CN107449909B - A kind of protein chip and kit for active tuberculosis diagnosis - Google Patents
A kind of protein chip and kit for active tuberculosis diagnosis Download PDFInfo
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Abstract
The invention discloses a kind of protein chips and kit for active tuberculosis diagnosis.Contain the specific antibody for 29 kinds of albumen on protein chip.The protein chip can specifically distinguish the tuberculosis patient after active tuberculosis patient and healthy population, non-tuberculous pneumonia patient and healing, and sensitivity with higher and specificity, high throughput, sample dosage are few, are convenient for common lab popularization and scale；The application in clinical diagnosis is conducive to faster more accurately find active tuberculosis patient simultaneously, it is expected to be used for the sieving and diagnosis of active tuberculosis and making a definite diagnosis for active tuberculosis, the physical examination of healthy population, the forecast assessment etc. of tuberculosis therapy curative effect provides strong technical support for the control of epidemic situation lungy.
Description
Technical field
The present invention relates to field of biomedicine technology more particularly to a kind of protein chip examinations for active tuberculosis diagnosis
Agent box.
Background technique
The World Health Organization in 2015 statistics indicate that, tuberculosis is the death as caused by single pathogen after the AIDS
The most infectious disease of number.How to reduce tuberculosis epidemic situation is that city's economy and society develop the key subjects faced.It causes to tie
The epidemic situation was severe that one of chief reason is a lack of effective early diagnosis technology for core disease.
For diagnosis of tuberculosis technology, go in century more than one, our today clinically still continue to use it is earliest
Diagnosis of tuberculosis technology: tulase smear and tulase culture.Although easy to operate, price economy, the sensibility of smear is not
More than 30%；The sensibility of culture is no more than 40%, and is always diagnosis because being the basis of strain idenfication and drug sensitive test
Goldstandard lungy, but it needs time-consuming 1-2 months, and many patients have begun treatment 2 months, or even longer time,
Tulase cultivation results can be obtained.Tulase diagnosis of molecular biology reagent, the especially research and development of gene Xpert are tuberculosis
Diagnosis provides important new technology.However, clinically considerable active tuberculosis patient does not have sputum specimen.Therefore, using base
In the etiological diagnosis technology of sputum specimen, even if all technologies all use, sensibility is not more than 50%.In other words,
The patient for having more than 50% can not find the aetology foundation of diagnosis of tuberculosis.
The undesirable wherein more prominent problem of diagnosis of tuberculosis method is the mark due to lacking specificity.It is many at present
Research team is dedicated to finding new breakthrough in terms of host, including new biological identification is found, is used for active tuberculosis
Diagnosis.Recently protein biochip technology is more highly sensitive because having, and can detect up to several hundred or even thousands of kinds of targets simultaneously
Albumen or polypeptide, are now widely used in molecular biology field and clinical detection, such as individualized treatment, drug development,
The research in the fields such as medical diagnosis on disease, prognosis detection, in the disease researches such as tumour, disease of cardiovascular system, neurological disease
Using.However, being used to the research such as diagnose or treat using the molecular marker of protein chip screening tuberculosis disease-specific, do not have also at present
There is relevant report.
Summary of the invention
Seriousness for existing tuberculosis epidemic situation and the deficiency to existing diagnostic technology, skill to be solved by this invention
Art problem is to provide a kind of highly sensitive, high specific protein chip kit for active tuberculosis diagnosis.
To achieve the above object, a kind of protein chip for active tuberculosis diagnosis of the present invention, on the protein chip
Contain the specific antibody for being directed to following albumen: integrin sample metalloproteinases and fibrin ferment 8, carbohydrate sulfotransferase
4, cytotoxic t lymphocyte-associated antigen 4, Cystatin E/M, folacin receptor 2, granular leukocyte colony stimulation
Biotic factor, hyaluronic acid proteoglycans connecting protein 1, interferon-' alpha '/beta receptor 2, insulin-like growth factor binding protein 5,
It is interleukin 1 R6, interleukin 11, IL-12p40, IL-21 R, Interleukin-23, white
Cytokine -2 9, interleukin 7, Kallikrein 14, lymphatic vessel endothelial hyaluronic acid receptor -1, matrix metal egg
White enzyme, marrow hematopoietic cell inhibiting factor, Platelet-derived growth factor A B, Platelet-derived growth factor BB, leucocyte are situated between
Plain -1 receptor 4, tissue factor approach inhibition factor, tumor necrosis factor b1, thrombomodulin, tissue metal proteases
Inhibitor -2, tumor necrosis factor b, thymic stromal lymphopoietin.
