CN109142334A - The kit and its detection method of spatial neighbor chemoluminescence method detection tumor necrosis factor - Google Patents
The kit and its detection method of spatial neighbor chemoluminescence method detection tumor necrosis factor Download PDFInfo
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- CN109142334A CN109142334A CN201811115053.2A CN201811115053A CN109142334A CN 109142334 A CN109142334 A CN 109142334A CN 201811115053 A CN201811115053 A CN 201811115053A CN 109142334 A CN109142334 A CN 109142334A
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- 108060008682 Tumor Necrosis Factor Proteins 0.000 title claims abstract description 66
- 238000001514 detection method Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 24
- 102000003390 tumor necrosis factor Human genes 0.000 title abstract 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims abstract description 64
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims abstract description 38
- HJCUTNIGJHJGCF-UHFFFAOYSA-N 9,10-dihydroacridine Chemical compound C1=CC=C2CC3=CC=CC=C3NC2=C1 HJCUTNIGJHJGCF-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000002671 adjuvant Substances 0.000 claims abstract description 26
- 239000003550 marker Substances 0.000 claims abstract description 25
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 18
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- 238000002372 labelling Methods 0.000 claims abstract description 9
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 9
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 7
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- 238000002360 preparation method Methods 0.000 claims description 21
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 15
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 230000009514 concussion Effects 0.000 claims description 8
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- 239000002994 raw material Substances 0.000 claims description 8
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
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- 239000012153 distilled water Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 239000007974 sodium acetate buffer Substances 0.000 claims description 4
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 4
- 239000012279 sodium borohydride Substances 0.000 claims description 4
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- 239000001509 sodium citrate Substances 0.000 claims description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 229910021538 borax Inorganic materials 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 3
- 206010054094 Tumour necrosis Diseases 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 238000004020 luminiscence type Methods 0.000 abstract description 5
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to the kits and its detection method of a kind of spatial neighbor chemoluminescence method detection tumor necrosis factor.Kit includes: enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；Wherein, calibration object includes the calibration object and 0.1M phosphate dilution of various concentration TNF-α antigen；Enzyme marker includes the TNF-α detection antibody and 0.05M phosphate buffer of peroxidase labelling；Luminous marker includes the TNF-α capture antibody and 0.05M Tris buffer of acridan label；Adjuvant includes shine adjuvant and citrate buffer.The present invention uses reagent spatial neighbor luminesceence analysis detection technique, as a kind of homogeneous chemistry luminescence technology truly, without cleaning, is not necessarily to carrier, is not coated with process, detection sensitivity is high, high specificity, so that testing result is more genuine and believable；Reaction time and reaction step are optimized simultaneously, keeps operation simpler.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of spatial neighbor chemoluminescence method detection tumor necrosis factor
Kit and its detection method.
Background technique
1975, after E.A.Carswell et al. has found the mouse bacterial injection lipopolysaccharides of bcg vaccination, meeting in serum
There is a kind of substance that can make kinds of tumors that hemorrhagic necrosis occur, and is named as tumor necrosis factor (tumor
Necrosis factor, TNF).The eighties are it is found that it has played important function, also known as cachectin in consumption disease.
TNF is mainly generated by the macrophage activated, NK cell and T lymphocyte.What Shalaby in 1985 generated macrophage
TNF is named as TNF-α, and the lymphotoxin (lymphotoxin, LT) that T lymphocyte generates is named as TNF-beta.Although TNF-α
Only has about 30% homology with TNF-beta, but they possess common receptor.The biological activity of TNF-α accounts for TNF gross activity
70%~95%, therefore the TNF that often says refers to TNF-α more at present.
TNF-α is made of 157 amino acid, is a transmembrane protein, it can be cut into soluble by TACE enzyme
Molecule.The raising of Serum TNF-α and IL-6 content and the pathophysiological process of gestation hypertension are closely related.Bent column Neil energy
It is enough substantially reduced the level of hypertension of pregnancy mouse intracorporal TNF-α and IL-6, the gestation that can significantly inhibit hypoxia inducible is high
The Development process of blood pressure.Therefore, Serum TNF-α and the level of IL-6 can be used as a kind of potential predicting marker to be used for gestation
The clinical diagnosis and treatment of phase hypertension.
