CN107677826A - For sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit - Google Patents
For sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit Download PDFInfo
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- CN107677826A CN107677826A CN201710723658.9A CN201710723658A CN107677826A CN 107677826 A CN107677826 A CN 107677826A CN 201710723658 A CN201710723658 A CN 201710723658A CN 107677826 A CN107677826 A CN 107677826A
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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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- 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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- 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 invention discloses one kind to be used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, and tumor marker component is：CD10, death receptor and growth and differentiation factor 15；Include tumor marker monoclonal detection antibody, Chemoluminescent substrate, the concentrated cleaning solution of tumor marker standard items, the coated microwell plate of tumor marker monoclonal capture antibody, horseradish peroxidase or alkali phosphatase enzyme mark.Beneficial effects of the present invention are as follows：With high sensitivity accuracy it is strong the characteristics of, its sensitivity and accuracy reach more than 70%, can effectively reduce in addition testing cost, reduce operating procedure and reduce detection error.
Description
Technical field：
The present invention relates to the kit field of cancer diagnosis detection, is specially a kind of quick for sarcoma of uterus early metaphase
Diagnose chemical luminescence reagent kit.
Background technology：
Sarcoma of uterus is initiated by one kind of uterine smooth muscle, endometrial stroma (or epithelium) and non-epithelium blending constituent
Between leaf source sexual reproduction road malignant tumour, structural constituent is complicated, and clinical manifestation is similar to fibroid but lacks specific.Due to nothing
Specific clinical shows, and CT is difficult, and most common sign is uterus increase, and most increases are obvious, sub in short-term
Palace enclosed mass increase is very fast, abdominopelvic cavity enclosed mass, or has ascites, stomachache and pain in the back；Lump uneven surface or in nodal-like.Uterus increases
Symptom is preoperative to be often misdiagnosed as " fibroid ", and rate of missed diagnosis is up to more than 50% by mistake, therefore many patients receive inappropriate
Operative treatment, such as laparoscope Minimally Invasive Surgery, cause iatrogenic tumour spread.The sarcoma of uterus incidence of disease is relatively low, is disliked in whole uterus
Property tumour 2~5% or so, accounts for the 1%~3% of gynecologic malignant tumor.
Although sarcoma of uterus is more rare in clinic, its aggressivity is strong, and grade malignancy is high, easily recurrence, lacks large sample and grinds
Study carefully, be easy to local infiltration and DISTANT METASTASES IN, wherein with sent out in hematogenous metastasis, abdominopelvic cavity transfer for main path, Lung metastases compared with
To be common, larger difficulty is brought to clinical treatment.Even early stage patient, also extreme difference, its case fatality rate exceed all uterus for prognosis
The 15% of malignant tumour, overall 5 years survival rates are less than 30%.The sarcoma of uterus cause of disease is still not clear so far, and clinical manifestation is also without spy
The opposite sex, it is difficult to make a definite diagnosis in the preoperative, it is postoperative histopathological examination (biopsy) that it, which diagnoses goldstandard,.Pathological diagnosis is a kind of sampling
Check, the quality of sample is required, the quality of sample directly affects the quality of pathological section；Pathological diagnosis is needed by diagnosis
The experience and professional knowledge of doctor, there is certain subjectivity；Pathological diagnosis testing cost is higher.It there is no at present for uterus meat
The specific tumor marker of knurl.
Fibroid is intrauterine benign tumour, in the women of more than 30 years old, has 20% to 30% uterus muscle occurs
Knurl.Although fibroid can cause the symptoms such as dysmenorrhoea, there is no life danger, so typically being treated using the hormone of Reservations uterus
Method.And sarcoma of uterus is malignant tumour, if being diffused into its hetero-organization, 5 years survival rates of patient only have 10% to 20%, because
This early detection is extremely important.
Current diagnosis of uterine sarcoma goldstandard is postoperative histopathological examination (biopsy).Pathological diagnosis is a kind of sampling inspection
Look into, the quality of sample is required, the quality of sample directly affects the quality of pathological section；Pathological diagnosis is needed by diagnosis doctor
The experience and professional knowledge of teacher, there is certain subjectivity；Pathological diagnosis testing cost is higher, and may make a definite diagnosis too late.
In addition to pathological diagnosis, also Magnetic resonance imaging (MRI) or positron e mission computed tomography (PET) diagnose skill
Art.When carrying out PET diagnosis, what is utilized is the property that sarcoma of uterus cell can absorb more glucosan, but fibroid is sometimes
Many glucose are absorbed, so more difficult accurate judgement.Diffusion-weighted imaging (DWI) is to the position of tumour and qualitative has side
Help, but result waits to confirm.
