CN105842461A - Rapid diagnostic kit for uterine sarcoma in early and middle stages and preparation method of kit - Google Patents
Rapid diagnostic kit for uterine sarcoma in early and middle stages and preparation method of kit Download PDFInfo
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- CN105842461A CN105842461A CN201610287446.6A CN201610287446A CN105842461A CN 105842461 A CN105842461 A CN 105842461A CN 201610287446 A CN201610287446 A CN 201610287446A CN 105842461 A CN105842461 A CN 105842461A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57442—Specifically defined cancers of the uterus and endometrial
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
Abstract
The invention discloses a rapid diagnostic kit for a uterine sarcoma in early and middle stages .The kit is prepared from CD10, death reporters and growth differentiation factors-15 .The invention further discloses a preparation method of the rapid diagnostic kit for the uterine sarcoma in the early and middle stages .By means of the rapid diagnostic kit for the uterine sarcoma in the early and middle stages, the uterine sarcoma can be detected more accurately and comprehensively, benign and malignant pelvic masses can be better judged, differential diagnosis is better conducted on gynecological benign uterine masses and uterine sarcomas, and the sensitivity and the accuracy of the kit both reach 70%.
Description
Technical field:
The present invention relates to the test kit field of diagnostic detection, be specially a kind of sarcoma of uterus early metaphase quick
Diagnostic kit and preparation method thereof.
Background technology:
Sarcoma of uterus is initiated by uterine smooth muscle, endometrial stroma (or epithelium) and the mixing of non-epithelium
Sexual reproduction road, leaf source malignant tumor between one class of composition, component of organization is complicated, clinical manifestation and hysteromyoma
Similar but lack specificity.Owing to showing without specific clinical, preoperative diagnosis difficulty, modal sign
Being that uterus increases, and most increase is substantially, in short-term, uterus enclosed mass increases very fast, abdominopelvic cavity enclosed mass,
Or have ascites, stomachache and lumbago；Lump air spots or in nodal-like.Uterus increases the preoperative often quilt of symptom
Mistaken diagnosis is " hysteromyoma ", and rate of missed diagnosis is up to more than 50% by mistake, and therefore many patients receive inappropriate
Operative treatment, such as peritoneoscope Minimally Invasive Surgery, causes iatrogenic tumour spread.Sarcoma of uterus sickness rate is relatively low,
At whole uterine malignant tumours 2～about 5%, account for the 1%～3% of gynecologic malignant tumor.
Although sarcoma of uterus is more rare in clinic, but its aggressivity is strong, and grade malignancy is high, easily recurs, and lacks
Weary study on large sample, it is easy to local infiltration and metastasis, wherein to send out in hematogenous metastasis, abdominopelvic cavity
Transfer is main path, and Lung metastases is the most common, brings bigger difficulty to clinical treatment.Even in early days
Patient, prognosis also extreme difference, its case fatality rate exceedes the 15% of all uterine malignant tumours, existence in overall 5 years
Rate is less than 30%.The sarcoma of uterus cause of disease is still not clear so far, and clinical manifestation, also without specificity, is difficult in art
Before make a definite diagnosis, its diagnosis goldstandard be postoperative histopathological examination (biopsy).Pathological diagnosis is a kind of sampling
Checking, requiring the quality of specimen, the quality of specimen directly affects the quality of pathological section；Pathology is examined
Disconnected experience and the Professional knowledge needing to rely on diagnostician, has certain subjectivity；Pathological diagnosis detects
Costly.There is no the specific tumor marker for sarcoma of uterus at present.
Hysteromyoma is intrauterine benign tumor, in the women of more than 30 years old, has 20% to 30% meeting
Hysteromyoma occurs.Although hysteromyoma can cause the symptoms such as dysmenorrhea, but does not has life danger, so one
As use the hormonotherapy of Reservations uterus.And sarcoma of uterus is malignant tumor, if being diffused into other groups
Knitting, 5 years survival rates of patient only have 10% to 20%, and therefore early discovery is extremely important.
