CN104597239B - Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant - Google Patents
Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant Download PDFInfo
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- CN104597239B CN104597239B CN201410790793.1A CN201410790793A CN104597239B CN 104597239 B CN104597239 B CN 104597239B CN 201410790793 A CN201410790793 A CN 201410790793A CN 104597239 B CN104597239 B CN 104597239B
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Abstract
The invention provides an antigen stimulant for detecting mycobacterium tuberculosis infection, and a kit comprising the antigen stimulant. The invention also provides an application of the antigen stimulant in reagents for detecting mycobacterium tuberculosis infection. The antigen stimulant comprises at least one polypeptide or analogues thereof in polypeptides shown as the sequences 1-11 in a sequence table, wherein the polypeptides respectively come from tuberculosis specific antigen polypeptides ESAT-6 and tuberculosis specific antigen polypeptides CFP-10. According to the antigen stimulant provided by the invention, peripheral blood T lymphocytes of tuberculosis infection patients can be effectively stimulated to generate IFN-gamma, so that the tuberculosis infection can be diagnosed at high sensitivity and high specificity, and the influence on BCC inoculation or other underlying diseases can be avoided.
Description
Technical field
The invention belongs to biomedical inspection field, and in particular to a kind of antigen for m tuberculosis infection detection
Stimulant and its application and the kit containing the antigenic stimulus thing.
Background technology
Tuberculosis is the infectious disease of the class serious harm human health caused by Much's bacillus.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.According to estimates, the population in the whole world about 1/3 has infected Much's bacillus, and has 8,000,000 emerging infectious diseases every year
Example, has every year 2000000 people to die from tuberculosis.The tuberculosis epidemic situation and m tuberculosis infection situation of China is all quite serious, is
One of high burden country of 22, whole world tuberculosis, tuberculosis patient numerical digit occupies the second in the world.
Described m 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 Much's bacillus, shows corresponding
Clinical symptoms, such as heating, cough, expectoration, rabat be abnormal.Without corresponding clinical symptoms after most people infection, but machine
The Much's bacillus that body cannot clear all, as latent tuberculosis infects.Latent tuberculosis infects person is in hypoimmunity
When can develop into active tuberculosis.
It is lungy early diagnosis it is lungy for effective control progress and Much's bacillus propagation have it is important
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..Much's bacillus
T cell gamma interferon (IFN-γ) release test (interferon-gamma release assay, IGRA) that infection causes
It is 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 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
In vain -6KD Early insulin secretions targeting antigen (ESAT-6) and 10KD cultures filtration albumen (CFP-10) are stimulus, 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) containing many mycobacterial species (including pathogenic point in the Much's bacillus purified protein derivative (PPD) for being used
Branch bacillus, environment mycobacterium and BCG) common antigen molecule, therefore the poor specificity of PPD diagnosis of tuberculosis, it is impossible to it is accurate
Really distinguish the positive sensitization being because actually in BCG inoculations, contact environment after various non-tuberculous mycobacterias of PPD experimental results also
Caused by being real m tuberculosis infection.Sputum smears method is although simple, but a big chunk tuberculosis patient phlegm
The inside might not have Much's bacillus (so-called " phlegm is cloudy " tuberculosis).So, Sputum smears training approach has diagnosis positive
Rate is low, easy missing 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 survey not only faces the diagnosis predicament of " phlegm yin constipation core " but also program is complicated, false positive easily occur.Therefore, immunological technique
Just into the important method of m tuberculosis infection diagnosis.