Protein chip of the present invention for active tuberculosis diagnosis, is prepared by this step: will contain 0.02-2ng
Claim 1 described in specific antibody PBS buffer solution 100-1000pl and two kinds of various concentrations positive control with entirely from
Point sample instrument point sample is moved on slide；The positive control of every kind of antibody and two kinds of various concentrations has 4 weights in each chip
It is multiple；The good slide of point sample is put in and is stood overnight under room temperature, is then evacuated in drier 2 hours dry；After drying
Slide loads onto matched 16 hole frame and one slide is divided into 16 non-interfering cells；After adhesive film closed frame,
It is saved backup after whole chip is encapsulated with air-locked pouch in 2 DEG C to 8 DEG C, the PBS buffer solution is to contain 0.01-
The PBS solution of 10g/100ml bovine albumin；The positive control is the ox IgG of biotin labeling.
A kind of protein chip kit for active tuberculosis diagnosis of the present invention, contains above-mentioned protein chip.
In actual operation, serial different albumen marks are prepared into through gradient dilution after the standard items mixture of freeze-drying being redissolved
The mixed liquor of will object concentration, for making the standard curve of multiple sandwich ELISA method.After the polypeptide stimulant of freeze-drying is redissolved
It is diluted to the mixed liquor of certain concentration in proportion, is used as the differential stimulus of sample.
Kit of the present invention is able to detect 29 common albumen, can specifically by active tuberculosis patient with
Tuberculosis patient after healthy population, non-tuberculous pneumonia patient and healing distinguishes, and sensitivity with higher and spy
It is anisotropic, sample dosage is few, can promote in common lab and the advantages that scale.Application of the present invention in clinical diagnosis is advantageous
In faster more accurately finding active tuberculosis patient, the sieving and diagnosis for being expected to be used for active tuberculosis and active tuberculosis are really
It examines, the physical examination of healthy population, the forecast assessment etc. of tuberculosis therapy curative effect.
The present invention has screened a series of albumen specifically expressed on tuberculosis patient using protein chip, and will sieve
Protein combination is selected into small chip, is used for diagnosis lungy.The present invention is superior to other to detect multiple protein simultaneously
Technology, demand specimen amount is extremely low, (normal healthy controls, non-tuberculous differentiating active tuberculosis patient and inactive tuberculosis crowd
Pneumonia patient and cure after tuberculosis patient) realize preferable sensibility and specificity the advantages of.For it is lungy early diagnosis and
Early treatment provides new method.
Protein chip kit of the present invention is by the detection method similar to sandwich method ELISA, with the channel containing Cy3
Laser scanner slide that reaction is terminated be scanned imaging, select suitable Laser Scanning Parameters to make on chip highest letter
Number close to saturation, gained image is stored as tiff file.Then it is converted the fluorescence signal of each point to chip reading software
Digital signal.The signal of each protein marker is drawn by the digital signal of the standard sample of the protein marker of gradient dilution
With concentration standard curve, concentration of each albumen in unknown sample is then calculated by corresponding standard curve.
Protein chip of the present invention is post-stimulatory through tuberculosis specific polypeptide mixture using sample detected
Culture supernatant.Mixtures of polypeptides is that polypeptide sequence derives from the polypeptide fragment of CFP-10 and ESAT-6 protein sequence synthesis by equivalent
Mixing.Culture medium and specific polypeptide mixture (10ug/ml) is added in the whole blood sample of collection, after mixing well, is put into 36 DEG C
It is cultivated in carbon dioxide incubator, after culture 16 hours, 2000 revs/min are centrifuged 5 minutes, extract supernatant.
