CN103604933A - Kit for detecting active tuberculosis based on antigen-specific TNF-alpha-ELISA (enzyme linked immunosorbent assay) and application thereof - Google Patents
Kit for detecting active tuberculosis based on antigen-specific TNF-alpha-ELISA (enzyme linked immunosorbent assay) and application thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting active tuberculosis based on antigen-specific TNF-alpha-ELISA (enzyme linked immunosorbent assay) and an application thereof. The kit comprises an ELISA plate coating a TNF-alpha antibody, a TNF-alpha protein standard, protein liquid of ESAT-6 and CFP-10, a biotin-marked TNF-alpha antibody and avidin-marked horseradish peroxidase. By adopting the kit for detecting active tuberculosis based on TNF-alpha-ELISA, a method for distinguishing active tuberculosis infection from latent tuberculosis infection is established; the TNF-alpha released by the peripheral blood mononuclear cell under the stimulus of tuberculosis-specific antigens ESAT-6 and CFP-10 and the background TNF-alpha released by the peripheral blood mononuclear cell without stimulus are quantitatively detected through the ELISA technology, and the value of the antigen-specific TNF-alpha is obtained by subtracting the two so as to perform direct diagnosis on the active tuberculosis.
Description
Technical field
The present invention relates to ELISA kit application, refer to particularly a kind of based on antigen specific T NF-α-ELISA detected activity kit lungy and application thereof.
Background technology
Tuberculosis is one of major disease threatening human health, mainly causes pulmonary disease, and its M & M is all high.In history, tuberculosis was once widely current in the world, and several hundred million people are seized life.Since the 1950's, antituberculotic is constantly found, and makes popular certain control that obtained lungy.In recent years, due to the ignorance of some country to tuberculosis prevention and treatment work, add that floating population increases, the continuous factor such as appearances of HIV-positive's propagation and Resistance Mycobacterium Tuberculosis strain, make the popular rise to some extent of tuberculosis.The whole world increases case 8,000,000 every year newly at present, has every year 2000000 people to die from tubercle bacillus affection.Therefore, the World Health Organization (WHO) has announced to enter " the global tuberculosis emergency circumstance ", and will be decided to be " World Tuberculosis Prevention and Cure Day " on annual March 24.
In the face of so severe situation, we should take measures early.Early diagnosis lungy and effectively treatment are the most important things of tuberculosis prevention and treatment work, for controlling tulase propagation, have important clinical meaning.Most domestic hospital diagnoses active tuberculosis according to direct smear microscopy and Much's bacillus culture technique at present.Although tulase plate coating checking is simple and accuracy is high, positive rate is low.It is with a high credibility that tulase is cultivated testing result, but consuming time longer, can not meet clinical demand.In addition, tuberculin skin test (PPD) experiment is the Main Basis that current domestic diagnosis of tuberculosis bacterium infects.This method is simple and expense is low, but its disadvantage is that it is the antigen mixture that Much's bacillus is slightly carried that PPD tests antigen used, all there is common antigen composition with many non-tuberculous mycobacterias and Bacille Calmette-Guerin (BCG), thereby cause this method for diagnosis of tuberculosis poor specificity.China is tuberculosis height Endemic Area, due to extensive inoculation BCG, also causes PPD experiment false positive rate very high, so the reaction power that can only test skin according to PPD clinically gives auxiliary diagnosis.
Tulase gamma interferon (γ-IFN) release test of cellular immunity mediation is to adopt in recent years enzyme linked immunosorbent assay (ELISA) or enzyme linked immunological spotting method (ELISpot), γ-the IFN discharging under Much's bacillus specific antigen stimulates by quantitative detection person under inspection PERIPHERAL BLOOD MONONUCLEAR CELL, thus to hiding, infect and diagnose with active tuberculosis.The method principle is that in Th1 type excreted factor γ-IFN and body, tubercle bacillus antigen content is closely related.While again being run into similar antigen by the T cell of antigen of mycobacterium tuberculosis sensitization, can produce high-caliber γ-IFN, therefore be used to the diagnosis of tuberculosis infection.According to this principle, England Oxford immunological technique company has developed TSPOT-TB tuberculosis infection diagnostic reagent kit, this kit application tulase specific antigen early antigen target 6(ESAT-6), culturing filtrate protein 10 (CFP-10) stimulates mononuclearcell in person under inspection's peripheral blood, thereby by detecting γ-IFN level, tuberculosis infection is diagnosed.This experiment is not affected by BCG and non-tuberculous mycobacteria, and its detection sensitivity and specificity all high.Regrettably,, although the method based on gamma interferon release test can be carried out quick diagnosis to m tuberculosis infection, the method can not infect for distinguishing activity and latent tuberculosis.
