CN106053783A - Quick time-resolved fluorescence immunoassay kit for detection of T cells infected with tuberculosis and detection method of kit - Google Patents
Quick time-resolved fluorescence immunoassay kit for detection of T cells infected with tuberculosis and detection method of kit Download PDFInfo
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- CN106053783A CN106053783A CN201610392936.2A CN201610392936A CN106053783A CN 106053783 A CN106053783 A CN 106053783A CN 201610392936 A CN201610392936 A CN 201610392936A CN 106053783 A CN106053783 A CN 106053783A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Abstract
The invention discloses a quick time-resolved fluorescence immunoassay kit for detection of T cells infected with tuberculosis. The kit is characterized by comprising 1) a test reaction cup; 2) a calibrator reaction cup; 3) a blank control culture tube containing an anticoagulant substance inside; 4) a test culture tube; 5) a positive control culture tube containing an anticoagulant substance and cell agglutinin inside; 6) an experiment buffer solution and 7) a wash concentrate. The invention further discloses a detection method of the quick time-resolved fluorescence immunoassay kit for detection of T cells infected with tuberculosis. The kit adopts a gamma-interferon in-vitro release method, a biotin-avidin method and double-antibody sandwich time-resolved fluorescence immunoassay together, the experimental methods are simple and quick, automatic operation is realized, and the detection system is an open type operation system.
Description
Technical field
The invention belongs to medicine and hygiene fields, particularly relate to a kind of rapid time being applicable to the detection of tuberculosis infection T cell
Resolved fluorometric immunoassay kits and detection method thereof.
Background technology
Tuberculosis is by the mycobacterium tuberculosis (Mycobacterium in mycobacterium tuberculosis complex
Tuberculosis, MTB), Mycobacterium bovis (Mycobacterium bovis) and mycobacterium africanum (Mycobacterium
Africanum) chronic infectious disease caused, can involve each organ of whole body, especially the most common with pulmonary tuberculosis.Tuberculosis branch
Bacillus is usually and is passed through air droplet infection to new host by respiratory tract tuberculosis patient.Incubation period lungy is that several weeks are to number
Month, period major part the infected non-evident sympton.Tuberculosis serious harm public safety, the whole world has 2,000,000 people to die from every year
Tuberculosis.In China, there is the newly-increased tuberculosis patient of 1,000,000 every year.
The microscopy of mycobacterium tuberculosis is the method for the most frequently used diagnosis of tuberculosis, but the positive rate of the method is low.Knot
The cultivation of core mycobacteria determines that goldstandard lungy, but owing to the speed of growth of mycobacterium tuberculosis is slow, test week
Phase length (2～3 weeks), it is impossible to meet clinical requirement.It addition, tuberculosis can also pass through Chest X-rays, detection of nucleic acids, physical examination etc.
Method confirms.The infection of latent tuberculosis bacterium is a kind of special state after host infection mycobacterium tuberculosis, in the infected's body
Mycobacterium tuberculosis is in retention-state, it is impossible to is diagnosed as active tuberculosis, but has the wind developing into active tuberculosis
Danger.The high-risk group infecting latent tuberculosis bacterium carries out early diagnosis and suitably intervenes, and has actively in Tubercufosis control
Meaning.Tuberculin test is uniquely to diagnose the method that latent tuberculosis bacterium infects, after mycobacterium tuberculosis infection 2～10 weeks
Interior tuberculin test transfers the positive to.But, some the infected is responseless to tuberculin, both includes in these crowds
Those of a variety of causes causes immunosuppressant, also include that some do not have immunosuppressant people.Additionally, bcg vaccination, infect non-
Pathogenic mycobacterium, and other factors, can cause non-tubercle bacillus affection person's tuberculin test positive.
The gamma interferon release in vitro method of tuberculosis infection T cell (interferon-gamma release Assays,
IGRAs) detection is cell mediated immunity (the cell mediated caused by mycobacterium tuberculosis specific antigen
Immunity, CMI) reaction realizes.The antigen of the test kit use of listing at present or polypeptide or its analog mainly have early
Phase secretion antigen target protein 6 (early secreting antigen target 6, ESAT6), culturing filtrate protein 10
One or more in (culture filtrate protein-10, CFP-10) and TB7.7 (p4) etc. are as differential stimulus
Antigen, the non-tuberculous mycobacteria [mycobacterium kansasii (Mycobacterium of all of strain of BCG vaccine and the overwhelming majority
Kansasii), Suhl adds mycobacteria (Mycobacterium szulgai), Hai Shi mycobacteria (Mycobacterium
Marinum) except] do not contain these protein.The blood of the patient infecting mycobacterium tuberculosis complex usually contains energy
Enough identifying the lymphocyte of these protein and other antigen of mycobacterium, this identification process is along with the conjunction of gamma interferon
Become and secretion.IGRAs utilizes Specific Antigen of Mycobacterium Tuberculosis or polypeptide or its analog to stimulate the knot in people's whole blood sample
Core antigen of mycobacterium specific T-cells produces immunne response, then quantitative determines immunity in human serum by the method for immunoassay
The concentration of the gamma interferon that response produces, thus judge that the T cell immunity whether experimenter has mycobacterium tuberculosis special is anti-
Should, i.e. tuberculosis infection T cell detection.Gamma interferon is a kind of cytokine of Th1 emiocytosis, is not only able to reflect body
The Th1 cellular immunization situation of tuberculosis, also closely related with the antigenic content of internal tulase.By antigen of mycobacterium tuberculosis sensitization
T cell can produce high-caliber gamma interferon when running into cognate antigen again, be therefore used for the diagnosis of tuberculosis latent infection.
Summary of the invention
It is an object of the invention to provide a kind of rapid time resolved fluorometric immunity being applicable to the detection of tuberculosis infection T cell
Assay kit, have employed gamma interferon release in vitro method, combines the biotin-labeled pentylamine method that have employed, double-antibody sandwich time
Resolved fluorometric immunization, experimental technique is simple and quick, can automation mechanized operation, detecting system is open operating system.
The technical solution used in the present invention is: a kind of rapid time resolved fluorometric being applicable to the detection of tuberculosis infection T cell
Immunoassay kits, including: 1) test reaction cup, its endoperidium has Streptavidin and is combined on Streptavidin
Biotinylation gamma interferon monoclonal antibody, and the gamma interferon monoclonal antibody containing lanthanide series labelling；2) calibration object
Reaction cup, its endoperidium has Streptavidin and the biotinylation gamma interferon monoclonal anti being combined on Streptavidin
Body, and containing europium labelling gamma interferon monoclonal antibody and the calibration object of variable concentrations；3) blank culture tube, it includes
There is anticoagulative substance；4) test cultures pipe, it is contained within anticoagulative substance and can stimulate antigen of mycobacterium tuberculosis specificity T
Cell produces the stimulus object of gamma interferon, stimulus object be the antigen of mycobacterium tuberculosis and derivative epitope peptide thereof and it
Analog at least one；5) positive control culture tube, it is contained within anticoagulative substance and cell agglutinin；6) experiment
Buffer；And 7) concentrate washing liquid.
The principle of the test kit of the present invention is: use gamma interferon release in vitro method and Time-resolved Fluoimmunoassay inspection
Survey the cell mediated immune response that mycobacterium tuberculosis specific antigen is caused；By mycobacterium tuberculosis specific antigen, polypeptide
Or their analog stimulates the T lymphocyte specific of m tuberculosis infection person and makes it breed, discharges γ-interference
Element；Be coated with in the reaction cup of Streptavidin connection biotinylation gamma interferon monoclonal antibody, and containing europium labelling γ-
Interferon monoclonal antibody is as test reaction cup, and in one group of reaction cup, the gamma interferon antigen possibly together with variable concentrations is made
For calibration object reaction cup, add sample in the relevant position of test reaction cup, then in calibration object reaction cup with added with sample
Test reaction cup adds test buffer, oscillation incubation, washing, dry reaction cup, utilizes time resolved fluoro-immunoassay
Instrument detection fluorescent value, fluorescent value intensity is directly proportional to the gamma interferon content in calibration object or sample.