Protein chip kit of the present invention for active tuberculosis diagnosis, has the characteristics that and advantage:
(1) on the composition of chip, the present invention uses the coated normal structure slide of active epoxy group, and experiment shows
The coated slide of active epoxy group can more effectively adsorb coated antibody on the surface of chip, and make the stability of antibody
Increase.
(2) before chip point sample use specific frame structure, optimize chip dot matrix so that sample can batch detection,
Easy to operate, sample dosage is small, non-cross pollution.
(3) in the preparation process of chip, controlled at 70-75F, humidity 40-45%, such chip can change
The form of kind point sample, keeps distribution of the antibody on slide more uniform.Meanwhile to be conducive to antibody more effective for the overnight stand of room temperature
Ground is fixed on slide.In addition to this, vacuum suction is dried 2 hours and with the plastic pouch air extracting seal of vapour resistant and in 4
DEG C save.Experiment shows that the chip saved in this way makes antibody more effectively be fixed on chip surface, and effectively
Increase the stability that chip saves.
(4) on the dot matrix of chip microarray, the present invention uses double positive controls of various concentration, and considers while using
The varying strength signal of the two standardizes different microarrays.Experiment shows that the present invention can significantly improve the quick of chip
Sensitivity and repeatability.
(5) to realize kit in the specificity of diagnostic activities tuberculosis, the mixing of tuberculosis specific polypeptide is added in the present invention
Object handles sample, and the sample of protein chip detection is protein ingredient different in the post-stimulatory blood plasma through specific polypeptide.It is real
It tests and shows that the present invention can be the tuberculosis patient after active tuberculosis patient and healthy population, non-tuberculous pneumonia patient and healing
It distinguishes, and sensitivity with higher and specificity.
Protein chip kit is diagnosed using active tuberculosis of the invention, kit of the present invention is able to detect 29
A common albumen, can be specifically by active tuberculosis patient and healthy population, non-tuberculous pneumonia patient and after curing
Tuberculosis patient distinguishes, and sensitivity with higher and specificity, high-throughput, sample dosage is few, can be in routine experimentation
The advantages that room popularization and scale.Application of the present invention in clinical diagnosis is conducive to faster more accurately find active tuberculosis
Patient, is expected to be used for the sieving and diagnosis of active tuberculosis and making a definite diagnosis for active tuberculosis, the physical examination of healthy population, and tuberculosis therapy is treated
The forecast assessment etc. of effect provides strong technical support for the control of epidemic situation lungy.
Specific embodiment
The embodiment of the present invention is described below in detail, the examples of the embodiments are intended to be used to explain the present invention, and cannot
It is interpreted as limitation of the present invention.In the examples where no specific technique or condition is specified, described according to the literature in the art
Technology or conditions or carried out according to product description.Reagents or instruments used without specified manufacturer is that can lead to
Cross the conventional products of commercially available acquisition.
Embodiment 1: the determination of protein chip point sample condition
1, the point sample concentration of antibody is captured: by capture antibody (specific antibody of 29 kinds of albumen) in different buffers
(sterile water, PBS buffer solution, the PBS of the bovine albumin containing various concentration, the PBS buffer solution etc. of the glycerol containing various concentration) is with 2x's
Then concentration dilution compares its activity and stabilization with untouchable point sample instrument point in chip surface with sandwich ELISA method
Property.Experiment shows to dilute capture antibody with best linear rate with containing 0.01-10g/100ml bovine albumin PBS buffer solution
And stability.
2, the condition control of point sample: at room temperature capture antibody (specific antibody of 29 kinds of albumen) directly point in slide
When surface, the point for capturing antibody is easy to produce hollow dots, and different antibodies point activity difference is larger, and is ultimately producing
Image often finds to have trailing phenomenon.It is 70-75F that the present invention, which controls point sample temperature, and humidity is by the antibody put when 40-45%
Point has best shape, and it was found that and the good slide of point sample is put in and is stood overnight under room temperature, second day vacuum suction
It is 2 hours dry, then the whole air-locked pouch of chip is encapsulated, the slide capture antibody prepared in this way has most
Good activity and it can at least stablize six months or more at 4 DEG C.
