CN110514841A - Kit and protein chip for the diagnosis of tuberculosis latent infection - Google Patents
Kit and protein chip for the diagnosis of tuberculosis latent infection Download PDFInfo
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- CN110514841A CN110514841A CN201910655757.7A CN201910655757A CN110514841A CN 110514841 A CN110514841 A CN 110514841A CN 201910655757 A CN201910655757 A CN 201910655757A CN 110514841 A CN110514841 A CN 110514841A
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Abstract
The present invention provides a kind of kits and protein chip for the diagnosis of tuberculosis latent infection, by detection interleukin-6, tumor necrosis factor receptor I I, epiregulin and interferon inducible protein 10 these four albumen expression, tuberculosis latent infection person and healthy population can be distinguished, and sensitivity and specificity with higher.The corresponding specific antibody of these four albumen is fixed on substrate, protein chip is prepared into, the expression of four kinds of albumen can be detected simultaneously, and has the advantages that sample requirement amount is low, simple, quick.Application in the molecular biology research and clinical detection of tuberculosis latent infection is conducive to the early diagnosis of tuberculosis latent infection, can more accurately find tuberculosis latent infection person.
Description
Technical field
The invention belongs to field of biomedicine technology, in particular to a kind of kit for the diagnosis of tuberculosis latent infection
And protein chip.
Background technique
Tuberculosis is the infectious disease that the death toll as caused by single pathogen is most after AIDS, causes tuberculosis
The epidemic situation was severe that one of chief reason is a lack of effective early diagnosis technology for disease.
It is not that whole people can fall ill after tubercle bacillus affection, a part of crowd can lapse to as latent infection state, that is, tie
Core latent infection person.Persistently there is tulase, but the control due to immunity of organisms to tulase in vivo in this part population,
There is no clinical manifestations.When immunity of organisms decline after, hide the intracorporal tulase of machine will " revivable ", in vivo
Tulase can replicate again, finally be changed into tuberculosis patient.Latent infection person has 10% chance to be changed into tuberculosis in life
People.Tulase has been infected in the population of Chinese about a quarter, if can this part population be observed or be intervened, energy
Greatly reduce generation lungy.But at present tuberculosis infection also lack specificity diagnostic method, clinically have antibody and
Cellular immunity detection, cellular immunity inspection includes PPD skin test and IGRA technology.PPD skin test is easier, but has with BCG
Cross reaction, false positive rate is relatively high, and particularly in China in the case where extensive bcg vaccination (BCG), service efficiency is bright
It is aobvious limited.What IGRA technology detected is the expression of tuberculosis specificity IFNg, although specificity significantly improves, the current party
Method is relatively bothersome and time-consuming, and price is all relatively expensive, the serious popularization and use limited clinically.Therefore, having must
A kind of highly sensitive, high specific method and product for tuberculosis latent infection person diagnosis is provided.
Summary of the invention
Based on this, the purpose of the present invention is to provide a kind of highly sensitive, high specific for tuberculosis latent infection person
The kit of diagnosis.
To achieve the above object, specific technical solution of the present invention is as follows:
Detect the examination of interleukin-6, tumor necrosis factor receptor I I, epiregulin and interferon inducible protein 10
Application of the agent in kit of the preparation for the diagnosis of tuberculosis latent infection.
A kind of kit for the diagnosis of tuberculosis latent infection, including being capable of quantitative detection interleukin-6, tumour
The reagent of mecrosis factor receptors II, epiregulin and interferon inducible protein 10.
The present invention also provides a kind of protein chip for the diagnosis of tuberculosis latent infection, specific technical solution is as follows:
A kind of protein chip for the diagnosis of tuberculosis latent infection includes: substrate and fixed catching on the substrate
Obtain antibody；
The capture antibody includes: for the specific antibody of interleukin-6, for tumor necrosis factor receptor I I
Specific antibody, the specific antibody for epiregulin and the specific antibody for interferon inducible protein 10；
The substrate is the substrate for being coated with active epoxy group.
The present invention also provides a kind of preparation method of protein chip for the diagnosis of tuberculosis latent infection, particular techniques
Scheme is as follows:
A kind of preparation method of the protein chip for the diagnosis of tuberculosis latent infection, comprising the following steps:
Active epoxy group is coated on the substrate of protein chip；
Capture antibody is fixed on the substrate of the protein chip；
The capture antibody includes: for the specific antibody of interleukin-6, for tumor necrosis factor receptor I I
Specific antibody, the specific antibody for epiregulin and the specific antibody for interferon inducible protein 10.