Preferably, the gamma interferon monoclonal antibody of described lanthanide series labelling is europium labelling gamma interferon monoclonal anti
Body.
Preferably, described stimulus object be mycobacterium tuberculosis Rv0733, Rv1978, Rv1980 (MPT64), Rv1981c,
Rv1984(CFP-21)、Rv1985c、Rv2654c(TB7.7)、Rv3425、Rv3429、Rv3615c(ESPC)、Rv3873、
Rv3874 (CFP-10), Rv3875 (ESAT-6), Rv3878, Rv3879c antigen, their derivative epitope peptide, chimeric weight
At least one in group antigen and the like；Described stimulus object content in test cultures pipe is 1～10 μ g/mL, μ here
G/mL refers to the pre-whole blood added relative in every milliliter of test cultures pipe, and the content of described stimulus object is 1～10 μ g.
It is highly preferred that described stimulus object is Rv3874 (CFP-10) and the compositions of Rv3875 (ESAT-6)；Wherein,
The mass ratio of Rv3874 (CFP-10) and Rv3875 (ESAT-6) is 1:1.Rv3874 (CFP-10) and Rv3875 (ESAT-6) is
Special advantage stimulus object, using effect is more preferably.
Preferably, described anticoagulative substance is heparin sodium and/or Lithium acid heparin；Described blank pipe, test cultures pipe and
Containing heparin sodium or the Lithium acid heparin of 9.8～28IU/mL blood in positive control culture tube respectively, IU/mL blood here refers to relatively
The pre-whole blood added in every milliliter of test cultures pipe, the content of described anticoagulative substance is 9.8～28IU.
Preferably, described cell agglutinin is phytohemagglutinin and/or ConA (ConA), more preferably
Phytohaemagglutinin L.
Preferably, described cell agglutinin content in described positive control culture tube is 1～20 μ g/mL, μ here
G/mL refers to the pre-whole blood added relative in every milliliter of test cultures pipe, and the content of described cell agglutinin is 1～20 μ g.
Preferably, described test buffer is the sodium chloride containing 0.15mol/L, the tween 20 of 0.6mL/L, 30ppmPreservative, the 4-hydroxyethyl piperazine ethanesulfonic acid of 5mmol/L, pH7.8 of ethylenediaminetetraacetic acid of 100 μm ol/L
Buffer；
Described concentration washing liquid is the sodium chloride containing 1.125g/L, the tween 20 of 0.2mL/L, 30ppmThe tris-HCI buffer of the 50mmol/L of preservative, pH7.8.
Preferably, the preparation method of described test reaction cup is: described Streptavidin is diluted to concentration 0.1～10 μ
G/mL, joins in skip test reaction cup, hatches 20～24h, washing at 2～8 DEG C；By described biotinylation gamma interferon
Monoclonal antibody is diluted to concentration 0.1～10 μ g/mL, joins in the test reaction cup being coated with Streptavidin, 2～8 DEG C
Under hatch 20～24h, washing；The most each cup adds sealing coat solution, forms sealing coat after drying, finally on sealing coat
Add the gamma interferon monoclonal antibody of described lanthanide series labelling that working concentration is 100～400ng/mL, after drying and get final product
Described test reaction cup.It is highly preferred that the gamma interferon monoclonal antibody of described lanthanide series labelling is europium labelling γ-interference
Element monoclonal antibody.
It is highly preferred that the sealing coat solution that described sealing coat uses is the sodium chloride containing 0.12mol/L, 0.38g/L
Sodium azide, the cattle gamma Globulin of 10g/L, the bovine serum albumin of 50g/L, the trehalose of 50g/L, 0.8g/L aboriginal mouse IgG,
Degeneration mouse IgG, 2g/L casein of 0.05g/L, the trihydroxy methyl ammonia of 50mmol/L, pH7.8 of heparin sodium of 37.5U/mL
Methylmethane-hydrochloric acid.
It is highly preferred that sealing coat solution is dried refers to dried overnight under (36 ± 1) DEG C, 5% damp condition；Europium labelling
Gamma interferon monoclonal antibody is dried and refers to dry up under 60 DEG C of hot blasts.
It is highly preferred that wherein the solution of dilution Streptavidin and biotinylation gamma interferon monoclonal antibody is
The phosphate buffer of 50mmol/L, pH7.8.
It is highly preferred that wherein the solution of dilution europium labelling gamma interferon monoclonal antibody is the chlorination containing 150mmol/L
Sodium, the sodium azide of 0.5g/L, the deionization bovine serum albumin of 2g/L, 125g/L trehalose 50mmol/L, pH7.8 three
Hydroxymethyl aminomethane-hydrochloric acid.
It is highly preferred that europium labelling gamma interferon method for preparing monoclonal antibody therein is, labelling gamma interferon list
Liquid NAP-10 post in clonal antibody is replaced as the carbonate buffer solution of 50mmol/L, pH9.0, and makes the dense of traget antibody
Degree reaches 2～5mg/mL, and the 4-[2-(4-isothiocyanatophenyl) acetenyl]-2 of the europium with 3 times of quality, 6 pairs of { [double (carboxylics of N, N
Methyl)-amino] methyl } pyridine reagent is blended in oscillation incubation (22 ± 2) h, reactant liquor 50mmol/L, pH at 2～8 DEG C
Sepharose CL-6B post (1cm × 40cm) chromatography of 7.80 tris-HCI buffer balances, respectively
Albumen eluting peak is collected in A280 monitoring.
Preferably, the preparation method of described calibration object reaction cup is: described Streptavidin is diluted to concentration 0.1～10
μ g/mL, joins in blank calibration object reaction cup, hatches 20～24h, washing at 2～8 DEG C；By described biotinylation γ-interference
Element monoclonal antibody be diluted to concentration 0.1～10 μ g/mL, join in the calibration object reaction cup being coated with Streptavidin, 2～
20～24h are hatched, washing at 8 DEG C；The most each cup adds sealing coat solution, forms sealing coat after drying, on sealing coat
Add the gamma interferon monoclonal antibody of the described lanthanide series labelling that working concentration is 100～400ng/mL, formed after drying
The gamma interferon monoclonal antibody layer of lanthanide series labelling, the gamma interferon monoclonal of the lanthanide series labelling in each cup
Add the calibration object of variable concentrations on antibody layer, the most i.e. obtain calibration object reaction cup.It is highly preferred that the concentration of described calibration object
Be respectively 0,6,300,15000pg/mL；The every concentration of calibration object arranges 2, and vacuum is crossed and moulded packaging, and 2～8 DEG C save backup.Every
Absciss layer solution is dried and refers to dried overnight under (36 ± 1) DEG C, 5% damp condition；The gamma interferon Dan Ke of lanthanide series labelling
Grand antibody and calibration object are dried and refer to dry up under 60 DEG C of hot blasts.It is highly preferred that the gamma interferon list of described lanthanide series labelling
Clonal antibody is europium labelling gamma interferon monoclonal antibody.The preparation method of described calibration object is: with United Kingdom National biological product
Calibrating institute (National Institute for Biological Standards and Control, NIBSC) numbering 87/
The gamma interferon biological reference material (1IU/mL ≈ 50pg/mL) of 586 is comparison, by Human Lymphobl Toid Interferon antigen step
1) the sealing coat solution in is diluted to 0, the calibration object of 6,300,15000pg/mL.