Current diagnosis of uterine sarcoma goldstandard is postoperative histopathological examination (biopsy).Pathological diagnosis is one
Planting sampling check, require the quality of specimen, the quality of specimen directly affects the quality of pathological section；
Pathological diagnosis needs to rely on experience and the Professional knowledge of diagnostician, has certain subjectivity；Pathology is examined
Disconnected testing cost is higher, and may make a definite diagnosis too late.In addition to pathological diagnosis, also nuclear magnetic resonance, NMR
Imaging (MRI) or positron e mission computed tomography (PET) diagnostic techniques.Carry out PE
During T diagnosis, utilization is the sarcoma of uterus cell character that can absorb more glucosan, but hysteromyoma has
Time also absorb a lot of glucose, so more difficult accurate judgement.Diffusion-weighted imaging (DWI) is to swollen
The position of tumor and qualitative helpful, but result waits to confirm.
In recent years, some research institutions are also developing relevant examination by immunological method or genetics method
Agent box.Once it has been reported that the research worker of Japan Fukui university developed a kind of cooperation positive electricity in 2011
The inspection medicament of sub-emission tomography (PET), for differentiating that the tumor that intrauterine occurs is on earth
Optimum hysteromyoma or pernicious sarcoma of uterus.The research worker of Fukui University is noticed, uterus meat
The receptor accepting estrogen in oncocyte there will be exception, and the ability causing cell to absorb estrogen reduces.
Research group is conceived to this point, produces the medicament of entitled " FES " by processing estrogen.This
Medicament can focus in hysteromyoma rather than sarcoma of uterus, then utilizes PET imaging, and confirmation is son
Palace muscular tumor or sarcoma of uterus.
Owing to sarcoma of uterus sings and symptoms does not has specificity, there is no the tumor markers of sensitivity, iconography
Specificity is the highest, the preoperative diagnosis being often difficult to make sarcoma of uterus, and majority is even art in art
Rear pathology just diagnoses.Either ultrasonic examination or Positron Emission Computed Tomography (PET) scanning,
The most all it is difficult to differentiate the good pernicious of smooth muscle tumor, and operates complex, human body is had necessarily
Injury.Diffusion-weighted imaging (DWI) is to the position of tumor and qualitative helpful, but result is still
To be validated.Postoperative histopathological examination is a kind of sampling check, requires the quality of specimen, specimen
Quality directly affect the quality of pathological section；Pathological diagnosis needs to rely on experience and the specialty of diagnostician
Knowledge, has certain subjectivity；Pathological diagnosis testing cost is higher, and may make a definite diagnosis too late.
Summary of the invention:
It is an object of the invention to for not enough present on above-mentioned existing diagnosis of uterine sarcoma, it is provided that a kind of
Highly sensitive, accuracy rate is high, easy to use and low-cost sarcoma of uterus early metaphase quick diagnosis examination
Agent box.
Another object of the present invention is to provide the preparation side of a kind of sarcoma of uterus early metaphase quick diagnosis reagent kit
Method.
In order to realize the object of the invention, the present invention adopts the technical scheme that: sarcoma of uterus early metaphase is quick
Diagnostic kit, by including that CD10 (Cluster of Differentiation 10, CD10), death are subject to
Body 6 (Death receptor 6, DR6) and GDF-15 (growth differentiation
Factor-15, GDF-15) make.
Sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention, including capture antibody and detection antibody, institute
State capture antibody for including CD10 (Cluster of Differentiation 10, CD10), death receptor
6 (Death receptor 6, DR6) and GDF-15 (growth differentiation
Factor-15, GDF-15) respectively prepare monoclonal antibody.
Described detection antibody is for including CD10 (Cluster of Differentiation 10, CD10), dead
Die receptor 6 (Death receptor 6, DR6) and GDF-15 (growth differentiation
Factor-15, GDF-15) respectively prepare polyclonal antibody.
Described capture antibody is by CD10 (Cluster of Differentiation 10, CD10), death
Receptor 6 (Death receptor 6, DR6) and GDF-15 (growth differentiation
Factor-15, GDF-15) it is cloned into carrier for expression of eukaryon, and in mammalian cell, realize albumen
Expression, obtain corresponding antigen after purification, the corresponding Dan Ke obtained by described antigen immune mammal
Grand antibody.
The step that the preparation method of described capture antibody includes is as follows:
(1) preparation of antigen: the gene of CD10, death receptor 6 and GDF-15 is cloned into
Carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtain antigen after purification, this
Antigen also can be used as standard substance；
(2) preparation of antibody is captured: by above-mentioned antigen immune mammal, obtain corresponding monoclonal antibody
For capture antibody.
Described detection antibody is to be cloned into by the gene of CD10, death receptor 6 and GDF-15
Carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, resisted the most accordingly
Former, that described antigen immune mammal is obtained corresponding polyclonal antibody.
