CN109100508A - A kind of chronic fatigue syndrome diagnostic kit - Google Patents
A kind of chronic fatigue syndrome diagnostic kit Download PDFInfo
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- CN109100508A CN109100508A CN201810742523.1A CN201810742523A CN109100508A CN 109100508 A CN109100508 A CN 109100508A CN 201810742523 A CN201810742523 A CN 201810742523A CN 109100508 A CN109100508 A CN 109100508A
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- chronic fatigue
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- G—PHYSICS
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- 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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- 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
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Abstract
The invention discloses a kind of chronic fatigue syndrome diagnostic kits, including abzyme target, Biotin-Antibody, standard protein, serum dilution, Streptavidin-HRP, HRP chromogenic substrate, HRP chromogenic reaction terminator, negative controls and positive control solution；The abzyme target up to being coated with specific antibodies less, the specific antibodies include 18 kinds of monoclonal antibodies, specially TGF-β, Activin B, ICAM1, Resistin, Leptin, IFN-γ, CD40L, TNFSF10, M-CSF, PDGF-BB, interferon-inducible protein -10, IL-1RA, IL-7, IL-12p40, IL-12p70, IL-13, IL-17A and IL-17F.Compared with prior art, which has many advantages, such as that diagnosis effect is accurate and reliable and easy to operate.
Description
Technical field
The present invention relates to field of medicine diagnostic technology, and in particular to a kind of chronic fatigue syndrome diagnostic kit.
Background technique
Chronic fatigue syndrome is one kind using fatigue as cardinal symptom, with low-heat, sore-throat, lymph node pain, muscle
It is powerless, ache, arthralgia, headache, sleep disturbance and neuropsychic symptom such as irritability, amnesia, absent minded, depressed etc.
Syndrome.According to the tentative diagnosis standard of United States Medicine, which has following three big core symptoms: 1) working, educates, society
Or the ability of individual activity was continued to decline more than six months, and with serious, new fatigue symptom；2) tired after non-fatigue；
3) it cannot regain one's vigor after sleeping.Has at least one of following symptom: 1) cognitive function in addition, also needing to make a definite diagnosis during diagnosis
Obstacle, such as disturbance of perception, memory disorders, the disturbance of thought；2) cannot often the time it is upright, such as vasovagal syncope, orthostatic
Tachycardia complex disease, orthostatic hypotension, orthostatic hypertension etc..
In the prior art, since chronic fatigue syndrome has the clinical manifestation of multisystem discomfort, lack specificity, while mesh
It is preceding also without be used for clinical detection molecule diagnosis kit, be usually misdiagnosed as neurasthenia, climacteric syndrome, endocrine
Imbalance, neurosis etc., so that the treatment of patient is delayed.Therefore, there is an urgent need to a kind of the chronic tired of high accuracy at present
Labor syndrome molecular diagnostic techniques.
Summary of the invention
The technical problems to be solved by the present invention are: chronic fatigue syndrome can be fast and accurately diagnosed to be by providing one kind
Kit.
In order to solve the above-mentioned technical problem, a kind of the technical solution adopted by the present invention are as follows: chronic fatigue syndrome diagnosis examination
Agent box, including abzyme target, Biotin-Antibody, standard protein, serum dilution, Streptavidin-horseradish peroxidase
(Horse Radish Peroxidase, HRP), HRP chromogenic substrate, HRP chromogenic reaction terminator, negative controls and the positive
Comparison liquid；
The abzyme target includes 18 kinds of monoclonal antibodies, tool up to specific antibodies, the specific antibodies are coated with less
Body is transforming growth factor (transforming growth factor- β, TGF-β), recombined human activin (Activin
B), intercellular adhesion molecule-1 (intercellular cell adhesion molecule-1, ICAM1), phylaxin
(Resistin), people's leptin (Leptin), human interferon (Interferon- γ, IFN-γ), tumor necrosis factor associated activation
Albumen (CD40L), tumor necrosis factor (tumor necrosis factor (ligand) superfamily-10, TRAIL
Or TNFSF10), macrophage colony stimulating factor (Macrophage colony-stimulating factor, M-CSF),
Human platelet-derived growth factor (Platelet-derived growth factor-BB, PDGF-BB), interferon-inducible
Albumen -10 (CXCL10or IP-10), interleukin-1 receptor antagonist (Interleukin-1receptor
Antagonist, IL-1RA), interleukin 7 (Interleukin-7, IL-7), IL-12p40, IL-12p70, IL-13,
IL-17A and IL-17F.
