CN105891492A - Method for detecting antigens of circulating tumor cells by virtue of microsphere enrichment enhancement technique and kit - Google Patents
Method for detecting antigens of circulating tumor cells by virtue of microsphere enrichment enhancement technique and kit Download PDFInfo
- Publication number
- CN105891492A CN105891492A CN201610327849.9A CN201610327849A CN105891492A CN 105891492 A CN105891492 A CN 105891492A CN 201610327849 A CN201610327849 A CN 201610327849A CN 105891492 A CN105891492 A CN 105891492A
- Authority
- CN
- China
- Prior art keywords
- carrying
- her2
- hrp
- latex beads
- circulating tumor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- 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
- G01N33/57415—Specifically defined cancers of breast
-
- 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
- G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Zoology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting antigens of circulating tumor cells by virtue of a microsphere enrichment enhancement technique and kits. The method comprises the following steps: (1) adding latex microspheres into MES buffer solution, adding HER2 rabbit monoclonal antibodies, BSA, HRP and EDC, and uniformly mixing, blowing and beating; carrying out oscillating reaction at 45 DEG C for 1h; carrying out centrifuging, washing with washing liquid, and removing supernatant; fixing volume by virtue of confining liquid, carrying out vibration confining at 45 DEG C for 30 minutes, and carrying out centrifugal washing once; and finally carrying out constant volume storage for later use; and (2) adding cell suspension into a micropore reaction batten, fixing volume with PBS, adding biotin labeled EpCAm antibodies and antibody labeled latex microspheres, and carrying out oscillating reaction at the room temperature for 1h; adding streptavidin magnetic beads, and carrying out oscillating reaction at the room temperature for 1h; carrying out magnetic separation, washing, and removing supernatant; and carrying out color development reaction. One kit comprises the HRP and the HER2 antibody labeled 80nm latex microspheres. Another kit comprises the 80nm latex microspheres, the HER2 rabbit monoclonal antibodies and the HRP, wherein the HER2 rabbit monoclonal antibodies are used for labeling the latex microspheres. The method has high sensitivity, good accuracy and cost advantage and is therefore convenient to cost advantage and apply.
Description
Technical field
A kind of method that the present invention relates to immune detection circulating tumor cell antigen.
Background technology
In the world, breast carcinoma is modal malignant tumor in female group, in all cases, there are about 20-
30% is " the most dangerous breast carcinoma " HER2 positive breast cancer.Relative to other kinds of breast carcinoma, HER2 positive breast
Faster, grade malignancy is higher for the progression of disease speed of cancer, it is easier to recurring and shift, prognosis is the most poor.
The sickness rate of breast carcinoma of China in ascendant trend year by year, the big city such as the most in Beijing, Tianjin, Shanghai, breast carcinoma position
Occupy first of female malignant sickness rate.According to the biological characteristics of breast carcinoma, it can be divided into 4～5 molecule subgroups, no
Same subgroup has different prognosis and therapeutic strategy.Modern medicine has come into the epoch of breast cancer classification treatment, gets trouble clear
The molecule type of person is the premise of correct treatment, in all patient with breast cancers, belongs to HER2 gene overexpression person and accounts for 20%-
30%, compare with other subgroup breast carcinoma, the type patient with breast cancer is easier to recurrence and transfer occur.If only accepting routine
Comprehensive Treatment, the life span of HER2 breast cancer patients with positive is only the half of HER2 negative patient；On the contrary, if can be the most true
Determining HER2 state, use immunotherapy targeted autoantibody early, compared with ordinary breast cancer patient, the chance for survival of HER2 positive patient will
Close to HER2 negative patient.