Preferably, the preparation method of described blank culture tube is: add in clean test tube (plastics or glass)
The heparin sodium of 9.8～28IU/mL blood or Lithium acid heparin；Test tube is uprightly put in full-automatic vacuum-pumping capping machine, true in making test tube
Idling pressure.
Preferably, the preparation method of described test cultures pipe is: add in clean test tube (plastics or glass) 9.8～
The heparin sodium of 28IU/mL blood or Lithium acid heparin, and Rv0733, Rv1978, Rv1980c (MPT64) of 1～10 μ g/mL,
Rv1981c、Rv1984c(CFP-21)、Rv1985c、Rv2654c(TB7.7)、Rv3425、Rv3429、Rv3615c(ESPC)、
One or more antigens in Rv3873, Rv3874 (CFP-10), Rv3875 (ESAT-6), Rv3878, Rv3879c or polypeptide or
Its analog or its chimeric recombinant antigens；Test tube is uprightly put in full-automatic vacuum-pumping capping machine, makes negative pressure of vacuum in test tube.
Preferably, the preparation method of described positive control culture tube is: add in clean test tube (plastics or glass)
The heparin sodium of 9.8～28IU/mL blood or Lithium acid heparin, and the phytohemagglutinin (PHA) of 1～20 μ g/mL or Semen Canavaliae coagulate
Collection element (ConA).Test tube is uprightly put in full-automatic vacuum-pumping capping machine, makes negative pressure of vacuum in test tube.
The lid of culture tube contains Nei Sai and it mates outer safety helmet, it is preferable that described blank culture tube, test training
The outer safety helmet color supporting pipe and positive control culture tube is different.It is highly preferred that the outer peace of described blank culture tube
Full cap color is green；The outer safety helmet color of described test cultures pipe is yellow；The outer safety of described positive control culture tube
Cap color is red.
Present invention also offers the described rapid time resolved fluorometric immunoassay being applicable to the detection of tuberculosis infection T cell
The detection method of test kit, comprises the following steps:
(1) add entirely in blank culture tube, test cultures pipe and three kinds of culture tubes of positive control culture tube respectively
Blood sample, and reverse make the content in whole blood sample and three kinds of culture tubes mix for several times；
(2) three kinds of culture tubes added with whole blood sample are placed at 35～38 DEG C upright cultivation 16～24 hours；
(3), after cultivation terminates, by centrifugal, blood plasma in the whole blood sample of three kinds of culture tubes is separated；
(4) that presets in calibration object reaction cup tests buffering containing being separately added in the hole of the calibration object of variable concentrations
Liquid, and by step 3) in blood plasma in the whole blood sample that separates and test buffer add in test reaction cup；
(5) by calibration object reaction cup and test reaction cup at 30～40 DEG C, oscillation incubation 5～20min；
(6) washing, dried, use time-resolved fluorescence immunoassay instrument to carry out fluorescent value counting.
Preferably, in described step (3), it is centrifuged and refers to be centrifuged under the rotating speed of 3000～5000r/min 10～20min；
In described step (6), it is dried and refers to be dried under 50～70 DEG C of hot blasts 30～40s.
Relative to prior art, the invention have the benefit that
The present invention uses dry reagent rapid time resolved fluorometric immuno analytical method, solves conventional Conventional Time and differentiates
Fluorescence immunoassay technology reagent component is various, and liquid reagent is not sufficiently stable, complex operation, the longest shortcoming.And this
Invention solves during conventional tuberculosis infection T cell measures needs independent blood taking tube and culture tube, trivial operations, easily go out
The shortcomings such as mistake, automaticity are the highest；Present invention achieves blood taking tube and culture tube integration, negative pressure of vacuum is accurately taken a blood sample；3 kinds
Culture tube uses different colours label and cap to make a distinction；Avoid during whole blood cell culture blood taking tube uncap, subpackage blood sample,
The processes such as secondary marker, it is therefore prevented that sample is uncapped at blood taking tube, secondary pollution during subpackage blood sample, during decreasing this
Potential bio-safety risk, also prevent secondary marker process produce mistake, as long as and after whole blood cell culture routinely from
I.e. can be placed in after the heart and on fully automatic system, be sampled detection, reduce working strength and improve work efficiency.The present invention
Culture tube have blood taking tube and culture tube integration, blood collection procedure decreases consumptive material uses, vacuum is accurately taken a blood sample, be suitable to from
The features such as dynamicization operation.The test kit of the present invention has testing result accuracy, highly sensitive, high specificity, good stability, inspection
The features such as survey method is easy, efficient, are suitable for real-time test (point-of-care testing, POCT).
Accompanying drawing explanation
Fig. 1 is the gamma interferon detection by quantitative dose-response curve of test kit of the present invention.
Detailed description of the invention
For better illustrating the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Streptavidin in following example is purchased from Shenzhen Fei Peng limited company；Biotin labeling gamma interferon
Monoclonal antibody is purchased from MabTech company of Holland；Labelling gamma interferon monoclonal antibody is purchased from Xiamen Wan Taikairui biology skill
Art company limited；Rv0733、Rv1978、Rv1980c(MPT64)、Rv1981c、Rv1984c(CFP-21)、Rv1985c、
Rv2654c(TB7.7)、Rv3425、Rv3429、Rv3615c(ESPC)、Rv3873、Rv3874(CFP-10)、Rv3875(ESAT-
6), Rv3878, Rv3879c antigen or polypeptide or its analog or its chimeric recombinant antigens are purchased from Sigma Co., USA；Plant blood
Solidifying element L (phytohaemagglutinin-L, PHA-L) is purchased from Sigma Co., USA；4-[the 2-(4-isothiocyanatophenyl) of europium
Acetenyl]-2,6 pairs of { [double (the carboxymethyl)-amino of N, N] methyl } pyridine reagent are purchased from PerkinElmer company of the U.S.；Blank anti-
Cup is answered to be purchased from Nunc company of Denmark；The gamma interferon biological reference material of numbering 87/586 is purchased from United Kingdom National biological product
Calibrating institute；Agarose gel Sepharose CL-6B is purchased from Pharmacia Inc., Sweden；Washing liquid, test buffer, FWZ-I type
Trace shaker, the full-automatic Enzyme-linked washing-board of DEM-III type are provided by Guangzhou City Fenghua Biological Engineering Co., Ltd；
VictorTM2D1420 type TRFIA instrument is purchased from Perkin Elmer company of the U.S.；AQT90FLEX rapid immunoassay instrument is purchased from pellet
Wheat Radiometer company.Other reagent are domestic analytical pure；Tuberculosis infection T cell detection kit (exempt from by release in vitro enzyme connection
Epidemic disease method) it is purchased from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd..