Further, the specific antibodies can also include transforming growth factor (transforming growth
Factor- α, TGF- α), IL-4, people's eosinophil chemokine (Eotaxin, preferably CCL11) and human soluble wither
Die one of correlation factor ligand (Human soluble apoptosis related factor ligand, sFasL)
Or it is a variety of.
Further, the abzyme target is prepared by following methods: the phosphate buffer for being 8.2 with pH
(Phosphate Buffered Saline, PBS buffer solution) dilutes the concentration of the various antibody in the specific antibodies respectively
It to 20-100 μ g/ml, is added in 96 hole elisa Plates by the additional amount in 100 holes μ l/, was placed under conditions of temperature is 2-8 DEG C
Then night is washed with cleaning solution, the cleaning solution is the phosphate-buffered that pH is 7.4 containing 0.05% tween (Tween-20)
Liquid, up to the abzyme target after drying.
Further, the Biotin-Antibody the preparation method comprises the following steps: by 18 kinds of monoclonal antibodies respectively with activation
Biotin mixing is marked, up to the Biotin-Antibody after the unbonded biotin of dialysis removal.
Further, the serum dilution is composed of the following components: actrapid monotard 6U/ml, potassium dihydrogen phosphate 3g/L, phosphorus
Sour disodium hydrogen 3g/L, sodium sulphate 5g/L, sodium chloride 22g/L, NaTDC 12g/L, EDTAP dipotassium ethylene diamine tetraacetate
(dipotassium ethylene diamine tetraacetate, EDTA-2K) 0.5g/L, solvent is water.
Further, the pH value of the serum dilution is 6.4.
Preferably, the chromogenic substrate be tetramethyl benzidine (3,3', 5,5'-Tetramethylbenzidine,
TMB)。
Further, the concentration of the TMB is 0.4mg/ml.
Further, the HRP chromogenic reaction terminator is sulfuric acid or hydrochloric acid.
Further, the concentration of the sulfuric acid is 2mol/L, and the concentration of the hydrochloric acid is 1mol/L.
Further, the negative controls are the normal human serum without the specific antibodies；The positive control solution
For the normal human serum containing specific antibodies.
Further, the standard protein is that arbitrarily can be used for quantitative protein, such as bovine serum albumin(BSA) (Bovine
Serum Albumin, BSA) etc..
The beneficial effects of the present invention are: the present invention program diagnostic kit, by 18 kinds and chronic fatigue syndrome
The quantitative analysis of the relevant inflammation associated immune cells factor significantly improves and faces as the auxiliary diagnosis means of the syndrome
Bed diagnosis rate；Solves the problems, such as chronic fatigue syndrome diagnostic means scarcity in the prior art；The judgement knot of kit of the present invention
Fruit can provide objective laboratory foundation for the clinical diagnosis of chronic fatigue syndrome；The present invention program is in conventional enzyme linked immunological
On the basis of adsorption test, by the effect between biotin and Streptavidin-HRP, a kind of life of high sensitivity is established
Object element-enzyme-linked immunologic detecting kit, with measure the TGF-β in sample to be tested, Activin B, ICAM1, Resistin,
Leptin, IFN-γ, CD40L, TNFSF10, M-CSF, PDGF-BB, interferon-inducible protein -10, IL-1RA, IL-7,
The expression of IL-12p40, IL-12p70, IL-13, IL-17A and IL-17F totally 18 kinds of cell factors；Easily due to biotin
With the protein such as antibody with Covalent bonding together, therefore, have biotin in the monoclonal antibody of biotin labeling easily with strepto- parent
Reaction is generated with element-HRP, had both played the role of multistage amplification, simultaneously as enzyme can be sent out when encountering corresponding chromogenic substrate
It gives birth to catalytic action and develops the color, to achieve the purpose that detect target antigen or antibody molecule；The present invention program diagnostic kit energy
Enough contents for effectively, steadily measuring 18 kinds of cell factors in patients serum, high sensitivity, test repeatability are good；In addition, this
The expression of 18 kind inflammation-associated cytokine of the scheme of the invention kit due to can detecte chronic fatigue syndrome patient's body
Level is of great significance to the clinical detection rate for improving chronic fatigue syndrome, while also helping the early diagnosis of patient
With pre- post-processing, the present invention program kit all has good application in the prevention of chronic fatigue syndrome and therapeutic process
Prospect.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, it is explained below in conjunction with embodiment.