T-SPOT.TB kits and QuantiFERON-TB Gold kit complicateds, expensive, cost are high,
Seriously constraining it and being used for millions of active tuberculosis patient and hundreds of millions of tuberculosis latent infection crowds is carried out
Extensive tuberculosis examination and diagnosis.In addition, the Antigenic Peptide included in these kits it is extremely complex and and non-principal according 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 Chinese patent application of the A of Publication No. CN 102516356 filters out 8-11 even from antigen Rv3615c
Continuous polypeptide fragment is infected as stimulus using ELISpot test (ELISPOT) diagnosis of tuberculosis, but its sensitivity
Only 64%, much do not reach the requirement of commercial kit.The Chinese patent application of the A of Publication No. CN 102297968 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 is used
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 the complexity of its detection method and interpretation of result, and needs to use flow cytometer, increased detection
Cost, limit it and promote the use of.The Chinese patent application of the A of Publication No. CN 103604933 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, yet with the space conformation of whole piece albumen the identification of effective epitope and T lymphocytic cell surfaces may be hindered
Molecule is combined, and it contains other epitopes and may produce negative regulation, affects the secretion of cell factor, causes 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 a kind of for the antigenic stimulus thing of m tuberculosis infection detection and containing 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 such that it is able to highly sensitive and high special diagnosis of tuberculosis infection, and not be inoculated with or it by BCG
The impact of his underlying disease.
The present invention is to reach its purpose, and the technical scheme of employing is as follows：
First aspect present invention provides a kind of antigenic stimulus thing for detecting m tuberculosis infection, and it is included such as sequence
At least one polypeptide or its analog in polypeptide in list shown in sequence 1～11.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.
Preferably, polypeptide or its analog group of the antigenic stimulus thing by shown in sequence in sequence table 1 and sequence 7～8
Into；Or, polypeptide or its analog of the shown antigenic stimulus thing shown in sequence in sequence table 9～11 is constituted.
Highly preferred, polypeptide or its analog of the antigenic stimulus thing shown in sequence in sequence table 1～3 is constituted；
Or, polypeptide or its analog of the antigenic stimulus thing shown in sequence in sequence table 4～6 is constituted.Using this preferred side
Formula, its Detection results are optimal, and detection sensitivity is optimal, and to the detection positive coincidence rate of the doubtful sample of tuberculosis infection 89% is up to,
Negative match-rate is up to 100%.Using preferred antigenic stimulus thing, the sample of volunteer on a large scale is tested, Ke Yiyou
Effect avoids interference of the pulmonary infection to result caused by other non-tuberculous mycobacterias, so as to effectively recognize Much's bacillus 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
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 1 tubercular can go out from examination in 37 unknown humans, exclude tumour, other pulmonary infections,
The interference of the diseases such as influenza, the specificity with height and sensitivity, such that it is able to generally investigate suitable for large-scale crowd, the feature
It is particularly important for tubercular's examination is carried out in China, and its diagnosis sensitivity is significantly better than existing patent with specificity
Technology.
The analog, refers to that its characteristic is identical with the polypeptide, including it equally can be combined with antibody specificity,
This homeopeptide generally has at least 70% homology, and preferably at least 90%, 95% homology.Its is unusual to come from
The replacement of amino acid residue, insertion, disappearance and modify, can occur on the N-terminal of sequence, C-terminal or other any positions.
Polypeptide of the present invention can be synthesized by existing chemical synthesis, it is also possible to by those skilled in the art
The technique for gene engineering grasped is prepared, and a kind of representative method is that aforementioned polypeptides are obtained with solid-phase synthesis,
Its general principle is：It is first that the hydroxyl to be synthesized the hydroxyl end amino acid of peptide chain is same insoluble with the structure of covalent bond
Macromolecule resin is connected, and is then passed through as moiety using this amino acid combined on solid phase carrier and sloughs amino protecting group
And with excessive activated carboxyl component reaction, spreading peptide chain.Repeat (be condensed → washing → deprotect → neutralize and wash → next
Wheel condensation) operation, the peptide chain length to be synthesized is reached, finally peptide chain is cleaved from resin, process through purifying etc.,
Obtain final product desired polypeptide.What wherein alpha-amido BOC (tertbutyloxycarbonyl) was protected is referred to as BOC solid-phase synthesis, 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 applicable to the detection of m tuberculosis infection, its basic Cleaning Principle
It is：The antigenic stimulus thing provided by the present invention, stimulates the T cell of Much's bacillus host, then detects T cell release
Cell factor, to determine whether T cell recognizes these polypeptides or the like, so as to indirectly reflect whether host infected tuberculosis
Mycobacterium.