Active tuberculosis patient should treat as early as possible.Yet China is tuberculosis big country, there is a large amount of latent tuberculosis the infecteds, this class latent tuberculosis infection population may all not have clinical symptoms throughout one's life, does not need to carry out any treatment yet.Due to the high sensitivity of gamma interferon release test, except active tuberculosis the infected, it is also all positive that adopting said method detects latent tuberculosis the infected, therefore to clinical application, brings difficulty.This is also difficult to for gamma interferon release test carries out Diagnosis of Tuberculosis the major reason of promoting in China.If can develop, only have active tuberculosis patient just to show as the positive, and latent tuberculosis patient show as negative detection method ，Jiang Dui China diagnosis of tuberculosis and treat significant.
At present, the domestic also untapped like product that goes out to diagnose for active tuberculosis.
Summary of the invention
Technical matters to be solved by this invention has just been to provide a kind of based on antigen specific T NF-α-ELISA detected activity kit lungy and application thereof, this kit detects by elisa technique the TNF-alpha levels that person under inspection's PERIPHERAL BLOOD MONONUCLEAR CELL is secreted under different stimulated, thereby calculates antigen specific T NF-α value.
For solving the problems of the technologies described above, provided by the invention a kind of based on TNF-α-ELISA detected activity kit lungy, this kit comprises:
1) ELISA Plate of coated TNF-Alpha antibodies;
2) TNF-α protein standard substance;
3) protein liquid of ESAT-6 and CFP-10;
4) biotin labeling TNF-Alpha antibodies.
Further, described kit also comprises in PBS concentrated cleaning solution, phytohemagglutin phytolectin, substrate solution and stop buffer any one or a few.
Again further, the protein liquid concentration of described ESAT-6 and CFP-10 is 10 μ g/ml.
Again further, described kit also comprises the horseradish peroxidase of Avidin mark.
The application of kit described in the present invention also provides in detected activity tuberculosis, comprises the following steps:
1) protein liquid that adds ESAT-6 and CFP-10 in the person under inspection's PERIPHERAL BLOOD MONONUCLEAR CELL suspension obtaining to collection, induce, negative control for to add nutrient solution to induce, for detection of background TNF-alpha levels in the nutrient solution of examinate's PERIPHERAL BLOOD MONONUCLEAR CELL;
2) at 37 ℃, 5%CO
2incubator in cultivate 16～20 hours, collect the supernatant of nutrient solution;
3) adopt elisa technique to detect each hole TNF-α concentration in nutrient solution supernatant;
4) calculate antigen specific T NF-α value: the TNF-alpha levels of PERIPHERAL BLOOD MONONUCLEAR CELL secretion under antigen of mycobacterium tuberculosis ESAT-6 and CFP-10 stimulation deducts background TNF-alpha levels;
5) according to antigen specific T NF-α value size, judge whether patient exists active tuberculosis to infect.