Embodiment 1
The dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
A kind of dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection, this test kit
Including:
1) test reaction cup, its endoperidium has Streptavidin, connection to have biotinylation gamma interferon monoclonal antibody,
And containing europium labelling gamma interferon monoclonal antibody；
2) calibration object reaction cup, its endoperidium has Streptavidin, connection to have biotinylation gamma interferon monoclonal anti
Body, and containing europium labelling gamma interferon monoclonal antibody and the calibration object of variable concentrations；Calibration object contains 0,6,300,15000pg/
The gamma interferon antigen of mL；
3) blank culture tube, it is contained within the heparin sodium of 15IU/mL blood, and inside is vacuum negative pressure condition；
4) test cultures pipe, its be contained within the heparin sodium of 15IU/mL blood and the Rv3874 (CFP-10) of 5 μ g/mL and
The combination of Rv3875 (ESAT-6), and inside is vacuum negative pressure condition；
5) positive control culture tube, it is contained within the heparin sodium of 15IU/mL blood, and 10 μ g/mL phytohaemagglutinin L, and
Inside is vacuum negative pressure condition；
6) test buffer, it is contained within the sodium chloride of 0.15mol/L, the tween 20 of 0.6mL/L, 30ppmPreservative, the 4-hydroxyethyl piperazine ethanesulfonic acid of 5mmol/L, pH7.8 of ethylenediaminetetraacetic acid of 100 μm ol/L
Buffer；
7) concentrating washing liquid, it is contained within the sodium chloride of 1.125g/L, the tween 20 of 0.2mL/L, 30ppmThe tris-HCI buffer of the 50mmol/L of preservative, pH7.8.
The preparation method of described test reaction cup is: by dilute for the phosphate buffer of Streptavidin 50mmol/L, pH7.8
It is interpreted into optimum concentration 5 μ g/mL, takes 50 μ L and join in blank reaction cup, 2～8 DEG C of refrigerators are hatched (22 ± 2) h, washs 1 time.
Biotinylation gamma interferon monoclonal antibody 50mmol/L, the phosphate buffer of pH7.8 is diluted to optimum concentration 5 μ g/mL,
Take 50 μ L and join in the reaction cup being coated with Streptavidin, 2～8 DEG C of refrigerators in hatch (22 ± 2) h, wash 2 times.Then
Being carefully added into 50 μ L sealing coat solution in each cup, dried overnight under (36 ± 1) DEG C, 5% damp condition, then at sealing coat
On be carefully added into the europium labelling gamma interferon monoclonal antibody of 1 μ L the suitableeest working concentration 200ng/mL, immediately at the hot blast of 60 DEG C
Under dry up, and vacuum is crossed and is moulded packaging, and 2～8 DEG C save backup.
Described sealing coat use sealing coat solution be the sodium chloride containing 0.12mol/L, the sodium azide of 0.38g/L,
The cattle gamma Globulin of 10g/L, the bovine serum albumin of 50g/L, the trehalose of 50g/L, 0.8g/L aboriginal mouse IgG, 0.05g/L
Degeneration mouse IgG, 2g/L casein, the trishydroxymethylaminomethane-salt of 50mmol/L, pH7.8 of heparin sodium of 37.5U/mL
Acid.
The solution of dilution europium labelling gamma interferon monoclonal antibody therein be the sodium chloride containing 150mmol/L,
The sodium azide of 0.5g/L, the deionization bovine serum albumin of 2g/L, three hydroxyls of 50mmol/L, pH7.8 of trehalose of 125g/L
Aminomethane-hydrochloric acid.
Europium labelling gamma interferon method for preparing monoclonal antibody therein is that labelling is with in gamma interferon monoclonal antibody
Liquid NAP-10 post be replaced as the carbonate buffer solution of 50mmol/L, pH9.0, and make the concentration of traget antibody reach 2～
5mg/mL, and the 4-[2-(4-isothiocyanatophenyl) acetenyl]-2 of the europium with 3 times of quality, 6 pairs of { [double (carboxymethyl)-ammonia of N, N
Base] methyl } pyridine reagent is blended in oscillation incubation (22 ± 2) h at 2～8 DEG C, reactant liquor 50mmol/L, pH 7.80 3 hydroxyl first
Sepharose CL-6B post (1cm × 40cm) chromatography of base aminomethane-hydrochloride buffer balance, A280 monitoring is collected respectively
Albumen eluting peak.
Wherein, the outer safety helmet color of blank culture tube is green；The outer safety helmet color of test cultures pipe is yellow
Color；The outer safety helmet color of positive control culture tube is red.
The preparation method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The test kit preparation method of the present embodiment, comprises the following steps:
1) test reaction cup is prepared: use the dry reagent rapid time of the present embodiment tuberculosis infection T cell detection to differentiate glimmering
The preparation method of the test reaction cup in light immunoassay kits is prepared；
2) reaction cup of calibration object is prepared: with United Kingdom National biological products assay institute (National Institute for
Biological Standards and Control, NIBSC) the gamma interferon biological reference material of numbering 87/586
(1IU/mL ≈ 50pg/mL) for comparison, by Human Lymphobl Toid Interferon antigen step 1) in sealing coat solution be diluted to 0,6,
The calibration object of 300,15000pg/mL, in step 1) on the basis of test reaction cup in europium labelling gamma interferon monoclonal
Being carefully added into the calibration object of 10 μ L on antibody layer, dry up immediately under the hot blast of 60 DEG C, the every concentration of calibration object arranges 2 holes, and very
Empty mistake moulds packaging, and 2～8 DEG C save backup.
3) blank culture tube is prepared: in clean test tube (plastics or glass), add the heparin sodium of 15IU/mL blood.
Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and making negative pressure of vacuum in test tube is 1mL/ pipe.In the lid of culture tube contains
Plug and its outer safety helmet of coupling, the outer safety helmet color of blank culture tube uses green.
4) test cultures pipe is prepared: in clean test tube (plastics or glass), add the heparin sodium of 15IU/mL blood, and
The Rv3874 (CFP-10) of 5 μ g/mL and the combination of Rv3875 (ESAT-6).Full-automatic vacuum-pumping capping machine uprightly put into by test tube
In, making negative pressure of vacuum in test tube is 1mL/ pipe.The lid of culture tube contains Nei Sai and it mates outer safety helmet, outer safety helmet color
Use yellow.
5) positive control culture tube is prepared: in clean test tube (plastics or glass), add the heparin sodium of 15IU/mL blood,
And 10 phytohaemagglutinin L of μ g/mL.Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and in making test tube, negative pressure of vacuum is
1mL/ manages.The lid of culture tube contains Nei Sai and it mates outer safety helmet, and the outer safety helmet color of positive control culture tube uses
Red.
6) test buffer is prepared: the 4-hydroxyethyl piperazine ethanesulfonic acid buffer of 5mmol/L, pH7.8 contains
The sodium chloride of 0.15mol/L, the tween 20 of 0.6mL/L, 30ppmPreservative, the second two of 100 μm ol/L
Amine tetraacethyl.It is distributed into semi-finished product.
7) preparation concentrates washing liquid: contain in the tris-HCI buffer of 50mmol/L, pH7.8
The sodium chloride of 1.125g/L, the tween 20 of 0.2mL/L, 30ppmPreservative.It is distributed into semi-finished product.
8) subpackage test buffer and concentration washing liquid；
9) label.
10) finished product assembles.
Above-mentioned steps products obtained therefrom subpackage is semi-finished product.Extract 3 parts out through specificity, elaboration, sensitivity and to stablize
Property assay approval just can be assembled into tuberculosis infection T cell detection kit (release in vitro immunofluorescence).After being completed also
Need to inspect by random samples qualified after just can dispatch from the factory.
The detection method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The detection method of the test kit of the present embodiment comprises the following steps:
1) release in vitro of human gamma-interferon
1. press blank pipe N, testing tube T and the order of positive control pipe P, extract respectively in 3 kinds of culture tubes or add
Enter 1mL whole blood sample (heparin sodium or clarin anticoagulant), at once reverse mixing more than 6 times, make content in 3 kinds of culture tubes with
Blood fully mixes；
2. above 3 kinds of culture tubes added with whole blood are put into 37 (± 1) DEG C constant incubator after blood collection in 4 hours
Middle cultivation 16～24 hours.Incubation keep culture tube upright.