The antigenic stimulus thing that the present invention is provided can be carried out in the reagent for detecting m tuberculosis infection is prepared should
With.
The present invention also provides a kind of kit for detecting m tuberculosis infection, and it is anti-that it includes 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
Most individual T cells 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 Much's bacillus host contacts, and by the cell factor for detecting T cell secretion, determines that T cell is
It is no to recognize the antigenic stimulus thing, so as to indirectly reflect whether host infected Much's bacillus.
The host refers to people or other mammals, other mammals such as primate, ox, sheep, pig, little
The rodent such as mouse and rat.
, generally in vivo by the antigen presensitization from Much's bacillus, these are by antigen sensibilization for described T cell
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 cells, or CD8+T cells.In the process,
T cell can be made and contacted with peptide (antigenic stimulus thing) in vitro or in vivo, and can in vitro or in vivo determine whether T cell recognizes
Peptide.Generally, whether the state change or measure T cell by measure T cell in the presence of peptide is combined to determine that T is thin with peptide
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, such as cell factor, 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 with specific knot
Close agent to combine and detect the conjugate with the presence of the complex of these materials to detect described material.Specificity knot
It is usually antibody, such as monoclonal antibody or polyclonal antibody to close agent, and standard typically can be bought or used from market
It is prepared by technology.Determine cell factor method be typically in field commonly use method, such as ELISA (EUSA),
ELISPOT (ELISpot 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 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 combine, then the Streptavidin of horseradish peroxidase-labeled and biotin specifically bind, most
Afterwards spot is formed by the chromogenic reaction 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 or internal.In one embodiment, from periphery
Monocyte is separated in blood or other samples, will be that one kind can be by peptide submission including T cell and APC (antigen presenting cell), APC
To the cell of T cell.In one embodiment, during peptide to be directly added in itself 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 the need for the peptide by APC submissions, APC is not
It is required.
The time length that peptide is contacted with T cell can according to for determine peptide know method for distinguishing and change.It is representative
, in each experiment 10 are added5～107PMBC (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.
Antigenic stimulus thing and the kit containing this specific antigen stimulant that the present invention is provided, can effectively in body
Outer detection m tuberculosis infection, and do not affected by inoculation BCG (BCG vaccine), with higher Sensitivity and Specificity,
It is not only suitable for the m tuberculosis infection examination that m tuberculosis infection clinical diagnosis is also applied for large-scale crowd.This
The kit of bright offer is designed according to Chinese's major histocompatibility complex, examination checking Much's bacillus dominant antigen
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 promoting in China and developing country.
IFN-γ-ELISPOT the kits that the present invention is provided in clinical practice, 55 Much's bacillus height after testing
Degree suspected infection person's blood preparation, is diagnosed as the patient 41 of m tuberculosis infection, and Jing Sputum smears, Sputum bacterial culture,
Rabat and clinical symptoms are integrated into being diagnosed as patient lungy 46, 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, Jing 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 with good clinical applicability, and specific good, sensitivity is high, high with the coincidence rate of other diagnostic techniques.
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
Box tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1～3 stimulate the IFN-γ-ELISPOT results after cell, and (spot number is
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
Plus the ground control of cell.
Fig. 2：For the preferred polypeptide the result of IFN-γ-ELISPOT Diagnosis of Tuberculosis kits of the present invention：, A is in Fig. 2
Polypeptide antigen SEQID NO.1 are this kit of this product with SEQID NO.7～8 combined stimulation result (spot number is 160), B
Tuberculosis specific polypeptide Antigenic Peptide SEQ IDNO.9～11 stimulate result after cell (spot number is 146), and C is negative control (spot
Count as 0), D is positive control (PHA) results of stimulation (spot number is 783), 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：It is shown
For the diagnostic result of this IFN-γ-ELISPOT kits peptide sequence 1,7～11 of a tuberculosis suspected patient.In 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 is 4), Fig. 4 (C) is that tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1 stimulate thin with SEQID NO.7～8
IFN-γ-ELISPOT results (spot number is 38) after born of the same parents, Fig. 4 (D) is this product tuberculosis specific polypeptide Antigenic Peptide SEQ ID
NO.9～11 stimulate the IFN-γ-ELISPOT results after cell (spot number is 43), and Fig. 4 (E) is that PHA positive controls stimulate carefully
IFN-γ-ELISPOT results after born of the same parents (spot number is 330).