. as preferred version, the application of kit in detected activity tuberculosis, comprises the following steps:
1) in four holes of common 96 hole reaction plates, add 100 μ l PERIPHERAL BLOOD MONONUCLEAR CELL suspension to be measured;
2) in above-mentioned four holes, add following reagent: negative control hole adds 50 μ l nutrient solutions; Positive control hole adds 50 μ l phytohemagglutin phytolectins; Two are detected hole and add respectively the ESAT-6 protein liquid of 50 μ l and the CFP-10 protein liquid of 50 μ l;
3) 96 orifice plates are placed in to 37 ℃, 5%CO
2incubator in cultivate 16～20 hours;
4) take out 96 orifice plates in step 3), collect each hole nutrient solution supernatant;
5) after being dissolved with PBS cleansing solution, TNF-α protein standard substance carries out doubling dilution: 800pg/ml, 400pg/ml, 200pg/ml, 100pg/ml, 50pg/ml and six concentration gradients of 25pg/ml;
6) get respectively 100 μ l step 4) and obtain the solution that cleer and peaceful step 5) on nutrient solution obtains six concentration gradients, add respectively in the hole of ELISA Plate of coated TNF-Alpha antibodies, blank hole adds the PBS cleansing solution of 100 μ l; Continuation adds respectively 50 μ l biotin labeling TNF-Alpha antibodies in above each hole;
7) ELISA Plate of coated TNF-Alpha antibodies is placed under 37 ℃ of conditions and is hatched 90 minutes;
8) take out the ELISA Plate of coated TNF-Alpha antibodies, abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
9) to the Avidin horseradish peroxidase bond that adds respectively 100 μ l in corresponding ELISA Plate hole; ELISA Plate is placed under 37 ℃ of conditions and is hatched 30 minutes;
10) take out ELISA Plate and abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
11) to the substrate solution that adds respectively 100 μ l in corresponding ELISA Plate hole, under lucifuge condition, incubated at room is 10～30 minutes;
12) again to the stop buffer that adds respectively 100 μ l in corresponding ELISA Plate hole, in 10 minutes, by microplate reader, detect 450nm place light absorption value;
13) by each concentration of TNF-α standard items and light absorption value drawing standard curve, and each detection sample light absorption value is converted into and is respectively detected sample TNF-α concentration by typical curve.
The principle of the invention:
The present invention can activate rapidly while being again subject to tuberculosis antigen induction according to tuberculosis infection patient body internal specific effector cell, and a large amount of secrete inflammatory cytokines TNF-α (tumor necrosis factor-alpha), and tuberculosis specificity T NF-α secretion hide and active tuberculosis infected patient in there is significant difference.So, compare with gamma interferon release test, for the method for detectable antigens specificity T NF-α, when distinguishing latency and active tuberculosis infection, there is advantage.
Beneficial effect of the present invention is:
The present invention's application is based on TNF-α-ELISA detected activity kit lungy, can set up a kind of method of distinguishing activity and latent tuberculosis infection, only active tuberculosis patient shows as the positive, the method quantitatively detects by elisa technique the TNF-α that under tuberculosis specific antigen ESAT-6 and CFP-10 incentive condition, PERIPHERAL BLOOD MONONUCLEAR CELL discharges, and the background TNF-α that in non-stimulation situation, PERIPHERAL BLOOD MONONUCLEAR CELL discharges, by above both subtract each other and obtain antigen specific T NF-α value, thereby active tuberculosis is directly diagnosed.
The method the present invention relates to and kit have good performance parameter when distinguishing latency and active tuberculosis infection, diagnostic activities tuberculosis infection susceptibility reaches 80%, and when applying this kit and detecting latent tuberculosis patient, non-tubercular and normal healthy controls person, the overwhelming majority is all negative, illustrates that this kit has good specificity equally.In addition, what the present invention applied is the Method And Principle based on ELISA, therefore has quick, economic dispatch advantage simultaneously, and above explanation this method is significant to early diagnosis lungy.
Accompanying drawing explanation
Fig. 1 is each concentration of TNF-α standard items and light absorption value drawing standard curve map;
Fig. 2 is the antigen specific T NF-α value distribution plan of ESAT-6 induction;
Fig. 3 is the antigen specific T NF-α value distribution plan of CFP-10 induction.
Embodiment
In order to explain better the present invention, below in conjunction with specific embodiment, further illustrate main contents of the present invention, but content of the present invention is not only confined to following examples.