Embodiment 2: the preparation of the protein chip kit of the present invention for active tuberculosis diagnosis.
In order to which, with the presence or absence of 29 kinds of albumen in the present invention, preparation, which is fixed with, is directed to following 29 kinds of albumen in test sample
Specific antibody slide: integrin sample metalloproteinases and fibrin ferment 8 (ADAM8), carbohydrate sulfotransferase 4
(CHST4), cytotoxic t lymphocyte-associated antigen 4
(CTLA4), Cystatin E/M (Cystatin E/M), folacin receptor 2 (FOLR2), granulocyte
Colony-stimulating biotic factor (GM-CSF), hyaluronic acid proteoglycans connecting protein 1 (HAPLN1), interferon-' alpha '/beta receptor 2
(IFNab R2), insulin-like growth factor binding protein 5 (IGFBP-5), interleukin 1 R6 (IL-1R6), leucocyte are situated between
- 11 (IL-11) of element, IL-12p40 (IL-12p40), IL-21 R (IL-21R), Interleukin-23
(IL-23), IL-29 (IL-29), interleukin 7 (IL-7), Kallikrein 14 (Kallikrein 14),
Lymphatic vessel endothelial hyaluronic acid receptor -1 (LYVE-1), matrix metalloproteinase (MMP-8), marrow hematopoietic cell inhibit because
Son (MPIF-1), Platelet-derived growth factor A B (PDGF-AB), Platelet-derived growth factor BB (PDGF-BB), leucocyte
- 1 receptor 4 (IL-1R4) of interleukin, tissue factor approach inhibition factor (TFPI), tumor necrosis factor b1 (TGFb1), blood plasma thrombus
Regulatory protein (Thrombomodulin), tissue inhibitor of metalloproteinase -2 (TIMP-2), tumor necrosis factor b (TNFb),
Thymic stromal lymphopoietin (TSLP).
1, the source of antibody:
Using the corresponding capture antibody of the specific proteins for listed protein in table 1 and detect antibody purposes,
Source, concentration are described in detail in table 1.The specific method is as follows: by detection antibody to be marked in a large amount of 1xPBS buffer (1
PBS of the milliliter antibody at 1 liter) in dialysis three times, at least 6 hours every time.Every milligram of antibody addition 80 is pressed after measuring antibody concentration
Microgram biotin DMSO solution mixes, and reacts 4 hours at room temperature.It is carried out with antibody of the PBS solution to biotin labeling
Analysis, removes free biotin and demarcates the detection antibody concentration of biotin labeling.
1 specific antibody title of table, the purposes of antibody, source, concentration information table
2, the preparation and preservation of protein chip
The PBS buffer solution of the specific antibody containing 0.02-2ng of 100-1000pl (is contained into 0.01-10g/100ml ox
Albumin) with full-automatic point sample instrument point sample on slide.The ox IgG of biotin labeling is as positive control.Every kind of antibody is every
There are 4 repetitions and the positive control of two kinds of various concentrations there are 4 repetitions in each chip in a chip.By point sample
Good slide is put in be stood overnight under room temperature, is then evacuated in drier 2 hours dry.Slide after drying, which is loaded onto, matches
One slide is divided into 16 non-interfering cells by 16 hole frames of set.After adhesive film closed frame, whole chip
It is saved backup with after the encapsulation of air-locked pouch in 2 DEG C to 8 DEG C.
Wherein, in the present embodiment, full-automatic point sample instrument is the product of Scienion company of Germany production；Slide is U.S.'s health
Peaceful Products.Certainly, in the above-mentioned steps of inventive technique scheme, the use of instrument and material is not limited to the present embodiment
Enumerate, but to be able to solve technical problem of the invention, and realize that corresponding technical effect is foundation.
3, the source of the protein standard substance detected:
It is described in detail in table 2 using the title of listed recombinant protein and source in table 2 is directed to:
The title of recombinant protein, source table in 2 protein standard substance of table
According to a certain amount after using the phosphate buffer containing 0.1% bovine albumin to dilute above each recombinant protein
It mixes, with freeze-drying drying and in -80 DEG C of preservations after packing.In the present embodiment, for doing standard curve use
Every kind of recombinant protein it is final using concentration it is as shown in table 4.But in fact, in other embodiments, for doing standard curve
Recombinant protein concentration can select different sections, it is not limited to the example of table 4.