Since Slamon in 1987 etc. are found that HER2 effect in human breast cancer pathogenesis, and classics is anti-
HER2 target therapeutic agent Herceptin applied for more than 10 years through U.S. FDA approval from 1998 since clinic the most,
Think that breast carcinoma HER2 state is the important prognostic factor of patient with breast cancer and the predictor of targeting medicament curative effect.Numerous studies
Data and clinical practice have made everybody reach certain common recognition at aspects such as anti-HER2 mechanisms of therapeutic action and clinical efficacies.And mesh
Before detect HER2 state clinically and be all based on patient's primary tumor or the detection of metastasis tissue, and these detections are often subject to
To impacts such as tumor tissues misoperations such as (draw materials) fixing and preservations, detection method, Tumor Heterogeneity or variations, cause trouble
The HER2 state of person cannot determine, particularly some relapse and metastasis patient, and the conventional tumor tissues holding time is longer or turns
Move stove and biopsy cannot obtain the situations such as tissue, more cannot over the course for the treatment of patient be monitored dynamically.For HER-2 sun
Property patient with breast cancer targeted therapy, current clinical guidelines is: 1. HER2 positive breast cancer postoperative adjuvant therapy suggestion use
Trastuzumab targeted therapy 1 year is standard care；2. progressive stage HER-2 positive breast cancer suggestion Trastuzumab uses at least one year, and
Recommending until being in progress, even, more renewing on the basis of may continue to Trastuzumab targeted therapy after targeting maintaining treatment is in progress
Chemotherapy.
Seek peace from the biological characteristics of HER-2 breast cancer patients with positive the curative effect of targeted therapy, the most as early as possible, accurately carry out
The detection of HER2 state, could carry out risk of recurrence degree accurate evaluation and take correct immunotherapy targeted autoantibody (Trastuzumab patient
Deng targeted drug).Paraffin-embedded tissue is used conventional immunohistochemical method and FISH (Fluorescence in situ hybridization) method to group by tradition
Knitting preparation of specimen and preserve higher requirement, therefore, once these are performed the operation or biopsy specimen can not meet detection and want
Asking, the HER-2 state of patient just cannot determine, tumor cells typing and Classification treatment just cannot be carried out, and sees this kind of the most repeatly
Patient, before as oversize in the Saving specimen time, staging tomography, sample reception is very little, conventional detection method is nonstandard, be repeatedly detected
HER-2 belongs to critical state, Patients on Recurrence transfer is not inconsistent with former tumor biological behavior but metastasis cannot biopsy etc. again, face
The HER-2 state that on bed, fubaritic patient is real；The heart that HER-2 targeted drug Trastuzumab itself is fairly expensive, the most potential
Dysentery, this make clinician patient use in the decision-making of targeted therapy in a dilemma, be difficult to accept or reject.Additionally, except visitor
Seeing outside efficient evaluation, HER-2 breast cancer patients with positive is in stable or complete incidence graph feelings during accepting targeted therapy
Under condition, the most also it cannot be carried out the most dynamically examination of curative effect, circulating tumor label and circulating tumor are thin
Born of the same parents (CTC) detection can only play a role to a certain extent, but and HER-2 positive breast cancer cells dynamic in non-real antimer
State change and curative effect.
Circulating tumor cell (CTCs) detection be generally acknowledge at present can monitor tumor patient in-vivo tumour load very well
Perfected process, in treatment of cancer, doctor is often desirable to as monitoring and circulating tumor cell level is adjusted treatment and prognosis
Diagnosis basis, but traditional detection method often cannot accurately determine CTCs level in the patient, CellSearch blood
Middle circulating tumor cell system is the technology that Veridex company is proprietary, by U.S. FDA access be applied to prediction have turn
The progresson free survival rate of breast carcinoma, colorectal cancer or the carcinoma of prostate of shifting property and total survival rate.This method of inspection can be carried out at any time,
It is easy to this type of cancer patient is continuously monitored.In Europe, CellSearch is also identified an effective diagnostic check
Instrument.China has been completed the clinical research of this technology, applies for official written reply to country FDA, and result of study is same with external
Class research is basically identical.The operation principle of CellSearch detection is, have employed a kind of antibody (title that can combine with iron granules
For ferrofluid), this antibody/ferrofluid has extremely strong specific binding capacity with CTCs.Strong magnetic is reapplied after in conjunction with
These CTCs are extracted from blood sample by body, reach the effect of cell enrichment, permissible after carrying out biological or chemical dyeing
CellSearch detector accurately identifies CTCs and counts.Swell it is now recognized that advanced breast cancer patient detects circulation in blood
Oncocyte system CTC marginal value is >=5CTCs, research it has been proved that before Breast Cancer Patients Treated >=5CTCs person PFS (gets nowhere
Life span) shorter, if the circulating tumor cell number of patient is reduced to less than 5 after the treatment, then its PFS will obtain substantially
Extend.Therefore, CTCs detection is particularly significant for patient with breast cancer's prognosis and curative effect evaluation, it is possible to improve controlling of advanced breast cancer
Treat strategy.The price of CellSearch detector about 4,000,000 yuan in the market, disregard detectable, general curative mechanism
Cannot introduce this detection technique, once introduce, the price of its detection is the most high.Therefore, general patient cannot bear its conduct
Conventional sense, more cannot function as the dynamic monitoring during treatment.