Embodiment 1
Based on TNF-α-ELISA detected activity kit lungy, this kit comprises:
1) ELISA Plate of coated TNF-Alpha antibodies;
2) TNF-α protein standard substance;
3) protein liquid of ESAT-6 and CFP-10;
4) biotin labeling TNF-Alpha antibodies;
Kit also comprises in PBS concentrated cleaning solution, phytohemagglutin phytolectin, substrate solution and stop buffer any one or a few.
Kit also comprises the horseradish peroxidase of Avidin mark.
The protein liquid concentration of ESAT-6 and CFP-10 is 10 μ g/ml.
. the application of kit in detected activity tuberculosis, comprises the following steps:
1) in four holes of common 96 hole reaction plates, add 100 μ l PERIPHERAL BLOOD MONONUCLEAR CELL suspension to be measured;
2) in above-mentioned four holes, add following reagent: negative control hole adds 50 μ l nutrient solutions; Positive control hole adds 50 μ l phytohemagglutin phytolectins; Two are detected hole and add respectively the ESAT-6 protein liquid of 50 μ l and the CFP-10 protein liquid of 50 μ l;
3) 96 orifice plates are placed in to 37 ℃, 5%CO
2incubator in cultivate 16～20 hours;
4) take out 96 orifice plates in step 3), collect each hole nutrient solution supernatant;
5) after being dissolved with PBS cleansing solution, TNF-α protein standard substance carries out doubling dilution: 800pg/ml, 400pg/ml, 200pg/ml, 100pg/ml, 50pg/ml and six concentration gradients of 25pg/ml;
6) get respectively 100 μ l step 4) and obtain the solution that cleer and peaceful step 5) on nutrient solution obtains six concentration gradients, add respectively in the hole of ELISA Plate of coated TNF-Alpha antibodies, then in blank hole, add the PBS cleansing solution of 100 μ l; Continuation adds respectively the biotin labeling TNF-Alpha antibodies of 50 μ l in above each hole;
7) ELISA Plate of coated TNF-Alpha antibodies is placed under 37 ℃ of conditions and is hatched 90 minutes;
8) take out the ELISA Plate of coated TNF-Alpha antibodies, abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
9) to the Avidin horseradish peroxidase bond that adds respectively 100 μ l in corresponding ELISA Plate hole; ELISA Plate is placed under 37 ℃ of conditions and is hatched 30 minutes;
10) take out ELISA Plate and abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
11) to the substrate solution that adds respectively 100 μ l in corresponding ELISA Plate hole, under lucifuge condition, incubated at room is 10～30 minutes;
12) again to the stop buffer that adds respectively 100 μ l in corresponding ELISA Plate hole, in 10 minutes, by microplate reader, detect 450nm place light absorption value;
13) by each concentration of TNF-α standard items and light absorption value drawing standard curve, and each detection sample light absorption value is converted into and is respectively detected sample TNF-α concentration by typical curve; Result is judged:
If positive control hole TNF-α concentration > 100pg/ml, sample meets the requirements.On the satisfactory basis of sample, if ESAT-6 detects hole or CFP-10 detects hole antigen specific T NF-α value >=135pg/ml, result is judged to be the positive (active tuberculosis infection); If ESAT-6 detects hole or CFP-10 detects hole antigen specific T NF-α value in 70～135pg/ml, result is judged to be suspicious; If ESAT-6 detects hole and CFP-10 detects hole antigen specific T NF-α value < 70pg/ml, result is judged to be feminine gender (infecting without active tuberculosis).
Article (horseradish peroxidase of ELISA Plate, TNF-α protein standard substance, biotin labeling TNF-Alpha antibodies and the Avidin mark of coated TNF-Alpha antibodies) used in kit of the present invention can bought on the market, directly use.
In real testing process, each ELISA Plate has a negative control, a positive control and one group of typical curve corresponding aperture at least, to guarantee the accuracy of each group experiment.
Embodiment 2
Use mentioned reagent box and corresponding method to detect 20 routine active tuberculosis patients, 20 routine latent tuberculosis collators, 10 routine non-tuberculars and 10 routine normal healthy controls persons.