3. cultivate after terminating, culture tube is centrifuged 10min with the rotating speed of 3000～5000r/min, takes blood plasma for inspection
Survey the concentration of gamma interferon in each culture tube.Not disturbance hemocyte is it must be particularly noted that when drawing plasma sample.As can not
Detection in time, it is necessary to supernatant blood plasma is taken out from culture tube, is placed in the clean centrifuge tube of 1.5mL, deposits 7 days for 2～8 DEG C, long
Phase storage should preserve under the conditions of at least-20 DEG C, and number of freezing and thawing should be not higher than 3 times.
2) detection by quantitative of human gamma-interferon
(1) reagent prepares
Test reaction cup and the calibration object reaction cup of reagent and requirement are balanced to room temperature (20～25 DEG C).Remaining
It is airtight and in 2～8 DEG C of preservations that reaction cup inserts valve bag in time.
(2) test operation
1. the calibration object of every concentration presets 2 holes, adds 30 μ L test buffer (calibration object concentration in calibration object reaction cup
Become 0,2,100,5000pg/mL), the test buffer drawing 10 μ L samples and absorption 20 μ l is sequentially added into test reaction cup
In.
2. reaction cup is at 37 DEG C, oscillation incubation 10min.
3., after hatching end, wash 4 times with cleaning mixture.
4. under 60 DEG C of hot blasts, it is dried 35s.
5. time-resolved fluorescence immunoassay instrument is used to carry out fluorescent value analysis of accounts.Fig. 1 be test kit of the present invention γ-
Interferon detection by quantitative dose-response curve.
Embodiment 2
The dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
A kind of dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection, this test kit
Including:
1) test reaction cup, its endoperidium has Streptavidin, biotinylation gamma interferon monoclonal antibody and europium labelling
Gamma interferon monoclonal antibody；
2) calibration object reaction cup, with embodiment 1；
3) blank culture tube, it is contained within the clarin of 9.8IU/mL blood, and inside is vacuum negative pressure condition；
4) test cultures pipe, its be contained within the clarin of 9.8IU/mL blood and the Rv3874 (CFP-10) of 1 μ g/mL and
The chimeric recombinant antibody of Rv3875 (ESAT-6), and inside is vacuum negative pressure condition；
5) positive control culture tube, it is contained within the clarin of 9.8IU/mL blood, and 1 μ g/mL ConA
, and inside is vacuum negative pressure condition (ConA)；
6) test buffer, with embodiment 1；
7) washing liquid is concentrated, with embodiment 1.
The preparation method of described test reaction cup is: by dilute for the phosphate buffer of Streptavidin 50mmol/L, pH7.8
It is interpreted into optimum concentration 0.1 μ g/mL, takes 50 μ L and join in blank reaction cup, 2～8 DEG C of refrigerators are hatched (22 ± 2) h, washs 1
Secondary.Biotinylation gamma interferon monoclonal antibody 50mmol/L, the phosphate buffer of pH7.8 is diluted to optimum concentration 0.1 μ
G/mL, takes 50 μ L and joins in the reaction cup being coated with Streptavidin, 2～8 DEG C of refrigerators in hatch (22 ± 2) h, wash 2 times.
The most each cup is carefully added into 50 μ L sealing coat solution, dried overnight under (36 ± 1) DEG C, 5% damp condition, then every
The europium labelling gamma interferon monoclonal antibody of 1 μ L the suitableeest working concentration 100ng/mL it is carefully added into, immediately at 60 DEG C on absciss layer
Drying up under hot blast, and vacuum is crossed and moulded packaging, 2～8 DEG C save backup.
The sealing coat solution that described sealing coat uses is with embodiment 1.
The solution of dilution europium labelling gamma interferon monoclonal antibody therein is with embodiment 1.
Europium labelling gamma interferon method for preparing monoclonal antibody therein is with embodiment 1.
Wherein, the outer safety helmet color of blank culture tube is green；The outer safety helmet color of test cultures pipe is yellow
Color；The outer safety helmet color of positive control culture tube is red.
The preparation method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The test kit preparation method of the present embodiment, comprises the following steps:
1) test reaction cup is prepared: use the dry reagent rapid time of the present embodiment tuberculosis infection T cell detection to differentiate glimmering
The preparation method of the test reaction cup in light immunoassay kits is prepared；
2) reaction cup of calibration object is prepared: with embodiment 1.
3) blank culture tube is prepared: in clean test tube (plastics or glass), add the heparin of 9.8IU/mL blood
Potassium.Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and making negative pressure of vacuum in test tube is 1mL/ pipe.The lid of culture tube contains
Interior plug and its outer safety helmet of coupling, the outer safety helmet color of blank culture tube uses green.
4) test cultures pipe is prepared: in clean test tube (plastics or glass), add the clarin of 9.8IU/mL blood, with
And 1 μ g/mL Rv3874 (CFP-10) and the chimeric recombinant antibody of Rv3875 (ESAT-6).Test tube is uprightly put into and is automatically taken out very
In empty capping machine, making negative pressure of vacuum in test tube is 1mL/ pipe.The lid of culture tube contains Nei Sai and it mates outer safety helmet, outer peace
Full cap color uses yellow.
5) positive control culture tube is prepared: in clean test tube (plastics or glass), add the heparin of 9.8IU/mL blood
Potassium, and the ConA (ConA) of 1 μ g/mL.Test tube is uprightly put in full-automatic vacuum-pumping capping machine, makes vacuum in test tube
Negative pressure is 1mL/ pipe.The lid of culture tube contains Nei Sai and it mates outer safety helmet, the outer safety helmet face of positive control culture tube
Color uses redness.
6) test buffer is prepared: with embodiment 1.
7) preparation concentrates washing liquid: with embodiment 1.
8) subpackage test buffer and concentration washing liquid；
9) label.
10) finished product assembles.
Above-mentioned steps products obtained therefrom subpackage is semi-finished product.Extract 3 parts out through specificity, elaboration, sensitivity and to stablize
Property assay approval just can be assembled into tuberculosis infection T cell detection kit (release in vitro immunofluorescence).After being completed also
Need to inspect by random samples qualified after just can dispatch from the factory.
The detection method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The detection method of the test kit of the present embodiment is with embodiment 1.
Embodiment 3
The dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
A kind of dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection, this test kit
Including:
1) test reaction cup, its endoperidium has Streptavidin, biotinylation gamma interferon monoclonal antibody and europium labelling
Gamma interferon monoclonal antibody；
2) calibration object reaction cup, with embodiment 1；
3) blank culture tube, it is contained within the heparin sodium of 28IU/mL blood, and inside is vacuum negative pressure condition；
4) test cultures pipe, its heparin sodium being contained within 28IU/mL blood and the Rv0733 of 10 μ g/mL, and inside is vacuum
Negative pressure state；
5) positive control culture tube, it is contained within the heparin sodium of 28IU/mL blood, and 20 μ g/mL ConAs
, and inside is vacuum negative pressure condition (ConA)；
6) test buffer, with embodiment 1；
7) washing liquid is concentrated, with embodiment 1.
The preparation method of described test reaction cup is: by dilute for the phosphate buffer of Streptavidin 50mmol/L, pH7.8
It is interpreted into optimum concentration 10 μ g/mL, takes 50 μ L and join in blank reaction cup, 2～8 DEG C of refrigerators are hatched (22 ± 2) h, washs 1
Secondary.Biotinylation gamma interferon monoclonal antibody 50mmol/L, the phosphate buffer of pH7.8 is diluted to optimum concentration 10 μ g/
ML, takes 50 μ L and joins in the reaction cup being coated with Streptavidin, 2～8 DEG C of refrigerators in hatch (22 ± 2) h, wash 2 times.So
Being carefully added into 50 μ L sealing coat solution in rear each cup, dried overnight under (36 ± 1) DEG C, 5% damp condition, then in isolation
The europium labelling gamma interferon monoclonal antibody of 1 μ L the suitableeest working concentration 400ng/mL it is carefully added into, immediately the heat of 60 DEG C on layer
Drying up under wind, and vacuum is crossed and moulded packaging, 2～8 DEG C save backup.