In acute intussusception in children infant, TNF-α, which increases, makes intestinal mucosa hyperemia, oedema, and enteron aisle can be made by persistently increasing
Smooth muscle spasm, contraction, cause stenosis of bowel, create conditions for entembole；Meanwhile high concentration TNF-α can make monokaryon
Cell and vascular endothelial cell etc. secrete relatively large interleukin-1 (IL-1) and IL-6, are made with the inflammation that the magnocell factor mediates
With, be further exacerbated by intestinal mucosa damage, aggravation intestinal smooth shrink.IL-6 is Th2 cytokines, in acute bowel set
In folded infant, IL-6 raising is immunoregulation effect, anti-inflammatory, antiviral effect, while the effect to anti-tnf-alpha, but TNF-α
Raising advantage causes intestinal smooth contracts last to lead to the generation of entembole in IL-6.In Acute intussusception infant serum
TNF-α and IL-6 are horizontal significantly raised.
Tumor necrosis factor-alpha (tumor necrosis factor- α, TNF-α) finds to cause in clinical studies
Blood-retina barrier (blood retinal barrier, BRB) is impaired and cooperates with the proliferation of VEGF.To diabetic microangiopathy
Become the measurement of serum TNF-alpha levels show the generation increased with diabetic microangiopathies of Serum TNF-α concentration with
Development has very close relationship.
Summary of the invention
For the defects in the prior art, it is an object of that present invention to provide a kind of spatial neighbor chemoluminescence methods to detect tumour
The kit and its detection method of necrosin.The method is as a kind of homogeneous chemistry luminescence technology truly, without carrying
Body is not coated with, washing process, and reagent constituents are few, and high sensitivity, reproducible, easy to operate.
To achieve the above object, technical solution provided by the invention are as follows:
In a first aspect, the present invention provides a kind of detection kit of tumor necrosis factor-alpha, kit includes: enzyme label
Object, luminous marker, adjuvant, triggering agent and calibration object；Wherein, the raw material components of enzyme marker include peroxidase labelling
TNF-α detect antibody, the raw material components of luminous marker include that the TNF-α of 9,10- acridan label captures antibody.
Preferably, the raw material components of enzyme marker further include 0.05M phosphate buffer；It is highly preferred that preparation method packet
It includes: weighing 5mg HRP and be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4, and 4 DEG C overnight；Add
20 μ L 0.2M pH, 9.5 carbonate buffer solution, addition 1mg anti-tnf-alpha monoclonal antibody room temperature, which is protected from light, immediately after is gently mixed
2h adds the 4mg/mL sodium borohydride liquid newly matched, and mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, to 0.15M pH
7.4PBS dialysis, 4 DEG C overnight；Dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Preferably, the raw material components of luminous marker further include 0.05M Tris buffer；It is highly preferred that preparation method packet
Include: the luminous substrate Acridan DMF of 500 μ L dissolves；The 41.3 μ L of Acridan of dissolution is drawn, it is slow that 0.05M Boratex is added
708.7 μ L of fliud flushing adds 250 μ L anti-tnf-alpha monoclonal antibodies, and overturning mixes 4~5 times, stands 30min at room temperature；It will
Label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, and taking-up addition qs glycerin is set -20 DEG C of refrigerators and saved.
Preferably, the component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；It is highly preferred that preparation
Method includes: to weigh citric acid 1.82g, and sodium citrate 10.45g adds pure water to dissolve and is settled to 1000mL, slow in citrate
Suitable luminous adjuvant is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, 0.05M pH 8.0Tris-HCl buffer is selected in triggering agent；It is highly preferred that preparation method includes: to claim
Tris 6.06g, sodium chloride 9g are taken, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes.It is added and spits in above-mentioned solution
- 20 2mL of temperature are settled to 1000mL after mixing, and dispense after mixing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, calibration object includes the calibration object and 0.1M phosphate buffer of various concentration TNF-α antigen；More preferably
Ground, preparation method include: to prepare calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds
Appropriate ultrapure water dissolution, 0.5~1mL of Proclin-300 after mixing, add ultrapure water to be settled to 1000mL, and as calibration object is dilute
Liquid is released, 2~8 DEG C store for future use；Prepare calibration object: the concentration of calibration object be 0,10pg/mL, 50pg/mL, 100pg/mL,
Purified tumor necrosin is diluted to respective concentration, 2~8 DEG C of storages with calibration object dilution by 500pg/mL, 1000pg/mL
It is spare.