One case is chosen
All patients and collator are all from HuaZhong Science University, TongJi medical school, TongJi Hospital.Four groups of crowds' the standard of including is as follows:
Active tuberculosis patient: the clinical symptoms such as have heating, night sweat or become thin, and clinical smear or cultivate positive.
Latent tuberculosis collator: without heating, night sweat or the clinical symptoms such as become thin, without tuberculosis Radiologic imaging, but tuberculosis infection T cell detection (T-SPOT) positive.
Non-tubercular: the clinical symptoms such as have heating, night sweat or become thin, but tuberculosis infection T cell detection (T-SPOT) feminine gender.
Normal healthy controls person: without heating, night sweat or the clinical symptoms such as become thin, without tuberculosis Radiologic imaging, and tuberculosis infection T cell detection (T-SPOT) feminine gender.
Two detection methods:
1, separated person under inspection's PERIPHERAL BLOOD MONONUCLEAR CELL
1) extract 4～6ml blood and add in aseptic anticoagulant blood-collecting pipe, take to put upside down and mix after blood, obtain anticoagulation;
2) take out an aseptic centrifuge tube of 15ml, add the aseptic PBS of 4ml anticoagulation and 4ml, obtain 8ml hemodilution product, put upside down and mix;
3) separately get an aseptic centrifuge tube of 15ml, add 4ml PERIPHERAL BLOOD MONONUCLEAR CELL parting liquid, then layer carefully adds 8ml dilute blood thereon, forms sharp interface, the centrifugal 25min of 1000g under room temperature condition;
4) the obvious cloud mononuclearcell of centrifugal rear visible one deck, draws this confluent monolayer cells to another aseptic centrifuge tube;
5) in the centrifuge tube of mononuclearcell is housed, add the aseptic PBS of 6-8ml, piping and druming mixes, the centrifugal 10min of 600g under room temperature condition;
6) abandon supernatant, repeating step (5);
7) abandon supernatant, add 200 μ l containing the RPMI-1640 re-suspended cell of 10% calf serum, get the cell of 10 μ l after resuspended and add in blood counting chamber, counting under the microscope, adds RPMI-1640 containing 10% calf serum by cell dilution to 2.5 * 10
6cell/ml, stand-by;
2, cell induction
1) in four holes of common 96 hole reaction plates, add 100 μ l PERIPHERAL BLOOD MONONUCLEAR CELL suspension to be measured (each detects sample and adds four holes);
2) in above-mentioned four holes, add following reagent: negative control hole adds 50 μ l nutrient solutions (for detection of the background TNF-alpha levels under non-incentive condition); Positive control hole adds 50 μ l phytohemagglutin phytolectins (PHA); Two are detected hole and add respectively the ESAT-6 protein liquid of 50 μ l and the CFP-10 protein liquid of 50 μ l;
3) 96 orifice plates are placed in to 37 ℃, 5%CO
2incubator in cultivate 16～20 hours;
3, detect
1) take out 96 orifice plates in the step 3) of above-mentioned cell induction, collect each hole nutrient solution supernatant;
2) after being dissolved with PBS cleansing solution, TNF-α protein standard substance carries out doubling dilution: 800pg/ml, 400pg/ml, 200pg/ml, 100pg/ml, 50pg/ml and six concentration gradients of 25pg/ml;
3) get respectively 100 μ l step 1) and obtain cleer and peaceful step 2 on nutrient solution) obtain the solution of six concentration gradients, add respectively in the hole of ELISA Plate of coated TNF-Alpha antibodies, blank hole adds the PBS cleansing solution of 100 μ l; Continuation adds respectively the biotin labeling TNF-Alpha antibodies of 50 μ l in above each hole;
4) ELISA Plate of coated TNF-Alpha antibodies is placed in 37 ℃ of incubators and is hatched 90 minutes;
5) take out the ELISA Plate of coated TNF-Alpha antibodies, abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
6) to the Avidin horseradish peroxidase bond that adds respectively 100 μ l in corresponding ELISA Plate hole; ELISA Plate is placed in 37 ℃ of incubators and is hatched 30 minutes;
7) take out ELISA Plate and abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
8) to the substrate solution that adds respectively 100 μ l in corresponding ELISA Plate hole, under lucifuge condition, incubated at room is 10～30 minutes;
9) again to the stop buffer that adds respectively 100 μ l in corresponding ELISA Plate hole, in 10 minutes, by microplate reader, detect 450nm place light absorption value;
10) by each concentration of TNF-α standard items and light absorption value drawing standard curve, and respectively all patients and 4 detection hole light absorption values of collator are converted into and are respectively detected sample TNF-α concentration by typical curve;
Standard items concentration and absorbance result are as follows:
According to standard items concentration and absorbance result drawing standard curve as Fig. 1:
The typical curve showing according to Fig. 1 converses all person under inspection ESAT-6 and detects hole and CFP-10 detection hole TNF-α concentration, and negative control hole is background TNF-α concentration, use above both subtraction calculations to go out that each sample ESAT-6 stimulates and the antigen specific T NF-α value of CFP-10 stimulation.