The step that the preparation method of described detection antibody includes is as follows:
(1) preparation of antigen: the gene of CD10, death receptor 6 and GDF-15 is cloned into
Carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtain antigen after purification, this
Antigen also can be used as standard substance；
(2) preparation of antibody is detected: by above-mentioned antigen immune mammal, obtain corresponding polyclonal antibody
For detection antibody.
Described capture antibody is coated in the hole of microtitration plate in advance, can simplify step, improves detection
Efficiency；The aperture in the hole of described titer plate is 0.5-5mm.
The preparation method of sarcoma of uterus early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: by CD10, death receptor 6 and the gene gram of GDF-15
Grand to carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtain required after purification
Antigen, wherein, described antigen also can be used as standard substance；
(2) preparation of antibody is captured: above-mentioned antigen immune mammal is obtained corresponding monoclonal anti
Body, described monoclonal antibody is as the capture antibody of this test kit；
(3) preparation of antibody is detected: above-mentioned antigen immune mammal is obtained corresponding Anti-TNF-α
Body, described polyclonal antibody is as the detection antibody of this test kit；
(4) capture antibody is coated:
1.. with carbonate/bicarbonate buffer (pH9.6) by capture antibody bag that concentration is 2 μ g/ml
By the hole of microtitration plate；2.. capping microtitration plate overnight incubation at 3 DEG C；3.. discard bag
By liquid (the capture antibody of carbonate/bicarbonate buffer dilution), and wash microtitration with cleaning mixture
Plate twice, adds 150 μ l PBST (phosphate Tween buffer), in micropore above tank every time
Whipping microtitration plate gently, removes cleaning mixture, pats microtitration plate, remove remaining on napkin
Drop, dry be put in 3 DEG C of environment standby.
(5) close and be loaded: 150 μ l Block buffer (1.2%BSA/PBS) are added in every hole, envelope
Closure is by protein binding site remaining in hole.
The detection method of sarcoma of uterus early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: by CD10, death receptor 6 and the gene of GDF-15
It is cloned into carrier for expression of eukaryon, and in mammalian cell, realizes the expression of albumen, obtain institute after purification
The antigen needed；
(2) preparation of antibody is captured: above-mentioned antigen immune mammal is obtained corresponding monoclonal anti
Body, described monoclonal antibody is as the capture antibody of this test kit；
(3) preparation of antibody is detected: above-mentioned antigen immune mammal is obtained corresponding Anti-TNF-α
Body, described polyclonal antibody is as the detection antibody of this test kit；
(4) capture antibody is coated:
1.. with carbonate/bicarbonate buffer (pH9.6) by capture antibody bag that concentration is 2 μ g/ml
By the hole of microtitration plate；
2.. capping microtitration plate overnight incubation at 3 DEG C；
3.. discard and be coated liquid (the capture antibody of carbonate/bicarbonate buffer dilution), and use cleaning mixture
Washing microtitration plate twice, adds 150 μ l PBST (phosphate Tween buffer) in micropore every time,
Whipping microtitration plate gently above tank, removes cleaning mixture, pats microtitration plate on napkin,
Remove remaining drop, dry be put in 3 DEG C of environment standby.
(5) close
1.. add 150 μ l Block buffer (1.2%BSA/PBS) in each hole of microtitration plate, envelope
Closure is by protein binding site remaining in hole；
2.. capping microtitration plate also hatches 1 hour at 37.5 DEG C；
(6) sample-adding
1.. add the sample of 100 μ l to each hole, hatch 60 minutes at 37.5 DEG C；Obtain accurately
Quantitative result, it is common practice that compare the signal of unknown sample and standard curve.Each ELISA Plate must
Palpus bioassay standard product (double mensuration or triplicate) and blank sample, to guarantee accuracy；
2.. discard sample, and wash microtitration plate three times, in micropore, add 150 μ l PBST every time
(phosphate Tween buffer)；
3.. add the detection antibody that 100 μ l concentration are 0.5 μ g/ml to each hole；
4.. capping microtitration plate also hatches 1 hour at 37.5 DEG C；
5.. wash microtitration plate four times with PBST；
6.. add 100 μ l labellings two and resist；
7.. capping microtitration plate also hatches 1 hour at 37.5 DEG C.
8.. wash microtitration plate four times with PBST.
(7) detection
1.. by TMB (3,3', 5,5'-tetramethylBenzidine) solution adds each hole to, hatches 15-30
Minute, add isopyknic stop buffer, at 450nm, then read optical density.