The sealing coat solution that described sealing coat uses is with embodiment 1.
The solution of dilution europium labelling gamma interferon monoclonal antibody therein is with embodiment 1.
Europium labelling gamma interferon method for preparing monoclonal antibody therein is with embodiment 1.
Wherein, the outer safety helmet color of blank culture tube is green；The outer safety helmet color of test cultures pipe is yellow
Color；The outer safety helmet color of positive control culture tube is red.
The preparation method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The test kit preparation method of the present embodiment, comprises the following steps:
1) test reaction cup is prepared: use the dry reagent rapid time of the present embodiment tuberculosis infection T cell detection to differentiate glimmering
The preparation method of the test reaction cup in light immunoassay kits is prepared；
2) reaction cup of calibration object is prepared: with embodiment 1.
3) blank culture tube is prepared: in clean test tube (plastics or glass), add the heparin sodium of 28IU/mL blood.
Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and making negative pressure of vacuum in test tube is 1mL/ pipe.In the lid of culture tube contains
Plug and its outer safety helmet of coupling, the outer safety helmet color of blank culture tube uses green.
4) test cultures pipe is prepared: in clean test tube (plastics or glass), add the heparin sodium of 28IU/mL blood, and
The Rv0733 of 10 μ g/mL.Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and making negative pressure of vacuum in test tube is 1mL/ pipe.Training
The lid supporting pipe contains Nei Sai and its outer safety helmet of coupling, and outer safety helmet color uses yellow.
5) positive control culture tube is prepared: in clean test tube (plastics or glass), add the heparin sodium of 28IU/mL blood,
And 20 ConAs (ConA) of μ g/mL.Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and in making test tube, vacuum is born
Press and manage for 1mL/.The lid of culture tube contains Nei Sai and it mates outer safety helmet, the outer safety helmet color of positive control culture tube
Use redness.
6) test buffer is prepared: with embodiment 1.
7) preparation concentrates washing liquid: with embodiment 1.
8) subpackage test buffer and concentration washing liquid；
9) label.
10) finished product assembles.
Above-mentioned steps products obtained therefrom subpackage is semi-finished product.Extract 3 parts out through specificity, elaboration, sensitivity and to stablize
Property assay approval just can be assembled into tuberculosis infection T cell detection kit (release in vitro immunofluorescence).After being completed also
Need to inspect by random samples qualified after just can dispatch from the factory.
The detection method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The detection method of the test kit of the present embodiment is with embodiment 1.
Embodiment 4
The dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
A kind of dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection, this test kit
Including:
1) test reaction cup, its endoperidium has Streptavidin, biotinylation gamma interferon monoclonal antibody and europium labelling
Gamma interferon monoclonal antibody；
2) calibration object reaction cup, with embodiment 1；
3) blank culture tube, it is contained within the clarin of 20IU/mL blood, and inside is vacuum negative pressure condition；
4) test cultures pipe, its clarin being contained within 20IU/mL blood and the Rv1978 of 6 μ g/mL, Rv1980,
The compositions of Rv1981c, Rv1984, Rv1985c and Rv2654c, and inside is vacuum negative pressure condition；
5) positive control culture tube, it is contained within the clarin of 20IU/mL blood, and 12 μ g/mL ConAs
, and inside is vacuum negative pressure condition (ConA)；
6) test buffer, with embodiment 1；
7) washing liquid is concentrated, with embodiment 1.
The preparation method of described test reaction cup is: by dilute for the phosphate buffer of Streptavidin 50mmol/L, pH7.8
It is interpreted into optimum concentration 6 μ g/mL, takes 50 μ L and join in blank reaction cup, 2～8 DEG C of refrigerators are hatched (22 ± 2) h, washs 1 time.
Biotinylation gamma interferon monoclonal antibody 50mmol/L, the phosphate buffer of pH7.8 is diluted to optimum concentration 6 μ g/mL,
Take 50 μ L and join in the reaction cup being coated with Streptavidin, 2～8 DEG C of refrigerators in hatch (22 ± 2) h, wash 2 times.Then
Being carefully added into 50 μ L sealing coat solution in each cup, dried overnight under (36 ± 1) DEG C, 5% damp condition, then at sealing coat
On be carefully added into the europium labelling gamma interferon monoclonal antibody of 1 μ L the suitableeest working concentration 300ng/mL, immediately at the hot blast of 60 DEG C
Under dry up, and vacuum is crossed and is moulded packaging, and 2～8 DEG C save backup.
The sealing coat solution that described sealing coat uses is with embodiment 1.
The solution of dilution europium labelling gamma interferon monoclonal antibody therein is with embodiment 1.
Europium labelling gamma interferon method for preparing monoclonal antibody therein is with embodiment 1.
Wherein, the outer safety helmet color of blank culture tube is green；The outer safety helmet color of test cultures pipe is yellow
Color；The outer safety helmet color of positive control culture tube is red.
The preparation method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The test kit preparation method of the present embodiment, comprises the following steps:
1) test reaction cup is prepared: use the dry reagent rapid time of the present embodiment tuberculosis infection T cell detection to differentiate glimmering
The preparation method of the test reaction cup in light immunoassay kits is prepared；
2) reaction cup of calibration object is prepared: with embodiment 1.
3) blank culture tube is prepared: in clean test tube (plastics or glass), add the heparin sodium of 20IU/mL blood.
Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and making negative pressure of vacuum in test tube is 1mL/ pipe.In the lid of culture tube contains
Plug and its outer safety helmet of coupling, the outer safety helmet color of blank culture tube uses green.
4) test cultures pipe is prepared: in clean test tube (plastics or glass), add the heparin sodium of 20IU/mL blood, and
The compositions of Rv1978, Rv1980c, Rv1981c, Rv1984c, Rv1985c and Rv2654c of 6 μ g/mL.Test tube is uprightly put into
In full-automatic vacuum-pumping capping machine, making negative pressure of vacuum in test tube is 1mL/ pipe.The lid of culture tube contains outside Nei Sai and its coupling
Safety helmet, outer safety helmet color uses yellow.
5) positive control culture tube is prepared: in clean test tube (plastics or glass), add the heparin sodium of 20IU/mL blood,
And 12 ConAs (ConA) of μ g/mL.Test tube is uprightly put in full-automatic vacuum-pumping capping machine, and in making test tube, vacuum is born
Press and manage for 1mL/.The lid of culture tube contains Nei Sai and it mates outer safety helmet, the outer safety helmet color of positive control culture tube
Use redness.
6) test buffer is prepared: with embodiment 1.
7) preparation concentrates washing liquid: with embodiment 1.
8) subpackage test buffer and concentration washing liquid；
9) label.
10) finished product assembles.
Above-mentioned steps products obtained therefrom subpackage is semi-finished product.Extract 3 parts out through specificity, elaboration, sensitivity and to stablize
Property assay approval just can be assembled into tuberculosis infection T cell detection kit (release in vitro immunofluorescence).After being completed also
Need to inspect by random samples qualified after just can dispatch from the factory.
The detection method of the dry reagent rapid time resolved fluorometric immunoassay kits of tuberculosis infection T cell detection
The detection method of the test kit of the present embodiment is with embodiment 1.
