 is washed 2 times, being resuspended in the RPMI1640 culture medium containing 10% fetal calf serum (FBS, Life company), (Life is public
Department), and calculate cell quantity.
(5) ELISPOT method detection T cell receives the IFN-γ discharged after stimulation, specific steps are as follows: in 96 with pvdf membrane
People's IFN-γ monoclonal antibody (ebioscience company) is coated on orifice plate (Millipore company) overnight, RPMI1640+
Every hole is added 2.5 × 10 after 10%FBS closing5Phytohemagglutin phytolectin (PHA) is added in a cell and polypeptide, positive controls, negative
Culture medium is added in control group or cell is not added as ground control in the solubilising reagent for dissolving polypeptide, another set.Incubator
It is middle to be incubated for 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, 3-amino-9-ethyl carbazole (AEC) substrate colour developing, forms macroscopic punctation on pvdf membrane.As a result
It is read on ELISPOT plate reading machine.Result judgement: spot number >=10, and be greater than 2 times of negative control hole spot numbers and be judged as sun
Property result；Spot number < 10, or less than 2 times negative control hole spot numbers are judged as negative findings.
(6) it interpretation of result: according to polypeptide to the frequency of positive findings after tuberculosis patient stimulate the reaction, finally screens to obtain 3
NO.1~3 dominant antigen epitope polypeptide SEQ ID ESAT-6 (its amino acid sequence can be found in sequence 1~3 in sequence table), 2
NO.7~8 preferred antigens epitope polypeptide SEQ ID ESAT-6 (its amino acid sequence can be found in sequence 7~8 in sequence table), 3
NO.4~6 dominant antigen epitope polypeptide SEQ ID CFP-10 (its amino acid sequence can be found in sequence 4~6 in sequence table), 3
NO.9~11 preferred antigens epitope polypeptide SEQ ID CFP-10 (its amino acid sequence can be found in sequence 9~11 in sequence table).
Fig. 1 is a typical IFN-γ-ELISPOT result of 1~6 screening of polypeptide.Wherein Fig. 1 (A) is that this kit tuberculosis is special
Property the polypeptide antigen peptide SEQ ID combined stimulation cell of NO.1~3 after IFN-γ-ELISPOT result (spot number be 123), Fig. 1
It (B) is the IFN-γ-ELISPOT after this kit tuberculosis specific polypeptide Antigenic Peptide SEQ ID combined stimulation cell of 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 that cell is not added.Fig. 2 is a typical IFN-γ-of the polypeptide screening of sequence 1,7~11
ELISPOT result.Fig. 2A is that (spot number is the polypeptide antigen peptide SEQ ID NO.1 and SEQID combined stimulation of NO.7~8 result
160), Fig. 2 B be this kit of this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID combined stimulation cell of NO.9~11 after tie
Fruit (spot number is 146), Fig. 2 C are negative control (spot number is 0), and Fig. 2 D is positive control (PHA) stimulus result (spot number
For 783), Fig. 2 E is the ground control that cell is not added (spot number is 0).
Embodiment 2: the IFN-γ-ELISPOT mycobacterium tuberculosis infection diagnostic reagent kit that the present invention is developed includes:
(1), antigenic stimulus object, 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 company；
(3), antibody is detected, is the antibody of the anti-human IFN-γ of biotin labeling；
(4), the Streptavidin of horseradish peroxidase-labeled；
(5), 3-amino-9-ethyl carbazole (AEC) chromogenic substrate；
(6), positive control stimulant, for phytohemagglutin phytolectin (PHA)；
(7), negative control object is culture medium (such as RPMI1640+10%FBS) or the solubilising reagent for dissolving polypeptide
(such as DMSO).
Embodiment 3: IFN-γ-ELISPOT kit of the invention is applied to mycobacterium tuberculosis infection suspected patient
Clinical diagnosis
Purpose: examine IFN-γ-ELISPOT mycobacterium tuberculosis infection diagnostic reagent kit of the invention 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) use Ficoll lymphocyte separation medium separating periphery blood monocytic cell (PBMC), PBMC through 5~10mlPBS or
After PRMI1640 is washed 2 times, it is resuspended in the RPMI1640 culture medium containing 10% fetal calf serum (FBS), and calculate cell quantity.