2.. the data obtained by serial dilutions draw standard curve, and concentration is marked on X-axis (logarithmic scale)
On, and absorbance is marked in Y-axis (lineal scale).On this standard curve, sample is drawn by interpolation
Product concentration.
The present invention, from numerous tumor markers, by various different permutation and combination, filters out 3
Tumor marker CD10, death receptor 6 and GDF-15 composition sarcoma of uterus early metaphase are fast
Speed diagnostic kit.Wherein, CD10 is a kind of neutral endopeptidase, and molecular weight is about 97kDa,
It is made up of 750 aminoacid, has been found to universally present in central nervous system and periphery various
In tissue, and there is numerous organizing specific sexual function, can induction of T cell apoptosis.Death receptor 6 (DR6)
It is a member of TNF death receptor family, in immune system and nervous system, plays critical function, extremely
Die receptor 6 (DR6) scalable neuronal cell and the degeneration and death of neural axis.Research display sarcoma of uterus
In patients serum, DR6 level is significantly more than healthy population.GDF-15 (GDF-15) is to convert
One remote branch of a clan of growth factor beta (transforming growth factor β, TGF-β) superfamily member, raw
Long differentiation factor-15 (GDF-15) is a kind of stress response protein, and under physiological conditions, GDF-15 is at prostate
With high expressed in Placenta Hominis, faint expression in its hetero-organizations most, great majority research show Growth and Differentiation because of
Son-15 (GDF-15) express rising in kinds of tumors.Owing to blood plasma extracting this 3 tumor markers
Yield poorly, complex steps, so we by use molecular cloning method produce in a large number.
Compared with prior art, beneficial effects of the present invention is as follows:
(1) CD10, DR6 and GDF-15 are combined the tumor markers index as sarcoma of uterus,
For the early metaphase Combining diagnosis of sarcoma of uterus, it is the most novel to design；More accurately can detect son all sidedly
Palace sarcoma, it is possible to preferably judge the good pernicious of pelvic lump, preferably optimum to gynecological image of uterine masses and
Sarcoma of uterus carries out Differential Diagnosis, and its sensitivity and accuracy all reach 70%；
(2) can be not only used for diagnosis of uterine sarcoma and health check-up examination, can be used for again sarcoma of uterus therapeutic effect
And prognosis recurrence monitoring, fill up domestic sarcoma of uterus early metaphase examination and the blank of diagnosis；
(3) production cost is low, the stability of testing result, and repeatability and accuracy are high.
Accompanying drawing explanation
Fig. 1 is the flow chart of sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention；
Fig. 2 is the schematic diagram of sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention；
Fig. 3 is the detection antibody best effort concentration of sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention
Comparative selection figure；
Fig. 4 is sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention before and after sarcoma of uterus is treated in blood
Change (the n=175 of CD10 detected；P < 0.05) comparison diagram；
Fig. 5 is sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention before and after sarcoma of uterus is treated in blood
Change (the n=175 of death receptor 6 detected；P < 0.05) comparison diagram；
Fig. 6 is sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention before and after sarcoma of uterus is treated in blood
Change (the n=175 of GDF-15 detected；P < 0.05) comparison diagram.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention is entered
The description explanation that row is detailed.
Sarcoma of uterus early metaphase quick diagnosis reagent kit, including capture antibody, capture antibody closes buffering
Liquid, standard substance, traget antibody, cleaning mixture and nitrite ion etc..Described traget antibody is selectedHRP labelling Two resist.Described capture antibody includes that CD10, death receptor 6 and GDF-15 prepare respectively
Monoclonal antibody；By to CD10, death receptor 6 and GDF-15 common quickly
Detection, can effectively improve Detection accuracy and sensitivity, effectively overcome only according to a certain tumor-marker
The deficiency that analyte detection causes, its sensitivity and accuracy all reach more than 70%.
Described capture antibody is CD10, death receptor 6 and GDF-15 are cloned into eukaryotic expression
Carrier, and in mammalian cell, realize the expression of albumen, obtain corresponding antigen after purification, by institute
State the corresponding monoclonal antibody that antigen immune mammal obtains.
The step that the preparation method of described capture antibody includes is as follows:
(1) preparation of antigen: by the method for molecular cloning CD10, death receptor 6 and growth point
Change the factor-15 gene and be cloned into carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen,
Obtaining antigen after purification, this antigen also can be used as standard substance；The preparation of described antigen can also use existing
Conventional method is had to prepare, the most burdensome at this.