Fig. 5：It is the result that using IFN-γ-ELISPOT kits of the present invention 37 volunteers are carried out with tuberculosis examination.
Fig. 6：It is positive tuberculosis to be prompted for shown by red block in Fig. 5 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).
Specific embodiment
Technical scheme is described further below in conjunction with the accompanying drawings.Experimental technique in following embodiments, such as
Without specified otherwise, the conventional method of the art is.Experiment material used if no special instructions, is city in embodiment
Routine biochemistry reagent is sold, the % in embodiment if no special instructions, is weight/mass percentage composition.
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
By bioinformatic analysis, and T cells examined in vitro immune response is designing and screen T cell dominant antigen
Epi-position, comprises the following steps：
(1) by bioinformatics technique and associated databases such as ProPred, MacVestor, Preotean etc., to RD1
The T cell antigen epi-position of albumen ESAT-6 and CFP-10 of area's coding is predicted, while analyzing it between different HLA partings
Identification relation.
(2) biological assays are combined, with the design of Overlap (overlap) method and preferably to go out 9 ESAT-6 more
Peptide and 11 CFP-10 polypeptides, each polypeptide includes 9～22 amino acid.
(3) tuberculosis patient blood preparation, per 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 medium separating periphery blood monocytic cells (PBMC), 5～10mlPBS of PBMC Jing or
After PRMI1640 washs 2 times, (Life is public to be resuspended in the RPMI1640 culture mediums containing 10% hyclone (FBS, Life company)
Department), and calculate cell quantity.
(5) IFN-γ that ELISPOT methods detection T cell discharges after receiving to stimulate, concretely comprises the following steps：With pvdf membrane 96
People's IFN-γ monoclonal antibody (ebioscience companies) is coated with orifice plate (Millipore companies) overnight, RPMI1640+
The addition 2.5 × 10 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, another set to be not added with cell as ground control.Incubator
Middle incubation 22 hours, sequentially adds the anti-human IFN-γ antibody of biotin labeling, the chain of horseradish peroxidase-labeled 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 it is judged as negative findings less than 2 times of negative control hole spot numbers.
(6) interpretation of result：According to frequency of the polypeptide to positive findings after tuberculosis patient IR, 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
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).Figure
1 is a typical IFN-γ-ELISPOT result of the examination of polypeptide 1～6.Wherein Fig. 1 (A) is this kit tuberculosis specificity
IFN-γ-ELISPOT results (spot number is 123) after the combined stimulation cell of polypeptide antigen peptide SEQ ID NO.1～3, Fig. 1
(B) it is 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) is negative control (spot number is 0), and for positive (PHA) control, (spot number is Fig. 1 (D)
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 polypeptide antigen peptide SEQ ID NO.1 and SEQID NO.7～8 combined stimulation result
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 m tuberculosis infection the diagnostic kits that the present invention is developed, comprising：
(1), antigenic stimulus thing, shown in sequence 1-11 in the amino acid sequence such as sequence table that filters out in embodiment 1
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, be purchased from ebioscience companies；
(3), detection antibody, 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 m tuberculosis infection suspected patient
Clinical diagnosis
Purpose：IFN-γ-ELISPOT m tuberculosis infection the diagnostic kits of the inspection present invention are in Diagnosis of Tuberculosis
Clinical applicability, specificity, sensitivity and the coincidence rate with other diagnostic techniques.
(1) 55 Much's bacillus height suspected infection person's blood preparations, per part of 2～5ml are collected.