(3) ELISPOT method detection T cell receives the IFN-γ discharged after stimulation, specific steps are as follows: in 96 with pvdf membrane
It is coated with people's IFN-γ monoclonal antibody on orifice plate to stay overnight, every hole is added 2.5 × 10 after RPMI1640+10%FBS closing5A cell
And polypeptide SEQ ID1~3 polypeptide (or preferred polypeptide combination, it is SEQ ID NO.1 polypeptide and SEQ ID NO.7~8 polypeptide
Combination) or ID4~6 polypeptide SEQ (or combination of NO.9~11 preferred polypeptide SEQ ID), positive controls addition plant blood
Solidifying element (PHA), negative control group are added 10%FBS/1640 culture medium or dissolve the solubilising reagent DMSO of polypeptide, and another set is not
Refinement born of the same parents are as ground control.It is incubated for 22 hours in incubator, the anti-human IFN-γ that biotin labeling is sequentially added after board-washing is anti-
Body, the Streptavidin of horseradish peroxidase-labeled, the colour developing of 3-amino-9-ethyl carbazole (AEC) substrate, the shape on pvdf membrane
At macroscopic punctation.As a result it is read on ELISPOT plate reading machine.Result judgement: spot number >=10, and it is greater 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 smear, Sputum bacterial culture, rabat with
And clinical symptoms are integrated into and are diagnosed as patient 46 lungy, are diagnosed as tying with IFN-γ-ELISPOT kit of the invention
The patient of core mycobacterial infections 41, therefore positive coincidence rate is 89.1%；It is diagnosed with this IFN-γ-ELISPOT kit
It is patient 9 of non-tuberculosis, through all negative knots of sputum smear, Sputum bacterial culture, rabat result and clinical symptoms diagnosis
Fruit, therefore negative match-rate reaches 100%.This IFN-γ-ELISPOT kit of tuberculosis suspected patient as an example of shown in Fig. 3
(the antigenic stimulus object that its used kit of the testing result contains is NO.1~3 polypeptide SEQ ID or polypeptide SEQ to diagnostic result
NO.4~6 ID).Fig. 3 (A) is negative control (spot number is 1), and Fig. 3 (B) is the nonimmune advantage polypeptide stimulation cell of ESAT-6
IFN-γ-ELISPOT result (spot number is 5) afterwards, Fig. 3 (C) is NO.1~3 tuberculosis specific polypeptide Antigenic Peptide SEQ ID
IFN-γ-ELISPOT result (spot number is 22) after combined stimulation cell, Fig. 3 (D) is that this product tuberculosis specific polypeptide is anti-
IFN-γ-ELISPOT result (spot number is 19) after the former peptide SEQ ID combined stimulation cell of NO.4~6, Fig. 3 (E) is PHA
Positive control stimulates the IFN-γ-ELISPOT result after cell (spot number is 475).Tuberculosis suspected patient as an example of shown in Fig. 4
The diagnostic result of preferred IFN-γ-ELISPOT kit (the antigenic stimulus object that its used kit of the testing result contains is
Polypeptide SEQ ID NO.1 and NO.7~8 SEQ ID are composed or NO.9~11 polypeptide SEQ ID are composed).Fig. 4 (A)
For negative control (spot number is 1), Fig. 4 (B) is the IFN-γ-ELISPOT after the nonimmune advantage polypeptide stimulation cell of ESAT-6
As a result (spot number is 4), Fig. 4 (C) is tuberculosis specific polypeptide SEQ ID NO.1 and SEQ ID NO.7~8 combined stimulation cell
IFN-γ-ELISPOT result (spot number is 38) afterwards, Fig. 4 (D) is this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID
IFN-γ-ELISPOT result (spot number is 43) after the combined stimulation cell of NO.9~11, Fig. 4 (E) is PHA positive control thorn
IFN-γ-ELISPOT result after swashing cell (spot number is 330).
Embodiment 4: the applicability of IFN-γ-ELISPOT kit of the invention in unknown crowd's tuberculosis screening
Purpose: examine IFN-γ-ELISPOT mycobacterium tuberculosis infection diagnostic reagent kit of the present invention (used in the embodiment
Kit, the antigenic stimulus object contained is combination or polypeptide SEQ ID NO.4~6 of NO.1~3 polypeptide SEQ ID
Combination) applicability, specificity and sensitivity in large-scale crowd tuberculosis screening.
(1) 37 negative volunteer blood's samples of HIV detection, every part of 2~3ml are collected.These inoculated BCG epidemic diseases per capita
Seedling.
(2) use Ficoll lymphocyte separation medium separating periphery blood monocytic cell (PBMC), PBMC through 5~10mlPBS or
After PRMI1640 is washed 2 times, it is resuspended in the RPMI1640 culture medium containing 10% fetal calf serum (FBS), and calculate cell quantity.