4, experimental result:
Antigen specific T NF-α value under antigen specific T NF-α value under four groups of person under inspection ESAT-6 stimulate and CFP-10 stimulate is expressed as follows respectively shown in Fig. 2 and Fig. 3:
According to following result judgment principle: if ESAT-6 detects hole or CFP-10 detects hole antigen specific T NF-α value >=135pg/ml, result is judged to be the positive (active tuberculosis infection); If ESAT-6 detects hole or CFP-10 detects hole antigen specific T NF-α value in 70～135pg/ml, result is judged to be suspicious; If ESAT-6 detects hole and CFP-10 detects hole antigen specific T NF-α value < 70pg/ml, result is judged to be feminine gender (infecting without active tuberculosis).
In 20 selected routine active tuberculosis patients, antigen specific T NF-α value (ESAT-6 detects hole or CFP-10 detection hole TNF-α concentration deducts negative control hole TNF-α concentration) is greater than 16 examples that have of 135pg/ml, and the susceptibility of using this method to detect reaches 80%;
In 20 selected routine latent tuberculosis contrasts, 18 routine antigen specific T NF-α values are all less than 135pg/ml, only have 2 routine results to be greater than 135pg/ml, are judged as the positive;
In 10 selected routine normal healthy controls, antigen specific T NF-α value is all less than 70pg/ml;
In 10 selected routine non-tuberculosis contrasts, antigen specific T NF-α value is less than 8 examples that have of 70pg/ml, and 1 example is suspicious, and 1 example is positive.
Above result shows, use detected activity tuberculosis infection of the present invention to have higher susceptibility and specificity, and experimental implementation is simple, within 24 hours, can report the result, and illustrates that kit of the present invention can be used as a kind of means of auxiliary diagnosis active tuberculosis.
Other unspecified part is prior art.Although above-described embodiment has been made detailed description to the present invention; but it is only the present invention's part embodiment; rather than whole embodiment, people can also obtain other embodiment according to the present embodiment under without creative prerequisite, and these embodiment belong to protection domain of the present invention.
Claims (7)
1. based on antigen specific T NF-α-ELISA detected activity kit lungy, it is characterized in that: this kit comprises:
1) ELISA Plate of coated TNF-Alpha antibodies;
2) TNF-α protein standard substance;
3) protein liquid of ESAT-6 and CFP-10;
4) biotin labeled TNF-Alpha antibodies.
2. kit according to claim 1, is characterized in that: described kit also comprises in concentrated PBS cleansing solution, phytohemagglutin phytolectin, substrate solution and stop buffer any one or a few.
3. kit according to claim 1 and 2, is characterized in that: the protein liquid concentration of described ESAT-6 and CFP-10 is 10 μ g/ml.
4. kit according to claim 1 and 2, is characterized in that: the label of described compatibility is horseradish peroxidase.
5. the application of the kit described in claim 1～4 any one in detected activity tuberculosis.