Embodiment 5
The positive cutoff value of the test kit of the present invention or the checking of reference interval
The positive cutoff value of tuberculosis infection T cell detection kit (release in vitro immunofluorescence) prepared by the present invention,
With reference to People's Republic of China's inspection and quarantining for import/export industry standard SN/T 3312-2012 mycobacterium tuberculosis gamma interferon
Result decision method in external detection method carries out interpretation, its interpretation method such as table 1.
Result decision method in table 1 mycobacterium tuberculosis gamma interferon external detection method
Note: the concentration of gamma interferon during N is blank pipe N in table, P is the dense of gamma interferon in positive control pipe P
Degree, T is the concentration of gamma interferon in testing tube T.
Checking test: collect whole blood sample totally 448 example.Wherein sample type is as follows: (1) passes through sputum smear or expectorant
Liquid cultivates the venous blood of the experimenter being defined as active tuberculosis, 77 examples；(2) feminine gender cultivated by sputum smear and sputum, but
(before and after diagnostic treatment, two chest films showed are effective by other means；Bronchial perfusate, chest X-ray inspection, pathology
Check, PPD strong positive person have two positives) venous blood of the person that confirms as m tuberculosis infection, 80 parts；(3) in pressing
Medical association of China clinic diagnosis guide tuberculosis fascicle requires the venous blood of the experimenter of the outer tuberculosis of lung of clinical definite, 25 examples；
(4) pulmonary carcinoma, non-tuberculosis respiratory tract disease and make a definite diagnosis other diseases patient 114 example；Chlamydia infection 5 example, pregnant 7 examples；(5) through asking
Volume investigation is from tubercule bacillus low exposure crowd, and all without clinical symptoms, without tuberculosis contact history, chest X-ray is normal,
Healthy population 134 example of having a medical check-up that tuberculin test is negative；(5) outpatient service follow-up case 7 example.Result such as table 2～table 3.According to
The result interpretation method of upper industry standard, with clinical diagnosis for " goldstandard ", the positive coincidence rate of tuberculosis group is 99.45%,
The negative match-rate of non-tuberculosis group is 96.54%, meets the gamma interferon release in vitro method of the tuberculosis infection T cell of report
The requirement of method of quality control: positive reference material coincidence rate is not less than 75%, and negative reference product coincidence rate is for being not less than 75%.
Interpretation knot can be carried out according to the result decision method in the mycobacterium tuberculosis gamma interferon external detection method of industry standard
Really.
The distribution of results situation of table 2 448 example sample
The result accordance of table 3 448 example sample
Embodiment 6
The analytical performance evaluation index of the test kit of the present invention
The Performance Evaluating Indexes of the tuberculosis infection T cell detection kit (release in vitro immunofluorescence) of the present invention is such as
Under:
1. blank limit: Parallel testing calibration object A 10 hole, calculates average (x) and the standard deviation (s) of its detection fluorescent value, profit
Calculating concentration value corresponding to " x+2s " with dose-response curve to limit for blank, blank limit is better than 2pg/mL.
2. negative reference product coincidence rate: detect 20 parts of fresh peripheral anticoagulants without the healthy volunteer of tuberculosis clinical symptoms
Blood sample, its negative reference product coincidence rate should be not less than 80%.Specific requirement: the healthy volunteer without tuberculosis clinical symptoms should
Comprise different grades of tuberculin skin test crowd, i.e. should comprise tuberculin skin test strong positive person (72h scleroma or blush diameter
>=15mm or with blister), tuberculin skin test positive (72h scleroma or blush diameter >=5mm) and tuberculin skin test the moon
Property person (72h scleroma or blush diameter ＜ 5mm) this 3 type.
3. positive reference material coincidence rate: detect 10 parts of mycobacterium tuberculosis smears and (or) cultivate positive tuberculosis patient
The fresh peripheral anticoagulation sample of (bacterium sun) and 10 parts of mycobacterium tuberculosis smears and (or) cultivate negative clinical definite tuberculosis
The fresh peripheral anticoagulation sample of patient's (bacterium is cloudy), its positive reference material coincidence rate should be not less than 80%.
4. precision: after application positive stimulus, blood plasma or enterprise's elaboration reference material detect as sample, parallel
Testing 10 holes, calculate the coefficient of variation (CV) value of concentration value, in batch, CV value should be not higher than 10.0%.3 batches of quantity-produced
Between test kit batch between CV value should be not higher than 20.0%.
The most linear: in the 2～5000pg/mL ranges of linearity, linearly dependent coefficient r should be not less than 0.9900.
6. accuracy: in the range of test kit calibration object B～F, with the test kit supporting calibration object detection world after calibration
The relative deviation of reference material or enterprise's reference material, the measured value of reference material and sign value should be in the range of ± 20.0%.
7. stability: preserve to finished product effect duration 2 months interior samples under the conditions of physical holding of the stock to blank limit, feminine gender
The indexs such as reference material coincidence rate, positive reference material coincidence rate, withinrun precision, linear and accuracy detect, testing result
Meet requirements above.
Embodiment 7
The interference test evaluation of the test kit of the present invention
The analysis Evaluation on specificity of the tuberculosis infection T cell detection kit (release in vitro immunofluorescence) of the present invention is such as
Under:
Detection is following disturbs sample, all without there is false positive or false negative result.Bilirubin 818 μm ol/L, hemoglobin
180g/L, the triglyceride 21.54mmol/L noiseless effect of testing result to this test kit.
Table 4 disturbs Sample size
Embodiment 8
The test kit of the present invention and the equivalence evaluation of reference kit
The equivalence of tuberculosis infection T cell detection kit (release in vitro immunofluorescence) prepared by the present invention is evaluated such as
Under:
In 340 example clinical trial samples, include 165 examples through living that the Diagnosis of Tuberculosis standard that this clinical trial specifies is made a definite diagnosis
Institute's tubercular's sample, 25 example outpatient service samples, 70 example pulmonary carcinoma and non-tuberculosis respiratory disease patient sample, 40 examples are had a medical check-up Healthy People
Sample, 40 example high-risk group's samples.With clinical diagnosis as goldstandard, reagent to be evaluated tuberculosis group sensitivity up to
85.71%, the specificity in non-tuberculosis and healthy population is up to 84.43%, and thick concordance rate is up to 85.19%.With Beijing ten thousand
The tuberculosis infection T cell detection kit (release in vitro euzymelinked immunosorbent assay (ELISA)) that safe biological Pharmaceutical limited company produces is comparison
Reagent, the results detailed in Table 5 and table 6.
Table 5 sample type and recall rate
Table 6 reagent to be evaluated and contrast agents testing result compare
Test kit to be evaluated has positive implicative (r with contrast agents box detection clinical sample resultp=0.70)；Positive symbol
Conjunction rate reaches 100%；Negative match-rate reaches 98.11%；Total coincidence rate P0It is 99.12%；Kappa index is 0.9824, for height
Unanimously (κ ＞ 0.75), it is believed that two system equivalences；The positive rate result difference of reagent to be evaluated and contrast agents is without statistics
Meaning (χ2=1.33 ＜ 3.84, P ＞ 0.05), it is believed that the assay of two kinds of methods is consistent.3 example reagent to be evaluated are with right
The sample not being inconsistent according to reagent, 1 example reagent to be evaluated is positive and specimen that contrast agents is negative is bacterium the moon pulmonary tuberculosis group patient in hospital
Sample, 2 example reagent to be evaluated are negative and specimen that contrast agents is positive is high-risk group person's sample, and reagent diplopore to be evaluated is rechecked
Result meets with result of the test for the first time, and PPD skin test is strong positive, but expectorant bacterium smear testing result is negative, and chest x-ray is examined
Look into no abnormality seen, may be latent tuberculosis mycobacterial infections person.Square grids table is set up, such as table 7 according to clinical test results.