(2) preparation of antibody is captured: by above-mentioned antigen immune mammal, obtain corresponding monoclonal
Antibody is capture antibody.Described mammal is mice and rabbit, preferably mice.Described capture antibody
Preparation can also use existing conventional method to prepare, the most burdensome at this.
It is many that described detection antibody includes that CD10, death receptor 6 and GDF-15 prepare respectively
Clonal antibody.
Described catch detection antibody be CD10, the gene of death receptor 6 and GDF-15 is cloned into
Carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, resisted the most accordingly
Former, that described antigen immune mammal is obtained corresponding polyclonal antibody.The preparation of described detection antibody
The step that method includes is as follows:
(1) preparation of antigen: by the method for molecular cloning CD10, death receptor 6 and growth point
Change the factor-15 gene and be cloned into carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen,
Obtaining antigen after purification, this antigen also can be used as standard substance；
(2) preparation of antibody is detected: by above-mentioned antigen immune mammal, obtain corresponding polyclone
Antibody is detection antibody.Described mammal is mice and rabbit, preferably rabbit.
Wherein, in the hole of the microtitration plate that described capture antibody can be coated in PVC material in advance,
Step can be simplified, improve detection efficiency.Wherein the aperture in the hole of microtitration plate is preferably 1-5mm.
The preparation method of sarcoma of uterus early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: by the method for molecular cloning by CD10, death receptor 6 and growth point
Change the factor-15 gene and be cloned into carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen,
Obtaining required antigen after purification, wherein, described antigen also can be used as standard substance；
(2) preparation of antibody is captured: above-mentioned antigen immune mammal is obtained corresponding monoclonal anti
Body, described monoclonal antibody is as the capture antibody of this test kit；
(3) preparation of antibody is detected: above-mentioned antigen immune mammal is obtained corresponding Anti-TNF-α
Body, described polyclonal antibody is as the detection antibody of this test kit；
(4) capture antibody is coated:
1.. with carbonate/bicarbonate buffer (pH9.6) by capture antibody that concentration is 2 μ g/ml
It is coated in the hole of microtitration plate；
2.. use bond plastic goods capping microtitration plate overnight incubation at 3 DEG C；
3.. discard and be coated liquid (the capture antibody of carbonate/bicarbonate buffer dilution), and with washing
Liquid washing microtitration plate twice, add 150 μ l PBST in micropore (phosphate tween buffers every time
Liquid), whipping microtitration plate gently above tank, remove cleaning mixture, napkin is patted microtitration
Plate, removes remaining drop, dry be put in 3 DEG C of environment standby；Described cleaning mixture is PBS (phosphoric acid
Salt buffer) a certain amount of Tween 20 of middle addition, the mass percent concentration of described Tween 20 is
0.05%；
(5) close
1.. add 150 μ l Block buffer in every hole of microtitration plate (for containing 1.2%BSA (cattle
Serum albumin) PBS (phosphate buffer)), be coated in hole remaining protein knot for closing
Close site；
2.. with bond plastic goods capping microtitration plate and hatch 1 hour at 37.5 DEG C.