Based on the above-mentioned technical proposal, the invention has the following advantages:
It was found by the inventors of the present invention that being adjusted by detection interleukin-6, tumor necrosis factor receptor I I, epidermis
The expression of element and these four albumen of interferon inducible protein 10 can distinguish tuberculosis latent infection person and healthy population
It comes, and sensitivity and specificity with higher.The corresponding specific antibody of these four albumen is fixed on substrate, is made
It is standby to detect the expression of four kinds of albumen simultaneously at protein chip, and have sample requirement amount low, simple, efficiently
Advantage.Application in the molecular biology research and clinical detection of tuberculosis latent infection is conducive to the morning of tuberculosis latent infection
Phase diagnosis, can more accurately find tuberculosis latent infection person.
Detailed description of the invention
Fig. 1 is the spotted area distribution schematic diagram of protein chip.
Specific embodiment
To facilitate the understanding of the present invention, it below with reference to embodiment to invention is more fully described, is given below
Presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to be retouched herein
The embodiment stated.Purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.Ying Li
Solution, in the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or is built according to manufacturer
The condition of view.Used various common agents, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff it is identical.It is specific to be intended merely to description for used term in the description of the invention
Embodiment purpose, it is not intended that in limitation the present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
A kind of protein chip for the diagnosis of tuberculosis latent infection of the invention includes: substrate and being fixed on the base
The capture antibody of on piece；
The capture antibody includes: for the specific antibody of interleukin-6, for tumor necrosis factor receptor I I
Specific antibody, the specific antibody for epiregulin and the specific antibody for interferon inducible protein 10.
It optionally, also include positive control on the protein chip.Preferably, the positive control is is fixed on
State the calf IgG on substrate.
Preferably, the positive control can be the calf IgG of various concentration.It is furthermore preferred that the calf IgG makes a living
The calf IgG of object element label.
Preferably, the substrate is the substrate for being coated with active epoxy group.The coated slide of active epoxy group can be with
More effectively absorption coated antibody is on the surface of chip, and increases the stability of antibody.
It preferably, include several non-interfering spotted areas on the substrate.
It is highly preferred that several described non-interfering spotted areas are obtained by detachable type framework apart.It is adopted before point sample
With specific frame structure, optimize chip dot matrix so that sample can batch detection, easy to operate, sample dosage is small, mutually not
Cross contamination.
It is further preferred that including 16~32 non-interfering point sample cells on the substrate.
As shown in Figure 1, point sample has the specific antibody for interleukin-6, for tumour in each point sample cell
The specific antibody of mecrosis factor receptors II, for epiregulin specific antibody and be directed to interferon inducible protein 10
Specific antibody and two kinds of various concentrations positive control calf IgG.
Specifically, every strain specific antibodies repeats point sample 4 times, and the positive control calf IgG of two kinds of various concentrations is also repeated
Point sample 4 times.
In some other embodiment, the number for repeating point sample can according to need reasonable set, such as repeatedly point sample
3~10 times.
A kind of preparation method of protein chip for the diagnosis of tuberculosis latent infection of the invention, comprising the following steps:
Capture antibody is fixed on the substrate of protein chip；
The capture antibody includes: for the specific antibody of interleukin-6, for tumor necrosis factor receptor I I
Specific antibody, the specific antibody for epiregulin and the specific antibody for interferon inducible protein 10.
Preferably, the capture antibody be fixed on the substrate of protein chip the following steps are included:
By the solution point sample of 100-1000pl capture antibody on the substrate, after standing overnight at room temperature, it is dried in vacuo
1-4 hours；
Bovine albumin containing 0.02-20ng capture antibody and 0.01-10g/100ml in the solution of the capture antibody.
Wherein, the overnight stand of room temperature is conducive to antibody and is more effectively fixed on slide.
Preferably, vacuum drying time is 1.5-2.5 hours.
It is highly preferred that the condition of the point sample are as follows: temperature 70-75F, humidity 40-45%.It, can under the conditions of such point sample
In the form of improving point sample, keep distribution of the antibody on slide more uniform.