The embodiment of the present invention are as follows: a kind of chronic fatigue syndrome diagnostic kit, including abzyme target, biotin-are anti-
Body, serum dilution, BSA, sulfuric acid, Streptavidin-HRP, HRP chromogenic substrate, negative controls and positive control solution；It is described
For abzyme target up to specific antibodies are coated with less, the specific antibodies include 18 kinds of monoclonal antibodies, specially TGF-β,
Activin B, ICAM1, Resistin, Leptin, IFN-γ, CD40L, TNFSF10, M-CSF, PDGF-BB, interferon inducer
Conductivity type albumen -10, IL-1RA, IL-7, IL-12p40, IL-12p70, IL-13, IL-17A and IL-17F.
The preparation process of above-mentioned chronic fatigue syndrome diagnostic kit is as follows:
The preparation of abzyme target: (any biotech firm is fixed from biotech firm's customization for 18 kinds of monoclonal antibodies as described above
System), each monoclonal antibody is diluted to 60 μ g/ml with the PBS buffer solution that pH is 8.2, is added by the additional amount in 100 holes μ l/
In 96 hole elisa Plates, temperature be 4 DEG C under conditions of stand overnight, then washed with cleaning solution, the cleaning solution be containing
The phosphate buffer that 0.05%Tween 20, pH are 7.4, drying, must be coated with 96 hole elisa Plates of anti-monoclonal antibody.
Steps are as follows for the use of above-mentioned chronic fatigue syndrome diagnostic kit:
1, it is loaded:
(1) it is coated with the 96 hole elisa Plates processing of monoclonal antibody: in 96 hole elisa Plates for being coated with monoclonal antibody
Positive control porose area, negative control porose area, sample to be tested porose area and blank porose area are divided, totally four groups of detection porose areas, positive control
It is added positive control solution in porose area, negative controls, 100 holes μ l/, sample to be tested is added in negative control porose area in 100 holes μ l/
Test serum sample is added in porose area, 100 holes μ l/, after sample adds, it is 0.5% that then the concentration of 50 μ l amounts, which is added, in every hole
The serum dilution of bovine serum albumin(BSA) and 50 μ l amounts, the serum dilution are as follows: actrapid monotard 6U/mL, potassium dihydrogen phosphate
3g/L, disodium hydrogen phosphate 3g/L, sodium sulphate 5g/L, sodium chloride 22g/L, NaTDC 12g/L, EDTA-2K 0.5g/L, add
It is 6.4 that water, which adjusts pH value,；Capping or overlay film on ELISA Plate, discard liquid after placing 2h under the conditions of 37 DEG C, use cleaning solution
Washing, drying；
(2) biotin-monoclonal antibody is added: 100 μ l biotins-monoclonal antibody being added in each detection hole, 37
Liquid is discarded after placing 1h under the conditions of DEG C, 350 μ l cleaning solutions are added in each detection hole and impregnate 2min, dries or lightly claps
Dry, washing, the movement dried are repeated 3 times；
(3) Streptavidin-horseradish peroxidase is added:
Streptavidin-horseradish peroxidase of 100 μ l is added in each detection hole, after placing 1h under the conditions of 37 DEG C
Liquid is discarded, 350 μ l cleaning solutions are added in each detection hole and impregnate 2min, dries or lightly pats dry, the movement washed, dried
It is repeated 5 times；
(4) chromogenic substrate is added:
It is 0.4mg/ml tetramethyl benzidine that a drop concentration is sequentially added in each detection hole, is kept away under the conditions of 37 DEG C
Light, colour developing；
(5) terminate liquid is added:
50 μ l terminate liquid (2mol/l sulphur are successively added in each detection hole according to the addition sequence of above-mentioned chromogenic substrate
Acid), reaction is terminated, terminates the liquid of reaction shown as in detection hole by blue fast transition yellowly.