The detection method of sarcoma of uterus early metaphase quick diagnosis reagent kit, it comprises the following steps:
(1) preparation of antigen: by the method for molecular cloning by CD10, death receptor 6 and growth
Differentiation factor-15 is cloned into carrier for expression of eukaryon, and realizes the expression of albumen in mammalian cell,
Obtaining required antigen after purification, wherein, described antigen also can be used as standard substance；
(2) preparation of antibody is captured: above-mentioned antigen immune mammal is obtained corresponding monoclonal anti
Body, described monoclonal antibody is as the capture antibody of this test kit；
(3) preparation of antibody is detected: above-mentioned antigen immune mammal is obtained corresponding Anti-TNF-α
Body, described polyclonal antibody is as the detection antibody of this test kit；
(4) capture antibody is coated:
1.. with carbonate/bicarbonate buffer (pH9.6) by capture antibody bag that concentration is 2 μ g/ml
By the hole of microtitration plate；
2.. use bond plastic goods capping microtitration plate overnight incubation at 3 DEG C；
3.. discard and be coated liquid (the capture antibody of carbonate/bicarbonate buffer dilution), and use cleaning mixture
Washing microtitration plate twice, add in micropore every time 150 μ l PBST (phosphate Tween buffer,
Can be the phosphate buffer of pH7.4 containing 0.05% tween 20), above tank, whipping is micro-gently
Amount titer plate, removes cleaning mixture, pats microtitration plate, remove remaining drop, dry on napkin
It is put in 3 DEG C of environment standby；Described cleaning mixture is that in PBS (phosphate buffer), addition is a certain amount of
Tween 20, the mass percent concentration of described Tween 20 is 0.05%；
(5) close
1.. 150 μ l Block buffer (1.2%BSA/PBS) are added in each hole of microtitration plate,
Closing is coated remaining protein binding site in hole；
2.. with bond plastic goods capping microtitration plate and hatch 1 hour at 37.5 DEG C；
(6) sample-adding
1.. the sample that 100 μ l suitably dilute (diluting 20 times) adds each hole to, at 37.5 DEG C
Under hatch 60 minutes；Obtain quantitative result accurately, it is common practice that compare unknown sample bent with standard
The signal of line.The necessary bioassay standard product (double mensuration or triplicate) of each ELISA Plate and blank sample,
To guarantee accuracy；
2.. discard sample, and wash microtitration plate three times, in micropore, add 150 μ l PBST every time
(phosphate Tween buffer)；
3.. add the detection antibody that 100 μ l concentration are 1 μ g/ml to each hole；
4.. with bond plastic goods capping microtitration plate and hatch 1 hour at 37.5 DEG C；
5.. wash microtitration plate four times with PBST；
6.. add 100 μ l labellings two and resist, its dilution 10000 times in PBS the most (1:
10000).Described labelling two is anti-can be HRP labelling goat-anti rabbit.
7.. with bond plastic goods capping microtitration plate and hatch 1 hour at 37.5 DEG C.
8.. wash microtitration plate four times with PBST.
(7) detection
1.. by TMB (3,3', 5,5'-tetramethylBenzidine) solution adds each hole to, hatches 15-30
Minute, add isopyknic stop buffer (2M H2SO4), then with enzyme-linked immunosorbent assay instrument at 450nm
Place reads optical density.
2.. the data obtained by serial dilutions draw standard curve, and concentration is marked on X-axis (logarithmic scale)
On, and absorbance is marked in Y-axis (lineal scale).On this standard curve, sample is drawn by interpolation
Product concentration.
Sarcoma of uterus early metaphase quick diagnosis reagent kit of the present invention includes CD10, death receptor 6
Raise the standard as diagnosis sarcoma of uterus, detection with in three indexs of GDF-15 two simultaneously
Principle is as shown in Figure 2；In CD10, death receptor 6 and three indexs of GDF-15 two with
Time decline as sarcoma of uterus therapeutic effect assessment standard.May be used for clinically by detection blood
The height of 3 tumor marker levels carries out dynamic evaluation to the therapeutic effect of sarcoma of uterus.Additionally also
May be used for relapse and metastasis and the application of Index for diagnosis clinically to sarcoma of uterus.
Test effect explanation
The selection of double-antibody sandwich elisa optimum experimental condition
It is coated mouse-anti people CD10, death receptor 6 and GDF-15 monoclonal antibody best effort concentration
Select: when determining that according to square formation method being coated concentration is 1ug/mL, the OD value of monoclonal antibody is 1.05,
So it is most preferably coated concentration is 1ug/mL.As it is shown on figure 3, CD10, death receptor 6 and growth
The selection of differentiation factor-15 multi-resistance best effort concentration: along with the increase of monoclonal antibody extension rate, son to be measured
Palace sarcoma case serum and normal human serum OD value have the trend successively decreased, when antibody concentration is 1:200
Time, positive control (positive control OD value deducts blank OD value) is (the most right with normal control
Blank OD value is deducted according to OD value) ratio of A450nm (being called for short P/N value) is higher, therefore select
Selecting rabbit anti-human antibody's best effort concentration is 1:200.The best effort concentration that serum is groped is 1:25.
It is 1.2%BSA that confining liquid gropes best effort solubility.
Clinical serum Samples detection
Have detected 500 parts of serum specimens altogether, with hospital through definitive pathological diagnosis for sarcoma of uterus patients serum as sun
Property matched group (180 examples (wherein in early days sarcoma of uterus 85 example, sarcoma of uterus in late period 95 example)), non-son
Palace sarcoma patients includes that cyclomastopathy, normal population serum are negative control group (320 example), and PBST is
Blank, carries out qualitative and detection by quantitative clinical serum specimen by above-mentioned double crush syndrome method.