Embodiment 3: the preparation of the mixtures of polypeptides of peripheral blood is stimulated
For guarantee stimulation polypeptide specificity, according to the sequence of two polypeptides of CFP-10 and ESAT-6 protein design, in detail
Carefully it is shown in Table 3.Peptide systhesis is completed by Shenzhen Han Yu biotech company, and Purity is more than 95%.
The source of 3 polypeptide of table and and sequence table
Embodiment 4: in the post-stimulatory blood supernatant of protein chip quantitative detection specific polypeptide of kit of the invention
The experiment of 29 kinds of protein expression levels.
1, peripheral blood stimulation and culture and the collection of supernatant
The sample diluting liquid (phosphate buffer of 0.1% bovine albumin) of 1.1 500 μ L of addition arrives mixtures of polypeptides (table 3
Polypeptide 1 and the equal mass mixings of polypeptide 2) tubule in, re-dissolve.It before opening tubule, is first rapidly centrifuged, lightly up and down
Lash dissolved powders.
1.2 anticoagulant heparin venous blood 250ml, are placed in sterile streaming pipe, and 250ml serum free medium is added and (reaches
Section is biotech company, DKW34-EHK0400) and different stimulated object: DMSO (10ug/ml, negative control pipe) and tuberculosis are special
Specific polypeptide mixture (10ug/ml, polypeptide stimulation pipe), after mixing well, is put into 36 DEG C of carbon dioxide incubators and cultivates.Culture
After 16 hours, 2000 revs/min are centrifuged 5 minutes, extract supernatant.
Polypeptide stimulates pipe to be completed by Shenzhen Han Yu biotech company, and Purity is more than 95%.
2, slide chip is completely dried
Slide chip is taken out from box, after equilibrium at room temperature 20-30min, packaging bag is opened, opens sealing
Then chip is placed on vacuum desiccator or drying at room temperature 1-2 hours by item.
3, the gradient dilution of protein standard substance
3.1 addition 500 μ L sample diluting liquid (phosphate buffer of 1M) to standard items mixture (be shown in Table 4 ingredient and
Concentration) tubule in, re-dissolve standard items.It before opening tubule, is first rapidly centrifuged, lightly lashes dissolved powders up and down,
Marking this tubule is Std1.
3.2 respectively 6 clean centrifuge tubes of label be Std2, Std3 to Std7, add 200 μ L sample diluting liquid to often
In a tubule.
The Std1 of 3.3 100 μ L of extraction, which is added in Std2, to be gently mixed, and 100 μ L are then extracted from Std2 and are added to Std
In 3, such gradient dilution to Std7.
3.4 extract the sample diluting liquid of 100 μ L into another new centrifuge tube, are labeled as CNTRL, as negative right
According to.
Note: because the initial concentration of every kind of albumen is different, after the gradient dilution of Std1 to Std7, each albumen
Series of concentrations is different.In this example, the concentration of gradient recombinant protein dilution is as shown in table 4.
Table 4 is after gradient dilution for making the concentration table (unit: Pg/ml) of the recombinant protein standard items of standard curve
4, chip operation process
The sample diluting liquid of 4.1 each 100 μ L of Kong Zhongjia is incubated for 30 minutes on room temperature shaker, closes Quantitative Western chip.
4.2 pump the sample diluting liquid in each hole, the titer and sample for adding 100 μ L respectively into different holes,
It is incubated overnight for 4 DEG C on shaking table.It will test sample dilution 1:1 dilution.
4.3 cleaning
The standard items or sample in each hole are pumped, IX washing lotion I (PBST, below herewith) is cleaned 5 times, each room 5min
Warm shaking table concussion, the IX washing lotion I of every 150 μ L of hole, cleaning will be cleaned washing lotion every time, dilute 20X washing lotion I with deionized water.