Further, it after vacuum suction is dry, saves with the plastic pouch air extracting seal of vapour resistant and in 0-8 DEG C.It is excellent
Selection of land is saved in (4 ± 0.5) DEG C.The chip saved in this way makes antibody more effectively be fixed on chip surface,
And effectively increase the stability that chip saves.
It preferably, include several non-interfering spotted areas on the substrate of the protein chip, by hole detachable type
Frame separates to obtain.
Preferably, the preparation method further include: the capture antibody of positive control is fixed on to the base of the protein chip
On piece, the positive control capture antibody is calf IgG.
Preferably, the positive control can be the calf IgG of various concentration.It is furthermore preferred that the calf IgG makes a living
The calf IgG of object element label.
Preferably, the present invention uses double positive controls of various concentration.It is marked simultaneously using the varying strength signal of the two
The different microarray of standardization can significantly improve the susceptibility and repeatability of chip.
It is highly preferred that the method that double positive controls are used to standardize microarray are as follows:
First calculate the positive control signal value POS=(POSl+4 × POS2)/2 of each microarray；Wherein POS1 is the
The signal value of the positive control of one concentration, POS2 are the signal value of the positive control of the second concentration；
Then all data are standardized with positive control signal value POS again: corrected value=original value ×
(sample average signal value POSave)/positive control signal value POS.
A kind of protein chip kit for the diagnosis of tuberculosis latent infection of the invention includes:
Protein chip as described above or the protein chip being prepared by preparation method as described above, and detection are anti-
Body；
The detection antibody includes: for the specific antibody of interleukin-6, for tumor necrosis factor receptor I I
Specific antibody, the specific antibody for epiregulin and the specific antibody for interferon inducible protein 10；
Wherein, for the different epitopes of the detection antibody of same protein and capture antibody difference identification of protein.
Preferably, the detection antibody has biotin, HRP or colloid gold label.
Preferably, the kit also includes: the Streptavidin of protein standard substance and/or fluorescein Cy3 label.
It is highly preferred that the protein standard substance is interleukin-6, tumor necrosis factor receptor I I, epiregulin and interferon
The mixture of inducible protein 10.
Wherein, in specific embodiments of the present invention, full-automatic point sample instrument is the product of Scienion company, Germany production；
The substrate of protein chip uses normal structure slide, and slide is Corning Incorporated's product.In some other embodiment,
Other feasible point sample instruments and substrate can also be used.
The protein relevant to latent infection is combined of embodiment 1
Take respectively sex hormone binding globulin (SHBG), interleukin 2 (IL-2), interleukin-6 (IL-6),
Interleukin 8 (IL-8), tumor necrosis factor receptor I I (TNF RII), epiregulin (Epiregulin), interferon
Inducible protein 10 (IP-10), serpin (SERPINA3), tumor necrosis factor a (TNFa) protein standard
Product after gradient dilution, by its fluorescent value of fluorescence spectrometry in the prior art, obtain standard curve fluorescence signal value and right
Concentration is answered, as shown in table 1.
1 protein standard substance standard curve fluorescence signal value of table and corresponding concentration
By upper table data, concentration and fluorescence signal intensity are made into Logarithm conversion respectively, standard curve is drawn, calculates to obtain line
Property regression equation, wherein x is Log (standard concentration), and Y is Log (fluorescence signal intensity), specific such as table 2.
2 protein standard substance standard curve of table
| Regression equation | R2 | |
| SHBG | Y=41.91x-8946.5 | 0.97 |
| IL2 | Y=1.4909x-1135.8 | 0.95 |
| IL-6 | Y=0.9522x+2.1817 | 0.97 |
| IL8 | Y=96.374x-2357 | 0.99 |
| TNF RII | Y=0.883x+1.8571 | 0.98 |
| Epiregulin | Y=0.7995x-0.5405 | 0.99 |
| IP-10 | Y=0.7323x+3.257 | 0.93 |
| SERPINA3 | Y=1.2738x-5829.2 | 0.95 |
| TNFa | Y=3.3867x-135.1 | 0.98 |
The peripheral blood serum for being clinically diagnosed as healthy person and latent tuberculosis infects patient is collected respectively, wherein health
Person (HC) 63, latent tuberculosis patient (LTBI) 21, is measured in serum respectively by fluorescence method in the prior art
SHBG, IL-2, IL-6, IL-8, TNF RII, Epiregulin, IP-10, the content of SERPINA3, TNFa.
And the concentration being calculated according to above-mentioned equation of linear regression, as shown in table 3.