In recent years, some research institutions are also developing related kit by immunological method or genetics method.Once
It has been reported that the researcher of Japan Fukui university developed a kind of cooperation positron emission tomography (PET) in 2011
Medicament is checked, for differentiating that the tumour that intrauterine occurs is benign fibroid or pernicious sarcoma of uterus on earth.Fukui
The researcher of university notices that exception occurs in the acceptor for receiving estrogen in sarcoma of uterus cell, causes cell to absorb female
The ability of hormone reduces.Research group is conceived to this point, and the medicament of entitled " FES " is produced by handling estrogen.It is this
Medicament can be focused in fibroid rather than sarcoma of uterus, be then imaged using PET, and confirmation is fibroid or uterus meat
Knurl.
It is special without the tumor markers of sensitivity, imaging diagnosis because sarcoma of uterus sings and symptoms is without specificity
Not high, the preoperative diagnosis for being often difficult to make sarcoma of uterus of property, majority is that even postoperative pathological just diagnoses in art.It is either super
Acoustic inspection or Positron Emission Computed Tomography (PET) scanning, all it is difficult to the good evil for differentiating smooth muscle tumor in the preoperative
Property, and operate complex, there is certain injury to human body.Diffusion-weighted imaging (DWI) to the position of tumour and
It is qualitative helpful, but result waits to confirm.Postoperative histopathological examination is a kind of sampling check, and having to the quality of sample will
Ask, the quality of sample directly affects the quality of pathological section；Pathological diagnosis needs the experience and professional knowledge by diagnostician,
With certain subjectivity；Pathological diagnosis testing cost is higher, and may make a definite diagnosis too late.
At present, in the market also comes out without the rapidly and efficiently diagnostic kit for sarcoma of uterus, seriously affects uterus
Sarcoma early detection and treatment.
The content of the invention：
The purpose of the present invention is to be directed to insufficient existing for above-mentioned existing diagnosis of uterine sarcoma, there is provided a kind of high sensitivity,
Accuracy is strong and easy to use is efficiently used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit.
In order to realize the object of the invention, the present invention adopts the technical scheme that：For sarcoma of uterus early metaphase quick diagnosis
Chemical luminescence reagent kit, tumor marker component are：CD10, death receptor and GDF-15.
The kit includes tumor marker standard items, tumor marker monoclonal captures the coated microwell plate of antibody,
The tumor marker monoclonal of horseradish peroxidase or alkali phosphatase enzyme mark detection antibody, Chemoluminescent substrate and concentration
Cleaning solution.
The Chemoluminescent substrate of the horseradish peroxidase includes A liquid and B liquid, needs during wherein A liquid/B liquid uses
1:1 mixing.
The Chemoluminescent substrate of the alkaline phosphatase is adamantane and its reinforcing agent.
Monoclonal antibody is as capture antibody made from the tumor marker difference.
Polyclonal antibody after HRP marks made from the tumor marker difference is as detection antibody.
The step of preparation method of the capture antibody includes is as follows：
(1) preparation of antigen：CD10, death receptor and GDF-15 gene cloning to protokaryon or eucaryon table
Up to carrier, antigen is obtained after realizing the expression and purification of albumen；It is for tumor marker calibration object.
(2) preparation of antibody is captured：By above-mentioned antigen immune mammal, it is anti-for capture to obtain corresponding monoclonal antibody
Body.
The step of preparation method of the detection antibody includes is as follows：
(1) preparation of antigen：CD10, death receptor and GDF-15 gene cloning to protokaryon or eucaryon table
Up to carrier, antigen is obtained after realizing the expression and purification of albumen；It is for tumor marker calibration object.
(2) preparation of antibody is detected：By above-mentioned antigen immune mammal, corresponding monoclonal antibody is obtained, then through peppery
Root peroxidase or alkali phosphatase enzyme mark are detection antibody.
The capture antibody is coated in the hole of microtiter plate in advance.It is anti-using the streptavidin system of biotin one coating
Body.
The horseradish peroxidase Chemoluminescent substrate includes A liquid and B liquid, wherein, Chemoluminescent substrate A liquid,
Including 13mM luminols, 0.6mM 4- xenols, 0.1mM4- iodobenzene boric acid.B liquid is 5mM urea peroxides.