The IX washing lotion I in each hole is pumped, IX washing lotion II (PBS, below herewith) is added and cleans 2 times, each room 5min
Warm shaking table concussion, the IX washing lotion II of every 150 μ L of hole, cleaning will be cleaned washing lotion every time, dilute 20X washing lotion II with deionized water.
The incubation of 4.4 detection antibody mixed liquors
Centrifugation detection antibody mixed liquor tubule, then be added 1.4ml sample diluting liquid, after mixing again quickly from
The heart.The detection antibody of 80 μ L is added into each hole, is incubated for 2 hours on room temperature shaker.
4.5 cleaning
The detection antibody in each hole is pumped, IX washing lotion I is cleaned 5 times, each 5min room temperature shaker concussion, every 150 μ L of hole
I X washing lotion I, cleaning will be cleaned washing lotion every time, I X washing lotion II is then added and cleans 2 times, the shake of each 5min room temperature shaker
It swings, the IX washing lotion II of every 150 μ L of hole, cleaning will be cleaned washing lotion every time.
The incubation of 4.6Cy3- Streptavidin
Be centrifuged Cy3_ Streptavidin tubule, then be added 1.4ml sample diluting liquid, after mixing again quickly from
The heart.The Cy3- Streptavidin of 80 μ L is added into each hole, slide is encased with aluminium-foil paper and is protected from light incubation, be incubated on room temperature shaker
I hour.
4.7 cleaning
The Cy3_ Streptavidin in each hole is pumped, I X washing lotion I is cleaned 5 times, each 5min room temperature shaker concussion, often
The IX washing lotion I of 150 μ L of hole, cleaning will be cleaned washing lotion every time.
4.8 fluorescence detection
I) slide frame is dismantled, not touch the one side of slide printing antibody with hand carefully.
Slide is placed in glass slide cleaning pipe, the IX washing lotion I of about 30ml is added, slide can be entirely covered, in room temperature
15min is shaken on shaking table, discards IX washing lotion I, adds the IX washing lotion II of about 30ml, 5min is shaken on room temperature shaker.
Remove the residual washing lotion of slide.Slide is placed in glass slide cleaning pipe/drying tube, not lid lid, in 1000rpm
It is centrifuged 3min.
Using laser scanner such as Innopsys scanning signal, using Cy3 or green channel (stimulating frequency=
532nm)。
The data of 4.9 chips are extracted and carry out data analysis with analysis software.
1) fluorescent value of biochip is read with Mapix software.13 (row) x12 (column) of the microarray parameter of chip,
The diameter 120um of point.
2) numerical value selected after reading is the median reading (F532Median- for removing local background
LocalBackground).It is bent come the standard for doing each recombinant protein with specific quantitatively chip software for calculation QAH-CUST-SW
Line.
It is done before the concentration for calculating different sample protein markers using two positive controls point identical on each chip
The standardization of reference system progress data.The signal value of two positive controls about differs 4 times.Before being standardized first
Calculate the positive control value POS=(POSl+4*POS2)/2 of each chip.Then all data are marked with the value again
Quasi-ization processing: corrected value=original value * (sample average POS)/POS.
5. Data Processing in Experiment
5.1, the concentration of 29 kinds of albumen in each sample is calculated by standard curve
By taking a clinical serum sample experiments result as an example, the fluorescence reading difference of eight standard samples is as shown in table 5.
Table 5: prominent photoreading (F532Median-Local Background) table of the standard items of three times gradient dilution
By taking IL-29 as an example, such as table of the relationship between the protein concentration of IL-29 and fluorescence signal is learnt according to the information of table 4 and 5
Shown in 6.
Relation table between the protein concentration and fluorescence signal of table 6:IL-29
Standard curve is drawn by table 6, calculates to obtain r2=0.9933, regression equation y=1.7556X
It is tested No. 1 Sample Negative control tube (1-1), signal strength 2619, calculating IL-29 content is 4597.92pg/
ml。
It is tested No. 1 sample stimulus pipe (1-2), signal strength 1984, calculating IL-29 content is 3483.11pg/ml.