Table 3
Calculate separately the SHBG of healthy person and latent tuberculosis patient, IL-2, IL-6, IL-8, TNF RII,
Epiregulin, IP-10, the average value of SERPINA3, TNFa expression quantity calculate p value, as shown in table 4.
The average value of 4 healthy person of table and each expressing quantity of latent tuberculosis patient
As it can be seen that healthy person and the SHBG of latent tuberculosis patient, IL2, IL-6, TNF RII, Epiregulin, IP-10,
SERPINA3, TNFa expression quantity have biological differences.
It is thin for detecting SHBG, IL2, IL-6, IL8, TNF RII, Epiregulin, IP-10, SERPINA3 and TNFa
The each group antibody conjugates test experience result of intracellular cytokine is as follows:
Table 5
As shown in table 5, SHBG coated antibody and TNF RII and IP-10 antibody have cross reaction；TNFa and SERPINA3
There is cross reaction with Epiregulin, IL8 antibody and the correlation of combination latent infection are lower, and could not match between antibody pair
Success is not suitable for use in conjunction detection.In conclusion final choice interleukin-6 (IL-6), tumor necrosis factor
This four groups of antibody of receptor II (TNF RII), epiregulin (Epiregulin), interferon inducible protein 10 (IP-10) are to group
At chip for detecting.
Preparation of the embodiment 2 for the protein chip kit of tuberculosis latent infection diagnosis
In order to which, with the presence or absence of 4 kinds of albumen in the present invention, preparation, which is fixed with, is directed to following 4 kinds of albumen in test sample
Specific antibody slide: interleukin-6 (IL-6), tumor necrosis factor receptor I I (TNF RII), epiregulin
(Epiregulin), interferon inducible protein 10 (IP-10).
1, the source of antibody
Using for interleukin-6 (IL-6), tumor necrosis factor receptor I I (TNF RII), epiregulin
(Epiregulin), the corresponding capture antibody of the specific proteins of interferon inducible protein 10 (IP-10) and detection antibody
Purposes, source, concentration are described in detail in table 6.Wherein, the different tables of capture antibody and detection antibody difference identification of protein
Position.
Biotin labeling is carried out to detection antibody, the specific method is as follows: by detection antibody to be marked a large amount of 1 ×
It dialyses in PBS buffer solution (PBS of the 1 milliliter of antibody at 1 liter) three times, at least 6 hours every time.Every milli is pressed after measuring antibody concentration
80 microgram biotin DMSO solutions are added in gram antibody, mix, and react 4 hours at room temperature.With PBS solution to biotin labeling
Antibody dialyse, remove free biotin and demarcate the detection antibody concentration of biotin labeling.
6 specific antibody title of table, the purposes of antibody, source, concentration information
2, the preparation and preservation of protein chip
(1), the point sample concentration for capturing antibody determines: by capture antibody, (sterile water, PBS are buffered in different buffers
Liquid, the PBS of the bovine albumin containing various concentration, the PBS buffer solution etc. of the glycerol containing various concentration) with 2 × concentration dilution, use is non-
Then contact point sample instrument point compares its activity and stability in chip surface with sandwich ELISA method.Experiment shows to use
Diluting capture antibody containing 0.01-10g/100ml bovine albumin PBS buffer solution has best linear rate and stability.
(2), the condition control of point sample: capture antibody is directly put in surface of glass slide at room temperature, captures the point of antibody
Hollow dots are easy to produce, and different antibodies point activity difference is larger, and often find to have to drag in the image ultimately produced
Tail phenomenon.It is 70-75F that the present invention, which controls point sample temperature, and the antibody point that humidity is 40-45% Shi Suodian has best shape,
And stood overnight under room temperature it was found that the good slide of point sample is put in, second day vacuum suction is 2 hours dry, then
Whole chip is encapsulated with air-locked pouch, and the slide capture antibody prepared in this way has optimal activity and can
At least to stablize six months or more at 4 DEG C.
(3), the PBS buffer solution of the antibody of the specificity containing 0.02-2ng of 100-1000pl (point sample: is contained into 0.01-
10g/100ml bovine albumin), using above-mentioned point sample condition, with full-automatic point sample instrument point sample on slide.Biotin labeling
Ox IgG is as positive control.Every kind of antibody has 4 repetitions and the positive control of two kinds of various concentrations in each chip
There are 4 repetitions in each chip.The good slide of point sample is put in and is stood overnight under room temperature, then in drier
Middle pumping is 2 hours dry.Slide after drying load onto matched 16 hole frame a slide be divided into 16 it is non-interfering
Cell.After adhesive film closed frame, saved backup after whole chip is encapsulated with air-locked pouch in 2 DEG C to 8 DEG C.