In recent years, before the deep combination international bio medical analysis technical field of bioassay technique and nanotechnology
Edge and hot issue.Compared with traditional material, nano level granule exists that specific surface area is big and the feature such as small-size effect.And magnetic
The research of property polymer microsphere starts from 20 century 70s, is that utilize that nanotechnology prepares a kind of has the micro-of magnetic properties
Ball.On the one hand, it has numerous characteristics of organic polymeric microspheres to this type of microsphere, such as by the approach such as copolymerization, surface modification,
Give the multiple reactive functional groups in its surface (such as hydroxyl, amino, carboxyl etc.), can be with anti-by the way of absorption or covalent bonding
The bioactive substance such as body, DNA, RNA combines；On the other hand, can make at externally-applied magnetic field easily owing to it has magnetic
Separate from medium under with.Compared with traditional isolation technics, separation and enrichment are incorporated into one by the method, and it is bigger
Specific surface area substantially increase the kinetic rate of the interphase interaction of reactant in separation process, have efficient, quick, non-
The advantage stained.Therefore, magnetic macromolecular microsphere is widely used work and separates material and carrier, as at clinical diagnosis, targeting
The fields such as medicine, cell separation, cell marking have broad application prospects.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides microsphere enrichment enhancement techniques detection circulating tumor cell and resist
Former method and test kit.
A kind of method of microsphere enrichment enhancement techniques detection circulating tumor cell antigen, step is as follows:
1) synthesis of HRP and HER2 antibody labeling latex beads:
Take 100ul solid content 10%w/v 80nm latex beads and add the piping and druming of MES buffer solution uniformly, add
0.614ug HER2 rabbit antibody mab, 90ugBSA, 9ugHRP, 5mg EDC also blows and beats uniformly, and total system is 1mL；Shake at 45 DEG C
Swing reaction 1h；It is centrifuged and washs with cleaning mixture, remove supernatant；Being settled to original volume 1mL with confining liquid, 30min is closed in 45 DEG C of vibrations,
It is centrifuged and washed once with confining liquid；Finally it is settled to 1mL with the confining liquid containing 0.5% sucrose；4 DEG C store for future use；
2) detection of circulating tumor cell antigen in peripheral blood:
Take 10-20uL cell suspension in micropore reaction batten in, be settled to 200uL with the PBS that pH is 2, add
The biotin labeling EpCAM antibody of 1uL0.04mg/mL and 50uL step 1) in the latex beads of antibody labeling, shaken at room temperature is anti-
Answer 1h；Add 2uL 1% Streptavidin MagneSphere, oscillating reactions 1h under room temperature；Magneto separate, washs and removes supernatant；Add
100 microlitre TMB and Substrate cocktail and 50 microlitre 1% carbamide peroxide solution, lucifuge 15min under room temperature condition, lucifuge terminates
Rear addition 50 microlitre sulphuric acid stop buffer；Magneto separate, takes 150 microlitre supernatants and measures OD450 in microplate reader.
A kind of test kit using described method, including the 80nm latex beads of HRP and HER2 antibody labeling.
A kind of test kit using described method, the HER2 rabbit including 80nm latex beads, for labelling latex beads is single
Anti antibody and HRP.
Accompanying drawing explanation
Fig. 1 is the standard curve of known low concentrations sample；
Fig. 2 is the standard curve of known high concentration sample.