(2) with Ficoll lymphocyte separation medium separating periphery blood monocytic cells (PBMC), 5～10mlPBS of PBMC Jing or
After PRMI1640 washs 2 times, the RPMI1640 culture mediums containing 10% hyclone (FBS) are resuspended in, and calculate cell quantity.
(3) IFN-γ that ELISPOT methods detection T cell discharges after receiving to stimulate, concretely comprises the following steps：With pvdf membrane 96
People's IFN-γ monoclonal antibody is coated with orifice plate overnight, every hole adds 2.5 × 105 cells after RPMI1640+10%FBS closings
And polypeptide SEQ ID1～3 polypeptide (or preferred polypeptide combination, it is SEQ ID NO.1 polypeptides and SEQ ID NO.7～8 polypeptide
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 the solubilising reagent DMSO of 10%FBS/1640 culture mediums or dissolving polypeptide, and another set is not
Plus cell is 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 be judged as less than 2 times of negative control hole spot numbers
Negative findings.
(4) interpretation of result：In 55 doubtful m tuberculosis infection patients, Jing Sputum smears, Sputum bacterial culture, rabat with
And clinical symptoms are integrated into being diagnosed as patient lungy 46, with the IFN-γ-ELISPOT kits of the present invention knot is diagnosed as
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, all negative knots of Jing 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 the testing result its used kit 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 result of preferred IFN-γ-ELISPOT kits (the antigenic stimulus thing that the testing result its used kit contains is
Polypeptide SEQ ID NO.1 are combined with SEQ ID NO.7～8 or polypeptide SEQ ID NO.9～11 combine).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 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 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
IFN-γ-ELISPOT results after sharp 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：Inspection IFN-γ-ELISPOT m tuberculosis infection diagnostic kits of the present invention are (used by the embodiment
Kit, combination or polypeptide SEQ ID NO.4～6 of the antigenic stimulus thing that it contains 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, per part of 2～3ml are collected.These inoculated BCG epidemic diseases per capita
Seedling.
(2) with Ficoll lymphocyte separation medium separating periphery blood monocytic cells (PBMC), 5～10mlPBS of PBMC Jing or
After PRMI1640 washs 2 times, the RPMI1640 culture mediums containing 10% hyclone (FBS) are resuspended in, and calculate cell quantity.
(3) IFN-γ that ELISPOT methods detection T cell discharges after receiving to stimulate, concretely comprises the following steps：With pvdf membrane 96
People's IFN-γ monoclonal antibody is coated with orifice plate overnight, the addition 2.5 × 10 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 is added cultivates
The solubilising reagent of base or dissolving polypeptide, another set is 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 develops 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 it is judged as negative findings less than 2 times of negative control hole spot numbers.
(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 stimulate after cell
IFN-γ-ELISPOT results (spot number is 6), Fig. 6 (C) is tuberculosis specific polypeptide Antigenic Peptide SEQ ID NO.1～3 joint
Stimulate the IFN-γ-ELISPOT results after cell (spot number is 17), 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,
Afterwards the volunteer is through further detection, it was demonstrated that for active tuberculosis mycobacterial infections, illustrate IFN-γ that the present invention develops-
ELISPOT kits are applicable to the generaI investigation of large-scale crowd tuberculosis and not by BCG vaccine inoculation or other underlying diseases etc.
Affect.In addition, 7～11 couples of similar unknown crowds of preferred polypeptide sequence have been also carried out with large-scale tuberculosis examination experiment, send out
Existing preferred polypeptide sequence 7～11 has specificity and the spirit that tuberculosis examination is carried out similar to 1～6 pair of large-scale crowd of peptide sequence
Quick property, will not be described here.
The above, is only presently preferred embodiments of the present invention, and any pro forma restriction is not done to the present invention, therefore
All contents without departing from technical solution of the present invention, any simply repair according to the technical spirit of the present invention to made for any of the above embodiments
Change, equivalent variations and modification, still fall within the range of technical solution of the present invention.