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 7 is directed to:
The title of recombinant protein, source in 7 protein standard substance of table
| Recombinant protein title | Source | Article No. |
| Interleukin-6 (IL-6) | Raybiotech | 230-00011 |
| Tumor necrosis factor receptor I I (TNF RII) | Raybiotech | 228-20264 |
| Epiregulin (Epiregulin) | Raybiotech | 230-10130 |
| Interferon inducible protein 10 (IP-10) | Raybiotech | 230-00211 |
According to certain after using the phosphate buffer containing 0.1% bovine albumin to dilute above each recombinant protein
Amount mixes, with freeze-drying drying and in -80 DEG C of preservations after packing.
4 kinds of protein expression levels in the blood supernatant of the protein chip quantitative detection of embodiment 3
1, the collection of peripheral blood supernatant
1.1 anticoagulant heparin venous blood 250ml, 2000 revs/min are centrifuged 5 minutes, extract supernatant, spare.
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
Sample diluting liquid (pH value 7.2, the dextran containing 5% mass percent, 1% matter of 3.1 500 μ L of addition
Measure the glycine of percentage, the Tris buffer of 5mM) into the tubule of standard items mixture, re-dissolve standard items.It opens
It before tubule, is first rapidly centrifuged, lightly lashes dissolved powders up and down, marking this tubule is Std 1.
3.2 respectively 6 clean centrifuge tubes of label be Std2, Std3 to Std7, add 200 μ L sample diluting liquid arrive
In each tubule.
The Std 1 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 Std 2 and are added to
In Std 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 egg
White series of concentrations is different.In the present embodiment, the concentration of gradient recombinant protein dilution is as shown in table 8.
The concentration of recombinant protein standard items of the table 8 after gradient dilution for doing standard curve is as follows
| Protein name | Control | Std1 | Std2 | Std3 | Std4 | Std5 | Std6 | Std7 | Unit |
| IL-6 | 0 | 2000 | 667 | 222 | 74 | 25 | 8 | 3 | Pg/mL |
| TNF RII | 0 | 40000 | 13333 | 4444 | 1481 | 494 | 165 | 55 | Pg/mL |
| Epiregulin | 0 | 400000 | 133333 | 44444 | 14815 | 4938 | 1646 | 549 | Pg/mL |
| IP-10 | 0 | 10000 | 3333 | 1111 | 370 | 123 | 41 | 14 | Pg/mL |
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 core
Piece.
4.2 pump the sample diluting liquid in each hole, and the titer and sample for adding 100 μ L are into hole, 4 on shaking table
It DEG C is incubated overnight.Sample is the serum being precipitated naturally after venous blood collection, is diluted using preceding with sample dilution 1:1.
4.3 cleaning
The standard items or sample in each hole are pumped, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shaker concussion, every hole
1 × washing lotion I of 150 μ L, cleaning will be cleaned washing lotion every time, dilute 20 × washing lotion I with deionized water.
1 × washing lotion I in each hole is pumped, 1 × washing lotion II is added and cleans 2 times, each 5min room temperature shaker concussion, often
1 × washing lotion II of 150 μ L of hole, cleaning will be cleaned washing lotion every time, dilute 20 × washing lotion II with deionized water.
The incubation of 4.4 detection antibody mixed liquors
Centrifugation detection antibody mixed liquor tubule, is then added the sample diluting liquid of 1.4ml, after mixing again quickly
Centrifugation.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, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shaker concussion, every 150 μ of hole
1 × washing lotion I of L, cleaning will be cleaned washing lotion every time, and 1 × washing lotion II is then added and cleans 2 times, each 5min room temperature shaker shake
It swings, 1 × washing lotion II of every 150 μ L of hole, cleaning will be cleaned washing lotion every time.
The incubation of 4.6 Cy3- Streptavidins
It is centrifuged Cy3- Streptavidin tubule, the sample diluting liquid of 1.4ml is then added, after mixing again quickly
Centrifugation.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, on room temperature shaker
It is incubated for 1 hour.