Kit of the present invention detects the content of tumor necrosis factor-alpha (TNF-α) in human serum using double antibody sandwich method.
By sample, the anti-tnf-alpha monoclonal antibody of horseradish peroxidase (HRP) label, acridan (Acridan) label
Anti-tnf-alpha monoclonal antibody one react, formed antigen-antibody sandwich complex so that horseradish peroxidase and 9,10-
Acridan (Acridan) is spatially able to close to each other, the luminous adjuvant of addition and triggering agent, generation flash type chemistry hair
Light；And unbonded free HRP labelled antibody and Acridan labelled antibody does not shine then.TNF-α content in sample is got over
The luminous value (RLU) of height, measurement is bigger.So luminous value is positively correlated with concentration of specimens within the scope of a certain concentration, pass through
The calibration object and its luminous value of known concentration draw working curve, can calculate TNF-α in sample according to the luminous value of sample
Content.
Second aspect, detection kit provided by the invention detect tumor necrosis factor-alpha in spatial neighbor chemoluminescence method
In application.
The third aspect, above-mentioned detection kit provided by the invention detect tumor necrosis factor in spatial neighbor chemoluminescence method
Method when son-α, comprising steps of S1: being separately added into the TNF-α detection antibody of 25 μ L standard items, 25 μ L peroxidase labellings
With 25 μ L luminous markers to reaction tube；S2: 15min is reacted in 37 DEG C of insulating boxs；S3: 5 μ L adjuvants are separately added into reaction
Pipe, concussion mix, and stand 1~2min；It is separately added into triggering 75 μ L of agent later, concussion is mixed, detected immediately, reads signal value；
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, calculate concentration of specimens by sample luminous value.
Technical solution provided by the invention, have it is following the utility model has the advantages that
(1) it is needed with traditional chemiluminescence using microwell plate or magnetic particle as carrier coated antibody or antigen phase
Than, carrier is not necessarily in kit provided by the invention and detection method, is not coated with, washing process, be it is a kind of truly
Homogeneous chemistry luminescence technology.
(2) traditional enzyme-catalyzed chemical luminescence technology is in the test analyte successively detection antibody with capture antibody and enzyme label
It must be cleaned after combining 2~3 times, to remove the unstable non-specific substance of be not associated with or combination；And the present invention is
After the combination of test analyte and two specific antibodies, the interference that shines is eliminated by adjuvant effect, triggering agent is added and generates
Luminous signal, whole process is without washing.
(3) traditional enzyme-catalyzed chemical luminescence technology needs substrate for enzymatic activity, 5~10 minutes or so detection luminous values；And this
Invention is flash type chemiluminescence, generates luminous signal immediately after triggering agent is added.
(4) reagent constituents provided by the invention are few, and production process is simple, and production cost reduces, and are easy to amplify production；And
Detection process is convenient, it is easy to accomplish full-automatic.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described.Implement below
Example is only used for clearly illustrating technical solution of the present invention, therefore is intended only as example, and cannot be used as a limitation and limit this hair
Bright protection scope.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantitative test in following embodiment,
Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.
The present invention provides a kind of detection kit of tumor necrosis factor-alpha, and kit includes: enzyme marker, luminescent marking
Object, adjuvant, triggering agent and calibration object；Wherein, the component of enzyme marker includes the TNF-α detection antibody of peroxidase labelling
With 0.05M phosphate buffer；The component of luminous marker include acridan label TNF-α capture antibody and
0.05M Tris buffer；The component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；Agent is triggered to select
0.05M pH 8.0Tris-HCl buffer；Calibration object include various concentration TNF-α antigen calibration object and 0.1M calibration object it is dilute
Release liquid.
One, the preparation of calibration object
(1) it prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, 0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, and 2~8 DEG C
It stores for future use.