(3) ELISPOT method detection T cell receives the IFN-γ discharged after stimulation, specific steps are as follows: in 96 with pvdf membrane
It is coated with people's IFN-γ monoclonal antibody on orifice plate to stay overnight, every hole is added 2.5 × 10 after RPMI1640+10%FBS closing5A cell
And phytohemagglutin phytolectin (PHA) is added in 4~6 polypeptide of 1~3 polypeptide of sequence or sequence, positive controls, culture is added in negative control group
Cell is not added as ground control in base or the solubilising reagent for dissolving polypeptide, another set.It is incubated for 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
The colour developing of base carbazole (AEC) substrate, forms macroscopic punctation on pvdf membrane.As a result it is read on ELISPOT plate reading machine
It takes.Result judgement: spot number >=10, and be greater than 2 times of negative control hole spot numbers and be judged as positive findings；Spot number < 10
It is a, 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 kit is applied to unknown crowd
Fruit, red block is shown in Fig. 5 prompts for positive result (visible figure for the detection of wherein an example IFN-γ-ELISPOT tuberculosis
6), wherein Fig. 6 (A) is negative control (spot number is 1), and Fig. 6 (B) is after the nonimmune advantage polypeptide of ESAT-6 stimulates cell
IFN-γ-ELISPOT result (spot number is 6), Fig. 6 (C) are tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1~3 joint
IFN-γ-ELISPOT result (spot number is 17) after stimulating cell, Fig. 6 (D) is this product tuberculosis specific polypeptide Antigenic Peptide
IFN-γ-ELISPOT result (spot number is 9) after the SEQ ID combined stimulation cell of NO.4~6, Fig. 6 (E) is that the PHA positive is right
According to the IFN-γ-ELISPOT result (spot number is 466) after stimulation cell, which prompts volunteer's IFN-γ-
The detection of ELISPOT tuberculosis is positive, and also illustrating has 1 in 37 volunteer's samples be in that the detection of IFN-γ-ELISPOT tuberculosis is positive,
The volunteer is by further detection afterwards, it was demonstrated that is active tuberculosis mycobacterial infections, illustrates the IFN-γ-that the present invention develops
ELISPOT kit is applicable to the generaI investigation of large-scale crowd tuberculosis and not by BCG vaccine inoculation or other underlying disease etc.
It influences.It is tested in addition, also having carried out large-scale tuberculosis screening to 7~11 couples of preferred polypeptide sequence similar unknown crowds, hair
Existing preferred polypeptide sequence 7~11, which has, is similar to specificity and spirit that 1~6 pair of large-scale crowd of polypeptide sequence carries out tuberculosis screening
Quick property, details are not described herein.
The above described is only a preferred embodiment of the present invention, limitation in any form not is done to the present invention, therefore
All contents without departing from technical solution of the present invention, it is made to the above embodiment according to the technical essence of the invention any simply to repair
Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
Claims (4)
1. a kind of antigenic stimulus object, which is characterized in that the antigenic stimulus object is as shown in sequence 1 in sequence table and sequence 7~8
Polypeptide composition.
2. antigenic stimulus object described in claim 1 is in preparation for detecting the application in the reagent that mycobacterium tuberculosis infects.
3. a kind of for detecting the kit of mycobacterium tuberculosis infection, which is characterized in that it includes as described in claim 1
Antigenic stimulus object, and the Streptavidin and 3- amino -9- ethyl of capture antibody, detection antibody, horseradish peroxidase-labeled
Carbazole chromogenic substrate.
4. kit according to claim 3, which is characterized in that the capture antibody is that the monoclonal of anti-human IFN-γ is anti-
Body, the detection antibody are the antibody of the anti-human IFN-γ of biotin labeling.
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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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| 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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| CN102297968A (en) * | 2010-06-28 | 2011-12-28 | 程小星 | Kit for assisted diagnosis of tuberculosis |
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| GB0215710D0 (en) * | 2002-07-05 | 2002-08-14 | Isis Innovation | Diagnostic method |
| WO2004099771A1 (en) * | 2003-05-08 | 2004-11-18 | Statens Serum Institut | A new specific epitope based immunological diagnosis of tuberculosis |
| CN101493454A (en) * | 2008-01-21 | 2009-07-29 | 范雄林 | Tuberculosis antigen specific whole blood IFN-gamma diagnosis kit, method for producing the same and method for using same |
| CN101419237B (en) * | 2008-09-03 | 2012-10-24 | 深圳市东湖医院 | ELISA fleck diagnosis kit for tubercle bacillus infect and method for preparing specific antigen |
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| CN107102137A (en) | 2017-08-29 |
| CN107064498B (en) | 2019-03-22 |
| CN104597239B (en) | 2017-04-26 |
| WO2016095273A1 (en) | 2016-06-23 |
| CN104597239A (en) | 2015-05-06 |
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