It is described to use the streptavidin system coated antibody of biotin one, including following coating step：A, it is affine to prepare strepto-
Plain microwell plate：0.05mol/L, pH9.6 carbonate buffer solution dilute Streptavidin to 15mg/mL, are added by 100uL/ holes micro-
In orifice plate, 4 DEG C are coated with overnight.Next day taking-up 0.01mol/L, pH7.4, containing 0.05% polysorbas20 PBS board-washings 3 times.Then use
Bovine serum albumin(BSA) confining liquid room temperature is closed 2 hours.
B, biotinylated mAb is prepared：Monoclonal antibody purification is diluted to 1mg/mL, Ran Houyong by 0.01M PBS
Antibody is adjusted to meta-alkalescence by 1.5M, pH 9.6CBS.0.5mg BNHS are weighed with 0.5mL dmso solutions, wait to mark in 1mL
Remember that antibody kind adds BNHS liquid 10uL, room temperature lucifuge, be gently mixed reaction 4h.Marked product was dialysed for 4 DEG C in 0.02M PBS
At night, it is standby to collect labelled antibody detection.
C, it is coated with biotinylated mAb：Biotinylated mAb is diluted to debita spissitudo, by 100uL/
Hole is added in Streptavidin microwell plate, and 37 DEG C are incubated 2 hours.D, close：With 0.01mol/L, pH7.4, containing 0.05% tween
20PBS board-washings 3 times, then be incubated 30 minutes with 37 DEG C of sucrose confining liquid, patted dry on blotting paper standby.
The present invention, by a variety of permutation and combination, filters out 3 tumor markers from numerous tumor markers
CD10, death receptor 6 and GDF-15 composition sarcoma of uterus early metaphase quick diagnosis reagent kit.Wherein, CD10 is one
Kind neutral endopeptidase, molecular weight is about 97kDa, is made up of, is had been found to universally present in maincenter 750 amino acid
In the various tissues of nervous system and periphery, and there are numerous organizing specific sexual functions, can induction of T cell apoptosis.Death receptor 6
(DR6) be TNF death receptors family a member, critical function, death receptor 6 are played in immune system and nervous system
(DR6) it can adjust the degeneration and death of neuronal cell and neural axis.Research shows that DR6 levels are bright in sarcoma of uterus patients serum
It is aobvious to exceed healthy population.GDF-15 (GDF-15) is transforming growth factor β (transforming growth
Factor β, TGF-β) superfamily member a remote branch of a clan, GDF-15 (GDF-15) is a kind of stress response protein,
GDF-15 high expression in prostate and placenta under physiological conditions, the faint expression in its most hetero-organization, most of research tables
Bright GDF-15 (GDF-15) expresses rise in kinds of tumors.Due to extracting this 3 tumor marker produce in blood plasma
Low, complex steps are measured, so we will largely be produced using the method for molecular cloning.
Compared with prior art, beneficial effects of the present invention are as follows：
(1) CD10, DR6 and GDF-15 are combined the tumor markers index as sarcoma of uterus, for sarcoma of uterus
Early metaphase Combining diagnosis, design are very novel；Sarcoma of uterus can be comprehensively accurately more detected, can preferably judge pelvic swelling
Block it is good pernicious, antidiastole preferably is carried out to the benign image of uterine masses of gynaecology and sarcoma of uterus, its sensitivity and accuracy are equal
Reach 70%；
(2) diagnosis of uterine sarcoma and physical examination examination are can be not only used for, can be used for sarcoma of uterus therapeutic effect and prognosis recurrence again
Monitoring, the blank of domestic sarcoma of uterus early metaphase examination and diagnosis is filled up；
(3) production cost is low, the stability of testing result, and repeatability and the degree of accuracy are high.
Brief description of the drawings
Fig. 1 is the schematic diagram that the present invention is used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit；
Fig. 2 is that the present invention is pretherapy and post-treatment in sarcoma of uterus for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit
Galectin-3 change comparison diagram is detected in blood；
Fig. 3 is that the present invention is pretherapy and post-treatment in sarcoma of uterus for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit
TTR change comparison diagram is detected in blood；
Fig. 4 is that the present invention is pretherapy and post-treatment in sarcoma of uterus for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit
HE4 change comparison diagram is detected in blood；
Fig. 5 is that the present invention is pretherapy and post-treatment in sarcoma of uterus for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit
CA125 change comparison diagram is detected in blood.
Embodiment
It is used to the chemiluminescence of sarcoma of uterus early metaphase quick diagnosis to the present invention below in conjunction with the drawings and specific embodiments try
Explanation is described in detail in agent box.