4.7 cleaning
The Cy3- Streptavidin in each hole is pumped, 1 × washing lotion I is cleaned 5 times, each 5min room temperature shaker concussion, often
1 × 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.
2) slide is placed in glass slide cleaning pipe, adds 1 × washing lotion I of about 30ml, can entirely covers slide, In
15min is shaken on room temperature shaker, discards 1 × washing lotion I, adds 1 × washing lotion II of about 30ml, 5min is shaken on room temperature shaker.
3) the residual washing lotion of slide is removed.Slide is placed in glass slide cleaning pipe/drying tube, not lid lid, In
1000rpm is centrifuged 3min.
4) use 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.3 (row) × 8 (column) of the microarray parameter of chip,
The diameter of point is with 120 μm.
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.
Identical two positive controls on each chip are used before the concentration of different albumen in calculating not same blood supernatant
Point does the standardization that reference system carries out data.The signal value of two positive controls about differs 4 times.
Normalizing steps are as follows:
First calculate the positive control signal value POS=(POSl+4 × POS2)/2 of each microarray；Wherein POS1 is the
The signal value of the positive control of one concentration, POS2 are the signal value of the positive control of the second concentration；
Then all data are standardized with positive control signal value POS again: corrected value=(original value
× sample average signal value POSave)/positive control signal value POS.Sample average signal value POSaveFor samples all in microarray
The average signal value of product.
Data Processing in Experiment of the embodiment 4 to 4 kinds of albumen in the supernatant of 3 quantitative detection blood of embodiment
1, the concentration of 4 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 of eight standard samples is respectively as shown in table 9.
The standard items of 9 three times gradient dilution of table dash forward photoreading (F532Median-Local Background)
By taking Epiregulin as an example, learn that the protein concentration of Epiregulin and fluorescence are believed according to the information of table 8 and table 9
Relationship between number is as shown in the table.
Relationship between the protein concentration and fluorescence signal of 10 Epiregulin of table
Standard curve is drawn by table 10, calculates to obtain r2=0.8589.Regression equation is y=0.3176x+12020
It is tested No. 1 sample (1-1), signal strength 4255, calculating Epiregulin content is 13371.388pg/
ml。
2, the judgement of testing result
After the Concentration Testing of albumen in each sample handles data according to above-mentioned mode, 4 in obtained each sample
The detectable concentration of albumen is established machine learning model support vector machines (SVM), and interpretation by R linguistic algorithm packet kernlab
The type of the sample.
The type of interpretation sample.Judgment criteria are as follows: as R > 0.23, be then judged as tuberculosis infection.Conversely, being then health
Control.
The sensibility and specificity of the resolution tuberculosis latent infection of embodiment 5
In order to which test kit detection is in the efficiency for differentiating tuberculosis latent infection person, prepared using the embodiment of the present invention 2
Kit has detected different clinical samples, judges it in the sensibility and specificity for differentiating tuberculosis latent infection.
It collects respectively and is clinically diagnosed as normal healthy controls HC (63) and latent tuberculosis infects patient LTBI (21),
Divide and collect peripheral blood serum, is utilized respectively the expression that kit detects 4 albumen, detection method and data processing method
Referring to embodiment 3- embodiment 4.The result and judging result of each sample are calculated after substitution SVM model, result value is greater than
0.23 is diagnosed as LTBI.It is detected in 63 HC using this kit as the result is shown, 57 are judged as HC, true positive rate 90%,
Illustrate the good sensibility of protein chip detection method of this discovery；In 21 LTBI of detection, 20 are judged as LTBI, false
Positive rate is 5.24%, illustrates that the protein chip detection method of this discovery is specific well.
11 different type clinical sample testing result of table
In conclusion the invention discloses a kind of protein chip kits diagnosed with tuberculosis latent infection.The kit
Use normal structure slide as topical carrier, the reaction of multiple sandwich ELISA can be completed in surface of glass slide.The knot of exploitation
Core latent infection diagnoses protein chip diagnostic kit can detect 4 kinds of albumen simultaneously on a protein chip.The kit
The sensitivity and specificity for detecting 4 kinds of albumen can achieve the level of single-factor ELISA.And by 4 kinds of protein combinations, research and develop
The protein chip diagnostic kit that can be used for diagnosis of tuberculosis latent infection person good to a kind of high sensitivity, specificity.This
Expression of the kind kit suitable for detecting multiple specific proteins from human peripheral, for the morning of tuberculosis latent infection
Phase diagnosis and the screening of healthy population.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to the above reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to of the invention
Protection scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. detect interleukin-6, tumor necrosis factor receptor I I, epiregulin and interferon inducible protein 10 reagent
Application in the kit of preparation tuberculosis latent infection diagnosis.