5.2, in each sample 29 protein concentrations conversion
Various kinds is originally obtained respectively in negative control pipe and positive control pipe supernatant after the concentration of 29 albumen, advanced before analysis
Row data conversion, conversion formula are as follows: log2 (x+1).X is the concentration that each sample corresponds to albumen, by the stimulation pipe numerical value after conversion
The numerical value of negative control pipe is subtracted to get the variation multiple (foldchange, FC) after stimulated with negative control: log2FC is gone out.
By taking above-mentioned IL-29 as an example, stimulate pipe log2 (x+1)=11.77, negative control pipe log2 (x+1)=12.17, phase
Subtracting rear numerical value is -0.40.
5.3, the judgement of testing result
After the Concentration Testing of albumen in each sample handles data according to above-mentioned mode, 29 in obtained each sample
The detectable concentration of albumen establishes machine learning model lasso trick/elasticity pessimistic concurrency control (LASSO) by R linguistic algorithm packet Glmnet,
Then the result (being indicated with R) of protein combination conversion is obtained.
The type of interpretation sample.Judgment criteria are as follows: as R > 0.35, be then judged as active tuberculosis.Conversely, being then non-live
Dynamic property tuberculosis.
Embodiment 6: the sensibility and specificity in resolution active tuberculosis of kit of the invention
In order to which test kit detection is in the efficiency for differentiating active tuberculosis, had detected not using kit of the invention
Same clinical sample judges it in the sensibility and specificity for differentiating active tuberculosis.
It is collected after being clinically diagnosed as active tuberculosis patient, normal healthy controls, non-tuberculous pneumonia patient and healing respectively
After tuberculosis patient (each 40) venous blood, stimulated using DMSO and mixtures of polypeptides (mixtures of polypeptides described in embodiment 3), then
Culture supernatant is collected, the expression of 29 albumen is detected using kit, the knot of each sample is finally calculated after substitution formula
Fruit and judging result, result value are greater than 0.35 and are diagnosed as active tuberculosis.
It is detected in 40 active tuberculosis patients using this kit as the result is shown, 34 are judged as active tuberculosis, very
Positive rate is 85%, illustrates that the protein chip detection method of this discovery is specific well；120 inactive tuberculosis of detection
In patient, 10 are judged as active tuberculosis, and false positive rate 8.33% illustrates that the protein chip detection method of this discovery is fine
Sensibility.
7 different type clinical sample testing result table of table
In conclusion the invention discloses a kind of protein chip kits diagnosed with active tuberculosis.The kit makes
It uses normal structure slide as topical carrier, the reaction of multiple sandwich ELISA can be completed in surface of glass slide.The activity of exploitation
Diagnosis of Tuberculosis protein chip diagnostic kit can detect 29 kinds of albumen simultaneously on a protein chip.Kit detection 29
The sensitivity and specificity of kind albumen can achieve the level of single-factor ELISA.In addition, being ground by the combination to 29 albumen
Having sent out one kind can be used for diagnostic activities protein chip diagnostic kit lungy.This kit is suitable for from people periphery
The expression of multiple specific proteins is detected in blood, for early diagnosis lungy, the judgement for the treatment of and the screening of crowd.
It should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than to the scope of the present invention
Limitation, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should understand that, can
With modification or equivalent replacement of the technical solution of the present invention are made, without departing from the spirit and scope of technical solution of the present invention.
SEQUENCE LISTING
<110>Shenzhen's galaxy Biotechnology Co., Ltd, Guangzhou Ray Biotechnology Co., Ltd.
<120>a kind of protein chip and kit for active tuberculosis diagnosis
<130> 1610836
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 92
<212> PRT
<213>artificial synthesized
<400> 1
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<211> 95
<212> PRT
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Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
20 25 30
Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser
35 40 45
Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu
50 55 60
Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly
65 70 75 80
Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala
85 90 95
Claims (3)
1. a kind of protein chip for active tuberculosis diagnosis, which is characterized in that the antibody on protein chip is for as follows
The specific antibody of albumen: integrin sample metalloproteinases and fibrin ferment 8, carbohydrate sulfotransferase 4, cytotoxic T
Lymphocyte-associated antigen 4, Cystatin E/M, folacin receptor 2, the granular leukocyte colony stimulating organism factor, thoroughly
Bright matter acid albumin glycan connecting protein 1, interferon-' alpha '/beta receptor 2, insulin-like growth factor binding protein 5, interleukins-
1R6, interleukin 11, IL-12p40, IL-21 R, Interleukin-23, interleukins-
29, interleukin 7, Kallikrein 14, lymphatic vessel endothelial hyaluronic acid receptor -1, matrix metalloproteinase, marrow
Hematopoietic cell inhibiting factor, Platelet-derived growth factor A B, Platelet-derived growth factor BB, interleukin 1 receptor 4,
Tissue factor approach inhibition factor, tumor necrosis factor b1, thrombomodulin, tissue inhibitor of metalloproteinase -2,
Tumor necrosis factor b and thymic stromal lymphopoietin.