As it is shown on figure 3, with P/N value > 2 it is double crush syndrome Positive judgement standards, with pathological diagnosis
Detecting clinical serum specimen for standard, the detection sensitivity of sarcoma of uterus result in early days (SN) is 65
%, sarcoma of uterus in late period detection sensitivity (SN) is 71%, and accuracy rate (SP) reaches 70%.
The clinical therapeutic effect to sarcoma of uterus carries out dynamic evaluation
For determining that can this test kit be used for the therapeutic effect to sarcoma of uterus and carry out dynamic evaluation, we collect
Serum before and after 175 one's share of expenses for a joint undertaking palace sarcoma patients treatments.The testing result of 175 patients sees 4-6,
CD10 in serum before and after result display sarcoma of uterus patient treatment, death receptor 6 and Growth and Differentiation because of
There were significant differences (P < 0.05) for the level of son-15.Effectively CD10, death receptor 6 in serum after treatment
Can be greatly reduced with the level of GDF-15, point out this test kit can be used for sarcoma of uterus
Therapeutic effect carries out dynamic evaluation.
The present invention uses euzymelinked immunosorbent assay (ELISA), by the correlation step such as antibody screening, purification and pairing, exploitation
Go out to have the sarcoma of uterus early metaphase diagnostic kit of 3 diagnosis indexs.This technology is examined relative to current
For disconnected technology, have highly sensitive, accuracy rate is high, easy to use and low-cost advantage.Cause
Rare and the histopathologic multiformity of sarcoma of uterus, the most still lacks therapeutic regimen and pre-with bad
The common recognition of rear relevant risk factor.The successful implementation of project, can increase sarcoma of uterus, ovary little carefully
The concern of the Rare Neoplasms diseases such as the clear cell carcinoma of born of the same parents' cancer, palace body and cervix uteri and research, drive these few
See the development of tumor disease external diagnosis reagent, formed from sieving and diagnosis treatment prognosis prison
Survey a complete industrial chain, power-assisted life science, ensure human health.
The present invention can simply, quickly and accurately to early metaphase sarcoma of uterus patient diagnose, and accomplishes early
Diagnosis, early treatment, improve survival rate.Traditional diagnosis of uterine sarcoma mode is comparatively laborious, costly,
Accuracy rate is the highest, and has certain injury to human body.The clinical practice of this Project Product, on the one hand fall
Low diagnosis cost, save substantial amounts of diagnoses and treatment expense for patient, decrease the injury to human body,
And accuracy rate is greatly improved；On the other hand, product can be also used for the monitoring of patient's Prognosis, periodically
Monitoring patient body is healthy, finds that recurrence sign is treated in time, escorts for patient health.
The invention belongs to the reagent that external diagnosis reagent is relevant to tumor-marker analyte detection.Use brand-new
Sarcoma of uterus mark is used for diagnosis and the examination of sarcoma of uterus early metaphase, relative to tumor-marker in early days
Analyte detection technology, has advantage highly sensitive, that accuracy rate is high, easy to operate, has started asymptomatic micro-
The new way of stove tumor, has great social meaning.
In-vitro diagnosis (In Vitro Diagnosis, IVD) refer to by sample (blood, body fluid, tissue) from
Carry out detecting and diagnosing, for in-vivo diagnostic after human body takes out.This several years in-vitro diagnosis
Technology fast development, from the gene sequencing of gene level, SNP examination, mutation gene diagnosis, to egg
The various biological marker analyte detection of white level, to cellular level circulating tumor cell detect (CTC),
Liquid-basedcytology detection (TCT), then to the PET/CT etc. in tissue level.Generally speaking, body
Outer diagnosis is to easier, faster, Noninvasive, and the direction of multi information is developed.
The method of present invention molecular cloning realizes the solubility expression of four kinds of albumen, purification in escherichia coli
Go out high-quality standard substance albumen.The three kinds of standard substance protein immune animals obtained with purification, preparation is corresponding
Monoclonal antibody；Strong the resisting of sensitivity high specific is selected again by substantial amounts of ELISA pairing testing sieve
Body is as diagnosis antibody, and is purified into efficient catch and detection antibody is standby.With preparation standard substance and
Diagnosis antibody assembles ELISA kit, and that gropes capture antibodies is coated concentration and the dilution times of detection antibody
Number, makes standardized test kit rule of operation；Carry out stability and destructive testing includes the most steady
Qualitative, high temperature accelerates the failure stability, transportation stability, upper machine stability.Collect 300 parts of serum,
Wherein normal person and each 150 parts of sarcoma of uterus patients serum；Carry out with 3 kinds of labels for standard substance
ELISA detects；Result is carried out statistical analysis, the sensitivity of assessment diagnostic kit and accuracy.