It is slow for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, including capture antibody, capture antibody closing
Fliud flushing, standard items, mark HRP detection antibody, cleaning solution and nitrite ion etc..Wherein tumor marker component is：CD10, death
Acceptor and GDF-15.Capturing antibody includes monoclonal antibody made from 3 kinds of labels difference；The kit bag
Include tumor marker standard items, tumor marker monoclonal capture antibody coated microwell plate, horseradish peroxidase or alkalescence
Tumor marker monoclonal detection antibody, Chemoluminescent substrate, the concentrated cleaning solution of phosphatase enzyme mark.The horseradish peroxidating
Thing enzymology luminous substrate liquid includes A liquid and B liquid, needs 1 during wherein A liquid/B liquid uses:1 mixing.Wherein, chemical luminous substrate
Liquid A liquid, including 13mM luminols, 0.6mM 4- xenols, 0.1mM 4- iodobenzene boric acid.B liquid is 5mM urea peroxides.It is described
Alkaline phosphatase Chemoluminescent substrate is adamantane and its reinforcing agent.
By detecting CD10, death receptor and GDF-15, make its sensitivity and accuracy reach 70% with
On.
The capture antibody is that CD10, death receptor and GDF-15 be cloned into carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, obtains corresponding antigen after purification, the antigen immune mammal is obtained
Corresponding monoclonal antibody.
The step of preparation method of the capture antibody includes is as follows：
(1) preparation of antigen：By the method for molecular cloning CD10, death receptor and GDF-15 gene
Carrier for expression of eukaryon is cloned into, and the expression of albumen is realized in mammalian cell, obtains antigen after purification, this antigen also may be used
Used as standard items；The preparation of the antigen can also be prepared using existing conventional method, no longer burdensome herein.
(2) preparation of antibody is captured：By above-mentioned antigen immune mammal, it is anti-for capture to obtain corresponding monoclonal antibody
Body.The mammal is mouse and rabbit, preferably mouse.The preparation of the capture antibody can also use existing conventional method
Prepare, it is no longer burdensome herein.
The detection antibody includes more after CD10, death receptor and HRP made from GDF-15 difference are marked
Clonal antibody.
The detection antibody is that CD10, death receptor and GDF-15 be cloned into carrier for expression of eukaryon, and
The expression of albumen is realized in mammalian cell, obtains corresponding antigen after purification, the antigen immune mammal is obtained
Progress HRP marks after polyclonal antibody.The step of preparation method of the detection antibody includes is as follows：
(1) preparation of antigen：Base by the method for molecular cloning CD10, death receptor and GDF-15
Because being cloned into carrier for expression of eukaryon, and the expression of albumen is realized in mammalian cell, obtain antigen after purification, this antigen
Standard items can be used as to use；
(2) preparation of antibody is detected：By above-mentioned antigen immune mammal, corresponding polyclonal antibody is obtained, then through horseradish
Peroxidase or alkali phosphatase enzyme mark are detection antibody；
The mammal is mouse and rabbit, preferably rabbit.HRP marks are carried out to the polyclonal antibody to be used
Conventional existing method at present.
Wherein, in the hole for the microtiter plate that the capture antibody can be coated in PVC material in advance, step can be simplified
Suddenly, detection efficiency is improved.
The capture antibody is coated in the hole of microtiter plate in advance.It is anti-using the streptavidin system of biotin one coating
Body.
The Chemoluminescent substrate of the horseradish peroxidase includes A liquid and B liquid, wherein, Chemoluminescent substrate A
Liquid, including 13mM luminols, 0.6mM 4- xenols, 0.1mM 4- iodobenzene boric acid.B liquid is 5mM urea peroxides.
It is described to use the streptavidin system coated antibody of biotin one, including following coating step：A, it is affine to prepare strepto-
Plain microwell plate：0.05mol/L, pH9.6 carbonate buffer solution dilute Streptavidin to 15mg/mL, are added by 100uL/ holes micro-
In orifice plate, 4 DEG C are coated with overnight.Next day taking-up 0.01mol/L, pH7.4, containing 0.05% polysorbas20 PBS board-washings 3 times.Then use
Bovine serum albumin(BSA) confining liquid room temperature is closed 2 hours.B, biotinylated mAb is prepared：0.01M PBS will purify Dan Ke
Grand antibody is diluted to 1mg/mL, and antibody then is adjusted into meta-alkalescence with 1.5M, pH 9.6CBS.0.5mg BNHS are weighed with 0.5mL
Dmso solution, the μ L of BNHS liquid 10 are added in 1mL antibody kinds to be marked, room temperature lucifuge, are gently mixed reaction 4h.Mark production
Thing 4 DEG C of dialysed overnights in 0.02M PBS, it is standby to collect labelled antibody detection.C, it is coated with biotinylated mAb：Will be raw
Thing elementization monoclonal antibody is diluted to debita spissitudo, is added by 100uL/ holes in Streptavidin microwell plate, and 37 DEG C of incubations 2 are small
When.D, close：With 0.01mol/L, pH7.4, containing 0.05% polysorbas20 PBS board-washings 3 times, then with sucrose confining liquid 37 DEG C be incubated 30
Minute, patted dry on blotting paper standby.