2. the protein chip described in claim 1 for active tuberculosis diagnosis, which is characterized in that be prepared by this step:
By the sun of the PBS buffer solution 100-1000pl of specific antibody described in the claim 1 containing 0.02-2ng and two kinds of various concentrations
Property control with full-automatic point sample instrument point sample on slide；The positive control of every kind of antibody and two kinds of various concentrations is in each chip
There are 4 repetitions；The good slide of point sample is put in and is stood overnight under room temperature, it is small that dry 2 are then evacuated in drier
When；Slide after drying loads onto matched 16 hole frame and one slide is divided into 16 non-interfering cells；Use adhesive film
After closed frame, saved backup after whole chip is encapsulated with air-locked pouch in 2 °C to 8 °C, the PBS buffer solution
For the PBS solution containing 0.01-10g/100ml bovine albumin；The positive control is the ox IgG of biotin labeling.
3. a kind of protein chip kit for active tuberculosis diagnosis, which is characterized in that contain egg described in claim 1
White chip.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710613324.6A CN107449909B (en) | 2017-07-25 | 2017-07-25 | A kind of protein chip and kit for active tuberculosis diagnosis |
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| CN201710613324.6A CN107449909B (en) | 2017-07-25 | 2017-07-25 | A kind of protein chip and kit for active tuberculosis diagnosis |
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| CN107449909A CN107449909A (en) | 2017-12-08 |
| CN107449909B true CN107449909B (en) | 2019-01-04 |
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| CN201710613324.6A Active CN107449909B (en) | 2017-07-25 | 2017-07-25 | A kind of protein chip and kit for active tuberculosis diagnosis |
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Families Citing this family (2)
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| CN108593939B (en) * | 2018-07-18 | 2023-11-14 | 瑞博奥(广州)生物科技股份有限公司 | Full-automatic protein chip and application thereof |
| CN109825567B (en) * | 2018-12-27 | 2022-06-10 | 博奥生物集团有限公司 | CircRNA biomarker for pneumonia diagnosis, application, primer and reagent |
Family Cites Families (4)
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| GB201213567D0 (en) * | 2012-07-31 | 2012-09-12 | Proteinlogic Ltd | Biomarkers |
| CN103869068B (en) * | 2012-12-18 | 2016-03-09 | 广州瑞博奥生物科技有限公司 | A kind of antibody chip kit for kinds of tumors diagnosis |
| CN103954754A (en) * | 2014-04-30 | 2014-07-30 | 广东省结核病控制中心 | Immunologic diagnosis kit of active tuberculosis |
| CN104459129A (en) * | 2015-01-05 | 2015-03-25 | 复旦大学附属华山医院 | Diagnostic kit for distinguishing active and latent mycobacterium tuberculosis infection |
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Address after: 518112 705, kelente R & D building, 1 ganliwu Road, Buji science and technology new town, Bulan Road, Longgang District, Shenzhen City, Guangdong Province Patentee after: SHENZHEN XINGXI BIOTECHNOLOGY Co.,Ltd. Patentee after: Reboo (Guangzhou) Biotechnology Co.,Ltd. Address before: 518112 705, kelente R & D building, 1 ganliwu Road, Buji science and technology new town, Bulan Road, Longgang District, Shenzhen City, Guangdong Province Patentee before: SHENZHEN XINGXI BIOTECHNOLOGY Co.,Ltd. Patentee before: RAYBIOTECH, Inc. |
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