2, result detects:
After terminate liquid is added in 15min, the light for detecting each detection hole under the wavelength condition of 450nm with enzyme-linked instrument is close
Spend OD value, the examination criteria of detection kit are as follows: using the concentration of standard items as ordinate (or logarithmic coordinates), OD value is abscissa
(or logarithmic coordinates), drawing standard curve using 1.30 software of curve expert, (best equation should be according to regression equation calculation
Obtained R2 value is determined, and is more leveled off to preferably 1 with R2 value), calculate the actual concentrations of sample.
The determination of chronic fatigue syndrome diagnostic kit multiple cytokine reference value is using the kit to 200
The testing result of normal population counts, and average value is defined with the Serum antibody concentrations value of 95% normal population, due to existing
Difference in crowds is only used as referring to.It is as shown in table 1 by detecting acquisition reference biomolecule concentration to normal population:
1 reference concentration of table
Above-mentioned chronic fatigue syndrome diagnostic kit is used for clinical trial experiment, specific as follows:
With the chronic fatigue syndrome diagnostic kit to the serum of an example chronic fatigue syndrome patient made a definite diagnosis
The ratio of the detection of progress inflammation-associated cytokine, each inflammatory cytokine concentration of the patients serum and reference concentration exists substantially
1 or so, the diagnosis of chronic fatigue syndrome can accurately be realized by being indicated above the present invention program.
Above-mentioned chronic fatigue syndrome diagnostic kit is subjected to clinical trial statistical analysis and clinical assessment experiment, specifically
It is as follows:
Patient by the way that 5 have been diagnosed as with chronic fatigue syndrome carry out after Serologic detection obtained corresponding cell because
Sub- concentration establishes clinical diagnosis evaluation system after for statistical analysis, the cell of 5 measured chronic fatigue syndrome patient because
Sub- concentration is specifically as shown in table 2 to the ratio of normal reference value, is changed to statistics reciprocal lower than 1.Exist in view of 18 kinds of cell factors
Mechanism of action in chronic fatigue syndrome is different, is united using weighting method to 5 experimental datas according to its importance
Meter analysis, obtains 5 weighted averages, then taking its average value is case average value 1.35 (P < 0.05).
2 CFS serum-concentration of table/reference concentration ratio and weighted average
The methods for clinical diagnosis of kit of the present invention are as follows: be higher than the patient of weighted average (such as 1.35), in conjunction with clinic
Symptom supports the diagnosis of chronic fatigue syndrome.It, will be according to gender, age, course of disease length, disease in view of the individual difference of patient
Situations such as feelings weight, gradually refines and improves diagnostic criteria.The present invention program kit is used for clinical detection, easy to operate, knot
Fruit is accurate and reliable.
In conclusion a kind of chronic fatigue syndrome diagnostic kit provided by the invention, which has diagnosis effect
The advantages that fruit is accurate and reliable and easy to operate.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright description are applied directly or indirectly in relevant technical field, are similarly included in this hair
In bright scope of patent protection.
Claims (10)
1. a kind of chronic fatigue syndrome diagnostic kit, it is characterised in that: including abzyme target, Biotin-Antibody, standard
Albumen, serum dilution, Streptavidin-HRP, HRP chromogenic substrate, HRP chromogenic reaction terminator, negative controls and the positive
Comparison liquid；
The abzyme target includes 18 kinds of monoclonal antibodies up to specific antibodies, the specific antibodies are coated with less, specially
TGF-β, Activin B, ICAM1, Resistin, Leptin, IFN-γ, CD40L, TNFSF10, M-CSF, PDGF-BB, interference
Plain inducible protein -10, IL-1RA, IL-7, IL-12p40, IL-12p70, IL-13, IL-17A and IL-17F.