The present invention, which is used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, includes CD10, death receptor and growth
Two standards raised simultaneously as diagnosis early metaphase breast cancer in three indexs of differentiation factor -15, Cleaning Principle such as Fig. 1 institutes
Show；Two in three CD10, death receptor and GDF-15 indexs decline as sarcoma of uterus therapeutic effect simultaneously
The standard of assessment.It can be used for clinically by treatment of the height of 3 tumor marker levels in detection blood to sarcoma of uterus
Effect carries out dynamic evaluation；It can be additionally used for the application of the clinically relapse and metastasis to sarcoma of uterus and Index for diagnosis.
Test effect explanation
The selection of double-antibody sandwich elisa optimum experimental condition
It is coated with the selection of the anti-human CD10 of mouse, death receptor and GDF-15 monoclonal antibody best effort concentration：According to side
When the tactical deployment of troops determines that it is 1ug/mL to be coated with concentration, the OD values of monoclonal antibody are 1.03, so its optimal coating concentration is 1ug/mL.
As shown in Fig. 2 rabbit-anti people CD10, death receptor and the how anti-best effort concentration of GDF-15 selection：With detection
The increase of antibody extension rate, sarcoma of uterus case serum and normal human serum OD values to be measured have the trend successively decreased, when antibody is dense
Spend for 1：When 200, (normal control OD values subtract positive control (positive control OD values subtract blank control OD values) with normal control
Blank control OD values) the ratio between A450nm (abbreviation P/N values) is higher, therefore it is 1 to select rabbit-anti human antibody best effort concentration：200.Blood
The best effort concentration groped clearly is 1：25.It is 1.2%BSA that confining liquid, which gropes best effort solubility,.
Clinical serum Samples detection
400 parts of serum specimens are have detected altogether, using hospital through being diagnosed as sarcoma of uterus I-II phases patients serum as positive control
Group (110), other benign patients and normal population serum be negative control group (290 (and normal 170, benign 120
Example)), PBST is blank control
The clinical therapeutic effect to sarcoma of uterus carries out dynamic evaluation
To determine that can this kit be used to carry out dynamic evaluation to the therapeutic effect of breast cancer, we have collected 102 parts
The pretherapy and post-treatment serum of sarcoma of uterus patient.As a result the testing result of 102 patients shows sarcoma of uterus patient referring to Fig. 3-5
CD10 in pretherapy and post-treatment serum, death receptor and GDF-15 horizontal there were significant differences (P<0.05).Effectively control
CD10, death receptor and GDF-15 level can be greatly reduced in serum after treatment, prompt this kit to be used for pair
The therapeutic effect of sarcoma of uterus carries out dynamic evaluation.
Preferred embodiment of the invention described in detail above, it will be appreciated that the ordinary skill of this area is without wound
The property made work can makes many modifications and variations according to the design of the present invention.Therefore, all technician in the art
According to present inventive concept in prior art basis by logic analysis, reasoning or according to the limited available technology of experiment
Scheme, should be among the protection domain determined by the claims.
Claims (8)
1. it is used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, it is characterised in that tumor marker component is：
CD10, death receptor and GDF-15.
2. according to claim 1 be used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, it is characterised in that
The kit includes tumor marker standard items, the coated microwell plate of tumor marker monoclonal capture antibody, horseradish peroxide
The tumor marker monoclonal of compound enzyme or alkali phosphatase enzyme mark detection antibody, Chemoluminescent substrate and concentrated cleaning solution.
3. according to claim 2 be used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, it is characterised in that
The horseradish peroxidase Chemoluminescent substrate includes A liquid and B liquid, needs 1 during wherein A liquid/B liquid uses:1 mixing.
4. according to claim 3 be used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, it is characterised in that
Alkaline phosphatase Chemoluminescent substrate is adamantane and its reinforcing agent.