(2) prepare calibration object: the concentration of calibration object be 0,10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL,
Purified tumor necrosin is diluted to respective concentration with calibration object dilution by 1000pg/mL, and 2~8 DEG C store for future use.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4,
4 DEG C overnight.
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, 1mg anti-tnf-alpha monoclonal antibody room is added immediately after
Temperature, which is protected from light, is gently mixed 2h, adds the 4mg/mL sodium borohydride liquid newly matched, and mixes, and in 4 DEG C of placement 2h, above-mentioned liquid is packed into bag filter
In, it dialyses to 0.15M pH 7.4PBS, 4 DEG C are overnight.
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved.
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, adds 250 μ L
Anti-tnf-alpha monoclonal antibody, overturning mix 4~5 times, stand 30min at room temperature.
(3) label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, taken out addition qs glycerin and set -20 DEG C
Refrigerator saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled
200mL。
It is examined using tumor necrosis factor-alpha detection kit in spatial neighbor chemoluminescence method the present invention also provides a kind of
Method when tumor necrosis factor-alpha is surveyed, comprising steps of
S1: the TNF-α detection antibody and 25 μ L luminescent markings of 25 μ L standard items, 25 μ L peroxidase labellings are separately added into
Object is to chemiluminescence reaction pipe.
S2: 15min is reacted in 37 DEG C of insulating boxs.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, calculate sample by sample luminous value
This concentration.
Technical solution provided by the invention is described further combined with specific embodiments below.
Embodiment one
The present embodiment provides a kind of detection kits of tumor necrosis factor-alpha, comprising: enzyme marker, luminous marker,
Adjuvant, triggering agent and calibration object；Wherein, calibration object includes the calibration object and 0.1M of no less than 6 various concentration TNF-α antigen
Phosphate buffer；Enzyme marker includes the TNF-α detection antibody and 0.05M phosphate buffer of peroxidase labelling；Hair
Signal object includes the TNF-α capture antibody and 0.05M Tris buffer of acridan label；The group subpackage of adjuvant
Include 6.0 citrate buffer of luminous adjuvant and pH；It triggers agent and selects 0.05M pH 8.0Tris-HCl buffer.
One, the preparation of calibration object
(1) it prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, Proclin-300 0.8mL after mixing, add ultrapure water to be settled to 1000mL, obtain calibration object dilution, and 4 DEG C of storages are standby
With.
(2) prepare calibration object: the concentration of calibration object be 0,10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL,
Purified tumor necrosin is diluted to respective concentration with calibration object dilution by 1000pg/mL, and 4 DEG C store for future use.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4,
4 DEG C overnight；
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, 1mg anti-tnf-alpha monoclonal antibody room is added immediately after
Temperature, which is protected from light, is gently mixed 2h, adds the 4mg/mL sodium borohydride liquid newly matched, and mixes, and in 4 DEG C of placement 2h, above-mentioned liquid is packed into bag filter
In, it dialyses to 0.15M pH 7.4PBS, 4 DEG C are overnight；
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved；
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, adds 250 μ L
Anti-tnf-alpha monoclonal antibody, overturning mix 5 times, stand 30min at room temperature；
(3) label reaction tube is placed on shaking table, is mixed at 4 DEG C overnight, taken out addition qs glycerin and set -20 DEG C of refrigerators
It saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Embodiment two
It is detected using tumor necrosis factor-alpha detection kit in spatial neighbor chemoluminescence method the present embodiment provides a kind of
Method when tumor necrosis factor-alpha, comprising steps of
S1: the TNF-α detection antibody and 25 μ L luminescent markings of 25 μ L standard items, 25 μ L peroxidase labellings are separately added into
Object is to chemiluminescence reaction pipe.
S2: 15min is reacted in 37 DEG C of insulating boxs.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, calculate sample by sample luminous value
This concentration.
The present invention detects tumor necrosis factor using spatial neighbor chemoluminescence method, and sensitivity can reach 5pg/mL.For
Septicemia and severe infection patient, the content of serum Tumor Necrosis Factor and prognosis are closely related, serum tumor necrosis because
The content of son is high, then poor prognosis.
Certainly, the case where being enumerated in addition to embodiment one and embodiment two, other conditions and parameter in preparation process etc.
It is possible.
Applicant has found after creative work: detection method provided by the invention is as a kind of truly equal
Phase chemiluminescence is not necessarily to carrier, is not coated with, washing process, and reagent constituents are few, and high sensitivity, reproducible, behaviour
Make simple.