2. chronic fatigue syndrome diagnostic kit according to claim 1, it is characterised in that: the abzyme target by
Following methods are prepared: being diluted the concentration of the various antibody in the specific antibodies respectively with the PBS buffer solution that pH is 8.2
It to 20-100 μ g/ml, is added in 96 hole elisa Plates by the additional amount in 100 holes μ l/, was placed under conditions of temperature is 2-8 DEG C
Then night is washed with cleaning solution, the cleaning solution is the Tween-20 for being 0.05% containing mass fraction, the phosphoric acid that pH is 7.4
Salt buffer obtains the abzyme target after dry.
3. chronic fatigue syndrome diagnostic kit according to claim 1, it is characterised in that: the Biotin-Antibody
Be marked the preparation method comprises the following steps: 18 kinds of monoclonal antibodies are mixed with the biotin of activation respectively, dialysis removal do not tie
The Biotin-Antibody is obtained after the biotin of conjunction.
4. chronic fatigue syndrome diagnostic kit according to claim 1, it is characterised in that: the serum dilution by
Following components composition: actrapid monotard 6U/ml, potassium dihydrogen phosphate 3g/L, disodium hydrogen phosphate 3g/L, sodium sulphate 5g/L, sodium chloride
22g/L, NaTDC 12g/L, EDTA-2K0.5g/L, solvent are water.
5. chronic fatigue syndrome diagnostic kit according to claim 4, it is characterised in that: the serum dilution
PH value is 6.4.
6. chronic fatigue syndrome diagnostic kit according to claim 1, it is characterised in that: the chromogenic substrate is
TMB。
7. chronic fatigue syndrome diagnostic kit according to claim 6, it is characterised in that: the concentration of the TMB is
0.4mg/ml。
8. chronic fatigue syndrome diagnostic kit according to claim 6, it is characterised in that: the HRP chromogenic reaction
Terminator is sulfuric acid or hydrochloric acid.
9. chronic fatigue syndrome diagnostic kit according to claim 1, it is characterised in that: the negative controls are
Normal human serum without the specific antibodies；The positive control solution is the normal human serum containing specific antibodies.
10. -9 described in any item chronic fatigue syndrome diagnostic kits according to claim 1, it is characterised in that: the mark
Quasi- albumen is BSA.
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| CN201810742523.1A CN109100508A (en) | 2018-07-09 | 2018-07-09 | A kind of chronic fatigue syndrome diagnostic kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110338878A (en) * | 2019-07-26 | 2019-10-18 | 珠海中科先进技术研究院有限公司 | A kind of thrombus acquisition equipment |
| CN110501501A (en) * | 2019-07-23 | 2019-11-26 | 武汉大学 | Lung cancer early diagnoses application and the kit of tumor markers |
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| CN105866418A (en) * | 2016-06-03 | 2016-08-17 | 广州华弘生物科技有限公司 | Triple test diagnostic kit for breast cancer |
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Non-Patent Citations (3)
| Title |
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| LIDBURY ET AL: "Activin B is a novel biomarker for chronic syndrome/myalgic encephalomyelitis (CFS/ME) diagnosis: a cross sectional study", 《J TRANSL MED》 * |
| MADY HORNIG ET AL: "Distinct plasma immune signatures in ME/CFS are present early in the course of illness", 《SCI. ADV.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110501501A (en) * | 2019-07-23 | 2019-11-26 | 武汉大学 | Lung cancer early diagnoses application and the kit of tumor markers |
| CN110338878A (en) * | 2019-07-26 | 2019-10-18 | 珠海中科先进技术研究院有限公司 | A kind of thrombus acquisition equipment |
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Application publication date: 20181228 |