5. according to claim 3 be used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, it is characterised in that
Monoclonal antibody is as capture antibody made from the tumor marker difference；
The step of preparation method of the capture antibody includes is as follows：
(1) preparation of antigen：CD10, death receptor and GDF-15 gene cloning to protokaryon or eukaryotic expression are carried
Body, antigen is obtained after realizing the expression and purification of albumen；It is for tumor marker calibration object；
(2) preparation of antibody is captured：By above-mentioned antigen immune mammal, corresponding monoclonal antibody is obtained as capture antibody.
6. according to claim 3 be used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, it is characterised in that
Polyclonal antibody after obtained HRP marks made from the tumor marker difference is used as detection antibody,
The step of preparation method of the detection antibody includes is as follows：
(1) preparation of antigen：CD10, death receptor and GDF-15 gene cloning to protokaryon or eukaryotic expression are carried
Body, antigen is obtained after realizing the expression and purification of albumen；It is for tumor marker calibration object；
(2) preparation of antibody is detected：By above-mentioned antigen immune mammal, corresponding monoclonal antibody is obtained, then through horseradish mistake
Oxide enzyme or alkali phosphatase enzyme mark are detection antibody.
7. according to claim 6 be used for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit, it is characterised in that
The horseradish peroxidase Chemoluminescent substrate includes A liquid and B liquid, wherein, Chemoluminescent substrate A liquid, including 13mM
Luminol, 0.6mM4- xenols, 0.1mM 4- iodobenzene boric acid.B liquid is 5mM urea peroxides.
8. the preparation method according to claim 6 for sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit,
Its feature is in, the streptavidin system coated antibody of biotin one, including following coating step：
A, Streptavidin microwell plate is prepared：0.05mol/L, pH9.6 carbonate buffer solution dilute Streptavidin to 15mg/
ML, added by 100uL/ holes in microwell plate, 4 DEG C are coated with overnight；Next day taking-up 0.01mol/L, pH7.4, containing 0.05% tween
20PBS board-washings 3 times；Then closed 2 hours with bovine serum albumin(BSA) confining liquid room temperature；
B, biotinylated mAb is prepared：Monoclonal antibody purification is diluted to 1mg/mL, Ran Houyong by 0.01M PBS
Antibody is adjusted to meta-alkalescence by 1.5M, pH 9.6CBS；0.5mg BNHS are weighed with 0.5mL dmso solutions, wait to mark in 1mL
Remember that antibody kind adds the μ L of BNHS liquid 10, room temperature lucifuge, be gently mixed reaction 4h.Marked product was dialysed for 4 DEG C in 0.02M PBS
At night, it is standby to collect labelled antibody detection；
C, it is coated with biotinylated mAb：Biotinylated mAb is diluted to debita spissitudo, added by 100uL/ holes
Enter in Streptavidin microwell plate, 37 DEG C are incubated 2 hours；
D, close：With 0.01mol/L, pH7.4, containing 0.05% polysorbas20 PBS board-washings 3 times, then with the 37 DEG C of incubations of sucrose confining liquid
30 minutes, patted dry on blotting paper standby.
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|---|---|---|---|
| CN201710723658.9A CN107677826A (en) | 2017-08-22 | 2017-08-22 | For sarcoma of uterus early metaphase quick diagnosis chemical luminescence reagent kit |
Applications Claiming Priority (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507424A (en) * | 2018-11-07 | 2019-03-22 | 国家纳米科学中心 | Based on thermophoresis to the detection system and method for EGFR receptor |
| CN112698041A (en) * | 2020-12-16 | 2021-04-23 | 深圳上泰生物工程有限公司 | Compound, growth differentiation factor 15 detection kit thereof and application |
| CN113238063A (en) * | 2021-05-31 | 2021-08-10 | 迈克生物股份有限公司 | Use of GDF15 to assess the progression of metabolic syndrome patients to cardiovascular disease |
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2017
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507424A (en) * | 2018-11-07 | 2019-03-22 | 国家纳米科学中心 | Based on thermophoresis to the detection system and method for EGFR receptor |
| CN112698041A (en) * | 2020-12-16 | 2021-04-23 | 深圳上泰生物工程有限公司 | Compound, growth differentiation factor 15 detection kit thereof and application |
| CN113238063A (en) * | 2021-05-31 | 2021-08-10 | 迈克生物股份有限公司 | Use of GDF15 to assess the progression of metabolic syndrome patients to cardiovascular disease |
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