6. application according to claim 5, is characterized in that: comprise the following steps:
1) in the person under inspection's PERIPHERAL BLOOD MONONUCLEAR CELL suspension obtaining to collection, add the protein liquid of ESAT-6 and CFP-10, induce, negative control for to add nutrient solution to induce in the nutrient solution of examinate's PERIPHERAL BLOOD MONONUCLEAR CELL;
2) at 37 ℃, 5%CO
2incubator in cultivate 16～20 hours, collect the supernatant of nutrient solution;
3) adopt elisa technique to detect each hole TNF-α concentration in nutrient solution supernatant;
4) calculate antigen specific T NF-α value: the TNF-alpha levels of PERIPHERAL BLOOD MONONUCLEAR CELL secretion under antigen of mycobacterium tuberculosis ESAT-6 and CFP-10 stimulation deducts background TNF-alpha levels;
5) according to antigen specific T NF-α value size, judge whether patient exists active tuberculosis to infect.
7. according to the application described in claim 5 or 6, it is characterized in that: comprise the following steps:
1) in four holes of common 96 hole reaction plates, add 100 μ l PERIPHERAL BLOOD MONONUCLEAR CELL suspension to be measured;
2) in above-mentioned four holes, add following reagent: negative control hole adds 50 μ l nutrient solutions; Positive control hole adds 50 μ l phytohemagglutin phytolectins; Two are detected hole and add respectively the ESAT-6 protein liquid of 50 μ l and the CFP-10 protein liquid of 50 μ l;
3) 96 orifice plates are placed in to 37 ℃, 5%CO
2incubator in cultivate 16～20 hours;
4) take out 96 orifice plates in step 3), collect each hole nutrient solution supernatant;
5) after being dissolved with PBS cleansing solution, TNF-α protein standard substance carries out doubling dilution: 800pg/ml, 400pg/ml, 200pg/ml, 100pg/ml, 50pg/ml and six concentration gradients of 25pg/ml;
6) get respectively 100 μ l step 4) and obtain the solution that cleer and peaceful step 5) on nutrient solution obtains six concentration gradients, add respectively in the hole of ELISA Plate of coated TNF-Alpha antibodies, then in blank hole, add the PBS cleansing solution of 100 μ l; Continuation adds respectively the biotin labeling TNF-Alpha antibodies of 50 μ l in above each hole;
7) ELISA Plate of coated TNF-Alpha antibodies is placed under 37 ℃ of conditions and is hatched 90 minutes;
8) take out the ELISA Plate of coated TNF-Alpha antibodies, abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
9) to the Avidin horseradish peroxidase bond that adds respectively 100 μ l in corresponding ELISA Plate hole; ELISA Plate is placed under 37 ℃ of conditions and is hatched 30 minutes;
10) take out ELISA Plate and abandon supernatant, add the PBS cleansing solution 2～3 times of 250～300 μ l, for the last time ELISA Plate is patted dry on filter paper;
11) to the substrate solution that adds respectively 100 μ l in corresponding ELISA Plate hole, under lucifuge condition, incubated at room is 10～30 minutes;
12) again to the stop buffer that adds respectively 100 μ l in corresponding ELISA Plate hole, in 10 minutes, by microplate reader, detect 450nm place light absorption value;
13) by each concentration of TNF-α standard items and light absorption value drawing standard curve, and each detection sample light absorption value is converted into and is respectively detected sample TNF-α concentration by typical curve; Result is judged.
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| CN105954521A (en) * | 2016-07-08 | 2016-09-21 | 广州华弘生物科技有限公司 | MTB (Mycobacterium Tuberculosis) infection diagnosis kit |
| CN106546737A (en) * | 2016-10-24 | 2017-03-29 | 广州迪澳医疗科技有限公司 | A kind of method of vitro detection active tuberculosis |
| CN106990252A (en) * | 2016-01-20 | 2017-07-28 | 厦门大学 | Method and kit for diagnostic activities tuberculosis |
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| CN110672857A (en) * | 2019-10-10 | 2020-01-10 | 四川大学华西第二医院 | TNF-alpha high-sensitivity Elisa detection kit and use method and application thereof |
| 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 |
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