The preferred embodiment of the present invention described in detail above, it will be appreciated that the common skill of this area
Art just can make many modifications and variations according to the design of the present invention without creative work.Therefore, all
Technical staff passes through logical analysis, reasoning according to present inventive concept on the basis of prior art in the art
Or according to the limited available technical scheme of experiment, all should determined by the claims
Protection domain among.
Claims (8)
1. sarcoma of uterus early metaphase quick diagnosis reagent kit, it is characterised in that by including that CD10, death receptor 6 and GDF-15 are made.
Sarcoma of uterus early metaphase quick diagnosis reagent kit the most according to claim 1, it is characterised in that including capturing antibody and detection antibody, described capture antibody is to include the monoclonal antibody that CD10, death receptor 6 and GDF-15 prepare respectively.
Sarcoma of uterus early metaphase quick diagnosis reagent kit the most according to claim 2, it is characterised in that described detection antibody is to include the polyclonal antibody that CD10, death receptor 6 and GDF-15 prepare respectively.
Sarcoma of uterus early metaphase quick diagnosis reagent kit the most according to claim 3, it is characterized in that, described capture antibody for being cloned into carrier for expression of eukaryon by CD10, death receptor 6 and GDF-15, and in mammalian cell, realize the expression of albumen, obtain corresponding antigen after purification, the corresponding monoclonal antibody obtained by described antigen immune mammal.
Sarcoma of uterus early metaphase quick diagnosis reagent kit the most according to claim 4, it is characterised in that the step that the preparation method of described capture antibody includes is as follows:
(1) preparation of antigen: the gene of CD10, death receptor 6 and GDF-15 is cloned into carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtaining antigen after purification, this antigen also can be used as standard substance；
(2) capture the preparation of antibody: by above-mentioned antigen immune mammal, obtain corresponding monoclonal antibody for capture antibody.
6. according to the arbitrary described sarcoma of uterus early metaphase quick diagnosis reagent kit of claim 3, it is characterized in that, described detection antibody is that the gene of CD10, death receptor 6 and GDF-15 is cloned into carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtain corresponding antigen after purification, the corresponding polyclonal antibody obtained by described antigen immune mammal.
Sarcoma of uterus early metaphase quick diagnosis reagent kit the most according to claim 6, it is characterised in that the step that the preparation method of described detection antibody includes is as follows:
(1) preparation of antigen: the gene of CD10, death receptor 6 and GDF-15 is cloned into carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtaining antigen after purification, this antigen also can be used as standard substance；
(2) detect the preparation of antibody: by above-mentioned antigen immune mammal, obtain corresponding polyclonal antibody for detection antibody.
8. the preparation method of the sarcoma of uterus early metaphase quick diagnosis reagent kit described in claim 3, its feature in, it comprises the following steps:
(1) preparation of antigen: CD10, death receptor 6 and GDF-15 gene are cloned into carrier for expression of eukaryon, and in mammalian cell, realize the expression of albumen, obtain required antigen after purification；
(2) preparation of antibody is captured: above-mentioned antigen immune mammal is obtained corresponding monoclonal antibody, and described monoclonal antibody is as the capture antibody of this test kit；
(3) preparation of antibody is detected: above-mentioned antigen immune mammal is obtained corresponding polyclonal antibody, and described polyclonal antibody is as the detection antibody of this test kit；
(4) capture antibody is coated:
1.. with carbonate/bicarbonate buffer, the capture antibody that concentration is 2 μ g/ml is coated the hole of microtitration plate；
2.. capping microtitration plate overnight incubation at 3 DEG C；
3.. discard and be coated liquid, and wash microtitration plate twice with cleaning mixture, in micropore, add 150 μ l PBST every time, remove cleaning mixture and remaining drop, dry and be put in 3 DEG C of environment；
(5) close: 150 μ l Block buffer are added in every hole, close and are coated remaining protein binding site in hole.
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| CN201610287446.6A CN105842461A (en) | 2016-04-30 | 2016-04-30 | Rapid diagnostic kit for uterine sarcoma in early and middle stages and preparation method of kit |
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