2. a kind of kit for the diagnosis of tuberculosis latent infection, which is characterized in that including being capable of quantitative detection interleukins-
6, the reagent of tumor necrosis factor receptor I I, epiregulin and interferon inducible protein 10.
3. kit according to claim 2 characterized by comprising
Specific antibody for interleukin-6, is directed to epidermis at the specific antibody for tumor necrosis factor receptor I I
Adjust the specific antibody of element and the specific antibody for interferon inducible protein 10.
4. kit according to claim 3 characterized by comprising
It is adjusted for the capture antibody of interleukin-6, for the capture antibody of tumor necrosis factor receptor I I, for epidermis
The capture antibody, the capture antibody for interferon inducible protein 10 of element；
It is adjusted for the detection antibody of interleukin-6, for the detection antibody of tumor necrosis factor receptor I I, for epidermis
The detection antibody and the detection antibody for interferon inducible protein 10 of element；
Wherein, for the different epitopes of the detection antibody of same protein and capture antibody difference identification of protein.
5. according to the described in any item kits of claim 2-4, which is characterized in that the kit further include: protein standard
The Streptavidin of product and/or fluorescein Cy3 label；
The protein standard substance is interleukin-6, tumor necrosis factor receptor I I, epiregulin and interferon induced protein
White 10 mixture.
6. a kind of protein chip for the diagnosis of tuberculosis latent infection, which is characterized in that include: substrate and being fixed on the base
The capture antibody of on piece；
The capture antibody includes: for the specific antibody of interleukin-6, for the spy of tumor necrosis factor receptor I I
Heterogenetic antibody, the specific antibody for epiregulin and the specific antibody for interferon inducible protein 10.
7. the protein chip according to claim 6 for the diagnosis of tuberculosis latent infection, which is characterized in that the substrate is
It is coated with the substrate of active epoxy group.
8. protein chip according to claim 6, which is characterized in that also include positive control on the protein chip；
The positive control is fixed calf IgG on the substrate.
9. a kind of preparation method of the protein chip for the diagnosis of tuberculosis latent infection, which comprises the following steps:
Active epoxy group is coated on the substrate of protein chip；
Capture antibody is fixed on the substrate of the protein chip；
The capture antibody includes: for the specific antibody of interleukin-6, for the spy of tumor necrosis factor receptor I I
Heterogenetic antibody, the specific antibody for epiregulin and the specific antibody for interferon inducible protein 10.
10. preparation method according to claim 9, which is characterized in that the capture antibody is fixed on the base of protein chip
On piece the following steps are included:
By the solution point sample of 100-1000pl capture antibody on the substrate, after standing overnight at room temperature, vacuum drying 1-4 is small
When；
Bovine albumin containing 0.02-20ng capture antibody and 0.01-10g/100ml in the solution of the capture antibody.
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| CN101833001A (en) * | 2010-04-21 | 2010-09-15 | 黄若磐 | Protein chip kit for detecting inflammatory factors and preparation method thereof |
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| CN102016552A (en) * | 2008-03-05 | 2011-04-13 | 神谷来克斯公司 | Methods and compositions for highly sensitive detection of molecules |
| CN101833001A (en) * | 2010-04-21 | 2010-09-15 | 黄若磐 | Protein chip kit for detecting inflammatory factors and preparation method thereof |
| CN104823052A (en) * | 2012-07-31 | 2015-08-05 | 蛋白逻辑有限责任公司 | Biomarkers for diagnosing and/or monitoring tuberculosis |
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Address after: 510530 No. 79 Ruihe Road, Luogang District, Guangzhou City, Guangdong Province Patentee after: Reboo (Guangzhou) Biotechnology Co.,Ltd. Patentee after: THE THIRD PEOPLE'S HOSPITAL OF SHENZHEN Address before: 510530 No. 79 Ruihe Road, Luogang District, Guangzhou City, Guangdong Province Patentee before: RAYBIOTECH, Inc. Patentee before: THE THIRD PEOPLE'S HOSPITAL OF SHENZHEN |