The contrast test evaluation result of 7 340 parts of clinical trial samples of table
The present invention uses dry reagent rapid time resolved fluorometric immuno analytical method, solves conventional Conventional Time and differentiates
Fluorescence immunoassay technology reagent component is various, and liquid reagent is not sufficiently stable, complex operation, the longest shortcoming.And this
Invention solves during conventional tuberculosis infection T cell measures needs independent blood taking tube and culture tube, trivial operations, easily go out
The shortcomings such as mistake, automaticity are the highest；Present invention achieves blood taking tube and culture tube integration, negative pressure of vacuum is accurately taken a blood sample；3 kinds
Culture tube uses different colours label and cap to make a distinction；Avoid during whole blood cell culture blood taking tube uncap, subpackage blood sample,
The processes such as secondary marker, it is therefore prevented that sample is uncapped at blood taking tube, secondary pollution during subpackage blood sample, during decreasing this
Potential bio-safety risk, also prevent secondary marker process produce mistake, as long as and after whole blood cell culture routinely from
I.e. can be placed in after the heart and on fully automatic system, be sampled detection, reduce working strength and improve work efficiency.The present invention
Culture tube have blood taking tube and culture tube integration, blood collection procedure decreases consumptive material uses, vacuum is accurately taken a blood sample, be suitable to from
The features such as dynamicization operation.The test kit of the present invention has testing result accuracy, highly sensitive, high specificity, good stability, inspection
The features such as survey method is easy, efficient, are suitable for real-time test (point-of-care testing, POCT).
Last institute is it should be noted that, the present invention is only protected by above example in order to technical scheme to be described
Protecting the restriction of scope, although being explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent, without deviating from the essence of technical solution of the present invention
And scope.
Claims (10)
1. the rapid time resolved fluorometric immunoassay kits being applicable to the detection of tuberculosis infection T cell, it is characterised in that:
Including: 1) test reaction cup, its endoperidium has Streptavidin and the biotinylation γ-interference being combined on Streptavidin
Element monoclonal antibody, and the gamma interferon monoclonal antibody containing lanthanide series labelling；2) calibration object reaction cup, its endoperidium
There are Streptavidin and the biotinylation gamma interferon monoclonal antibody being combined on Streptavidin, and containing europium labelling
Gamma interferon monoclonal antibody and the calibration object of variable concentrations；3) blank culture tube, it is contained within anticoagulative substance；4)
Test cultures pipe, it is contained within anticoagulative substance and antigen of mycobacterium tuberculosis specific T-cells can be stimulated to produce γ-interference
The stimulus object of element, stimulus object be in the antigen of mycobacterium tuberculosis and derivative epitope peptide thereof and their analog extremely
Few one；5) positive control culture tube, it is contained within anticoagulative substance and cell agglutinin；6) test buffer；And 7) dense
Contracting washing liquid.
Test kit the most according to claim 1, it is characterised in that: described stimulus object be mycobacterium tuberculosis Rv0733,
Rv1978、Rv1980c、Rv1981c、Rv1984c、Rv1985c、Rv2654c、Rv3425、Rv3429、Rv3615c、Rv3873、
In Rv3874, Rv3875, Rv3878, Rv3879c antigen, their derivative epitope peptide, chimeric recombinant antigens and the like
At least one.
Test kit the most according to claim 1, it is characterised in that: described stimulus object is the combination of Rv3874 and Rv3875.
Test kit the most according to claim 1, it is characterised in that: described anticoagulative substance is heparin sodium and/or Lithium acid heparin；
Described cell agglutinin is phytohemagglutinin and/or ConA.
Test kit the most according to claim 1, it is characterised in that: described test buffer is the chlorine containing 0.15mol/L
Change sodium, the tween 20 of 0.6mL/L, 30ppmPreservative, the ethylenediaminetetraacetic acid of 100 μm ol/L
The 4-hydroxyethyl piperazine ethanesulfonic acid buffer of 5mmol/L, pH7.8.
Test kit the most according to claim 1, it is characterised in that: described concentration washing liquid is the chlorination containing 1.125g/L
Sodium, the tween 20 of 0.2mL/L, 30ppmThe trihydroxy methyl amino of 50 mmol/L of preservative, pH7.8
Methane-hydrochloride buffer.
Test kit the most according to claim 1, it is characterised in that: the preparation method of described test reaction cup is: by described
Streptavidin is diluted to concentration 0.1～10 μ g/mL, joins in skip test reaction cup, hatches 20～24h at 2～8 DEG C,
Washing；Described biotinylation gamma interferon monoclonal antibody is diluted to concentration 0.1～10 μ g/mL, joins and be coated with strepto-
In the test reaction cup of Avidin, hatch 20～24h at 2～8 DEG C, washing；The most each cup adds sealing coat solution, is dried
Rear formation sealing coat, to prevent the gamma interferon monoclonal antibody of lanthanide series labelling to be adsorbed onto on test reaction cup, finally exists
The gamma interferon monoclonal antibody of the described lanthanide series labelling that working concentration is 100～400ng/mL is added on sealing coat, dry
Described test reaction cup is i.e. obtained after dry.
Test kit the most according to claim 7, it is characterised in that: the sealing coat solution that described sealing coat uses is for containing
The sodium chloride of 0.12mol/L, the sodium azide of 0.38g/L, the cattle gamma Globulin of 10g/L, the bovine serum albumin of 50g/L, 50g/L
Trehalose, degeneration mouse IgG, 2g/L casein of 0.8g/L aboriginal mouse IgG, 0.05g/L, the heparin sodium of 37.5U/mL
Trishydroxymethylaminomethane-the hydrochloric acid of 50mmol/L, pH7.8.
9. according to the rapid time resolved fluorometric being applicable to the detection of tuberculosis infection T cell according to any one of claim 1～8
The detection method of immunoassay kits, it is characterised in that: comprise the following steps:
(1) in blank culture tube, test cultures pipe and three kinds of culture tubes of positive control culture tube, whole blood is added respectively
This, and reverse make the content in whole blood sample and three kinds of culture tubes mix for several times；
(2) three kinds of culture tubes added with whole blood sample are placed at 35～38 DEG C upright cultivation 16～24 hours；
(3), after cultivation terminates, by centrifugal, blood plasma in the whole blood sample of three kinds of culture tubes is separated；
(4) that presets in calibration object reaction cup is separately added into test buffer containing in the hole of the calibration object of variable concentrations, and
By step 3) in blood plasma in the whole blood sample that separates and test buffer add in test reaction cup；
(5) by calibration object reaction cup and test reaction cup at 30～40 DEG C, oscillation incubation 5～20min；
(6) washing, dried, use time-resolved fluorescence immunoassay instrument to carry out fluorescent value counting.
Detection method the most according to claim 9, it is characterised in that: in described step (3), centrifugal refer to 3000～
10～20min it are centrifuged under the rotating speed of 5000r/min；
In described step (6), it is dried and refers to be dried under 50～70 DEG C of hot blasts 30～40s.
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| CN108548919A (en) * | 2018-04-04 | 2018-09-18 | 广州市雷德生物科技有限公司 | A kind of pre-coated elisa plate and its preparation method and application |
| CN110221063A (en) * | 2019-07-03 | 2019-09-10 | 广州市丰华生物工程有限公司 | A kind of rapid time resolved fluorometric immunoassay detection kit and its detection method |
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