Claims (4)
1. a kind of antigenic stimulus thing, it is characterised in that polypeptide group of the antigenic stimulus thing by shown in sequence in sequence table 1～3
Into.
2. application of the antigenic stimulus thing described in claim 1 in the reagent for detecting m tuberculosis infection is prepared.
3. a kind of kit for detecting m tuberculosis infection, it is characterised in that it is included 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 is that the monoclonal of anti-human IFN-γ resists
Body, the detection antibody is the antibody of the anti-human IFN-γ of biotin labeling.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710119273.1A CN107102137A (en) | 2014-12-17 | 2014-12-17 | Antigenic stimulus thing, kit and its application for detecting mycobacterium tuberculosis infection |
| CN201710119615.XA CN107121546B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
| CN201710122036.0A CN107064498B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
| CN201410790793.1A CN104597239B (en) | 2014-12-17 | 2014-12-17 | Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant |
| PCT/CN2014/095543 WO2016095273A1 (en) | 2014-12-17 | 2014-12-30 | Antigen stimulant for detecting mycobacterium tuberculosis infection, kit, and applications of antigen stimulant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410790793.1A CN104597239B (en) | 2014-12-17 | 2014-12-17 | Antigen stimulant and kit for detecting mycobacterium tuberculosis infection, and application of antigen stimulant |
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| CN201710122036.0A Division CN107064498B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
| CN201710119615.XA Division CN107121546B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
| CN201710119273.1A Division CN107102137A (en) | 2014-12-17 | 2014-12-17 | Antigenic stimulus thing, kit and its application for detecting mycobacterium tuberculosis infection |
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| CN104597239A CN104597239A (en) | 2015-05-06 |
| CN104597239B true CN104597239B (en) | 2017-04-26 |
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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 |
| 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 |
| CN201710122036.0A Expired - Fee Related CN107064498B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
| 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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| CN201710122036.0A Expired - Fee Related CN107064498B (en) | 2014-12-17 | 2014-12-17 | For detecting antigenic stimulus object, kit and its application of mycobacterium tuberculosis infection |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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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| 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 |
| AU2009294850B2 (en) * | 2008-09-22 | 2015-07-02 | Oregon Health & Science University | Methods for detecting a Mycobacterium tuberculosis infection |
| CN102297968B (en) * | 2010-06-28 | 2013-10-30 | 程小星 | Kit for assisted diagnosis of tuberculosis |
| CN102608333B (en) * | 2012-03-30 | 2013-06-05 | 中国科学院微生物研究所 | Tuberculosis diagnostic composition and application thereof |
| CN103604933A (en) * | 2013-11-27 | 2014-02-26 | 华中科技大学同济医学院附属同济医院 | Kit for detecting active tuberculosis based on antigen-specific TNF-alpha-ELISA (enzyme linked immunosorbent assay) and application thereof |
| CN103760345B (en) * | 2014-01-17 | 2016-03-02 | 北京旷博生物技术股份有限公司 | A kind of kit and application thereof utilizing peripheral blood detection m tuberculosis infection |
| CN104020297B (en) * | 2014-06-10 | 2016-12-07 | 上海交通大学医学院 | Test kit for m tuberculosis infection detection and clinical therapeutic efficacy monitoring and application thereof |
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2014
- 2014-12-17 CN CN201410790793.1A patent/CN104597239B/en active Active
- 2014-12-17 CN CN201710119615.XA patent/CN107121546B/en not_active Expired - Fee Related
- 2014-12-17 CN CN201710122036.0A patent/CN107064498B/en not_active Expired - Fee Related
- 2014-12-17 CN CN201710119273.1A patent/CN107102137A/en active Pending
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| Publication number | Publication date |
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| CN107102137A (en) | 2017-08-29 |
| CN107064498B (en) | 2019-03-22 |
| CN107064498A (en) | 2017-08-18 |
| WO2016095273A1 (en) | 2016-06-23 |
| CN107121546A (en) | 2017-09-01 |
| CN104597239A (en) | 2015-05-06 |
| CN107121546B (en) | 2019-03-22 |
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