It should be noted that unless otherwise indicated, technical term or scientific term used in this application should be this hair
The ordinary meaning that bright one of ordinary skill in the art are understood.Unless specifically stated otherwise, it otherwise illustrates in these embodiments
Component and opposite step, numerical expression and the numerical value of step are not limit the scope of the invention.It is illustrated and described herein
In all examples, unless otherwise prescribed, any occurrence should be construed as merely illustratively, not as limitation, because
This, other examples of exemplary embodiment can have different values.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover in protection scope of the present invention.
Claims (8)
1. a kind of detection kit of tumor necrosis factor-alpha, which is characterized in that the kit includes:
Enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；
Wherein, the raw material components of the enzyme marker include the TNF-α detection antibody of peroxidase labelling, the luminescent marking
The raw material components of object include the TNF-α capture antibody of acridan label.
2. the detection kit of tumor necrosis factor-alpha according to claim 1, it is characterised in that:
The raw material components of the enzyme marker further include 0.05M phosphate buffer；
Preferably, preparation method includes:
It weighs 5mg HRP to be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4, and 4 DEG C overnight；
20 μ L 0.2M pH, 9.5 carbonate buffer solution is added, 1mg anti-tnf-alpha monoclonal antibody room temperature is added immediately after and is protected from light
It is gently mixed 2h, adds the 4mg/mL sodium borohydride liquid newly matched, mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, it is right
0.15M pH 7.4PBS dialysis, 4 DEG C overnight；
Dialysate is taken out, adds qs glycerin to be placed on -20 DEG C of refrigerators and saves.
3. the detection kit of tumor necrosis factor-alpha according to claim 1, it is characterised in that:
The raw material components of the luminous marker further include 0.05M Tris buffer；
Preferably, preparation method includes:
The luminous substrate Acridan DMF of 500 μ L is dissolved；
The 41.3 μ L of Acridan of dissolution is drawn, 708.7 μ L of 0.05M sodium borate buffer liquid is added, adds 250 μ L anti-tnf-alpha
Monoclonal antibody, overturning mix 4~5 times, are stored at room temperature 30min；
Label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, addition qs glycerin is taken out and is placed on -20 DEG C of refrigerators
It saves.
4. the detection kit of tumor necrosis factor-alpha according to claim 1, it is characterised in that:
The component of the adjuvant includes the citrate buffer of luminous adjuvant and pH 6.0；
Preferably, preparation method includes:
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, in citrate buffer
It is middle that the adjuvant that shines is added, it is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
5. the detection kit of tumor necrosis factor-alpha according to claim 1, it is characterised in that:
0.05M pH 8.0Tris-HCl buffer is selected in the triggering agent；
Preferably, preparation method includes:
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added and spits later
- 20 2mL of temperature, are settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 200mL.
6. the detection kit of tumor necrosis factor-alpha according to claim 1, it is characterised in that:
The calibration object includes the calibration object and 0.1M calibration object dilution of various concentration TNF-α antigen；
Preferably, preparation method includes:
It prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, NaH2PO4·2H2O 3.0g, adds ultrapure water to dissolve,
0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, 2~8 DEG C of storages
It is spare；
Prepare calibration object: the concentration of calibration object is 0,10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, is used
The calibration object dilution is diluted to respective concentration for TNF-α is purified, and 2~8 DEG C store for future use.
7. the described in any item detection kits of claim 1~6 detect tumor necrosis factor-in spatial neighbor chemoluminescence method
Application in α.
8. the described in any item kits of claim 1~6 are when spatial neighbor chemoluminescence method detects tumor necrosis factor-alpha
Method, comprising steps of
S1: 25 μ L standard items, the TNF-α detection antibody of 25 μ L peroxidase labellings and 25 μ L luminous markers are separately added into extremely
Chemiluminescence reaction pipe；
S2: 15min is reacted in 37 DEG C of insulating boxs；
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；It is separately added into touching later
75 μ L of agent is sent out, concussion is mixed, detected immediately, reads signal value；
S4: concentration and luminous value to calibration object carry out tetra- parameter fitting of logistic, and it is dense to calculate sample by sample luminous value
Degree.
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