Detailed description of the invention
Owing to Nano-grade latex microsphere has bigger specific surface area, the excellent load of the materials such as protein nucleic acid can be become
Body.The present invention uses magnetic microsphere to separate and enrichment HER-2 positive breast cancer cells, and makees with the Nano-grade latex microsphere of little yardstick
For solid phase carrier coupled antibody and enzyme molecule simultaneously, substitute the enzyme labelled antibody in conventional ELISA, operational approach such as conventional ELISA
Operation, uses microplate reader to carry out reading.Compare traditional compound reaction process nano-carrier and can introduce more multienzyme molecule (as peppery
Root peroxidase HRP), bring and more significantly detect signal, reach the effect strengthened.
One, material and reagent
Table 1 is drug specifications, without particularly pointing out, for conventional reagent.
Table 1
| Nomenclature of drug | Purity |
| MES | High-purity |
| EDC | 99% |
| Carboxyl latex microsphere (80nm) | 10% (w/v) |
| HRP | Biochemical reagents BR/RZ-3 |
| HER2 antibody | 0.307mg/mL |
| EpCAM antibody | 0.5mg/mL |
| Immunomagnetic beads | 10mg/mL |
| TMB | Super pure |
| DMSO | AR |
| Carbamide peroxide | 97% |
| Concentrated sulphuric acid | GR |
| Disodium hydrogen phosphate | ACS |
| Potassium dihydrogen phosphate | ACS |
| Hydrochloric acid | AR |
| Sodium chloride | AR |
| Glycine | 99% |
PBS: 8gNaCl, 0.2gKCl, 1.42gNa2HPO4, 0.27gKH2PO4It is dissolved in 1L deionized water, regulation
PH；
MES buffer: 0.1562gMES is dissolved in 40mL deionized water, regulates pH；
Lavation buffer solution: 0.15g glycine is dissolved in 100mLMES buffer；
Confining liquid: 0.15g glycine, 1gBSA is dissolved in 100mLMES buffer；
R2: 0.5% (mass percent) sucrose is dissolved in confining liquid；
TMB solution: 3mgTMB is dissolved in 1mLDMSO；
Substrate dilution: 2.52g citric acid, 3.26gNa2HPO4, it is dissolved in 120mL deionized water；Regulation pH to 5.5；
1% carbamide peroxide solution: 10mg carbamide peroxide is dissolved in 1mL deionized water；
Stop buffer: 2mol/L sulphuric acid.
Two, experimental procedure
1) synthesis of HRP and HER2 antibody labeling latex beads:
Take 100uL solid content 10% (w/v) latex beads and add the piping and druming of appropriate MES buffer solution uniformly, add
0.614ugHER2 rabbit monoclonal antibodies, 90ugBSA, 9ugHRP, 5mgEDC also blow and beat uniformly；In metal bath, 45 DEG C of vibrations are anti-
Answer 1h；It is centrifuged and washs 3 times with cleaning mixture, remove supernatant；Being settled to 1mL with confining liquid, in metal bath, 45 DEG C of vibrations are closed
30min is centrifugal and washed once with confining liquid；Finally it is settled to 1mL with R2；4 DEG C store for future use.
2) detection of circulating tumor cell antigen in peripheral blood:
Take 10-20uL cell suspension in micropore reaction batten, be settled to 200uL with the PBS that pH is 2, add 0.04ug
Biotin labeling EpCAM Mus monoclonal antibody (0.04mg/mL) and the latex beads of 50uLHRP and HER2 antibody labeling, under room temperature condition
Oscillating reactions 1h；Add 2uL1% (w/v solid content) Streptavidin MagneSphere, oscillating reactions 1h under room temperature；Magneto separate, washs 3
Secondary and remove supernatant；The TMB solution and the 1.9ml substrate dilution that take 100uL 3mg/mL are made into TMB and Substrate cocktail, and
Addition 100uL TMB mixed liquor and 50uL 1% carbamide peroxide solution in above-mentioned precipitation, lucifuge 15min under room temperature condition,
Lucifuge adds 50uL sulphuric acid stop buffer after terminating；Magneto separate, takes 150uL supernatant in 96 clean orifice plates, and in microplate reader
Measure OD450.
Three, result
1, the range of linearity
Range of linearity assessed information is the important evidence evaluated and intend commercialized product effectiveness, is also the weight needed for equipment registration
Want one of declaration material.
When needing the concentration measuring solution, no matter the concentration of solution is the concentration of reagent itself, or reagent and sample
The concentration that (such as serum, quality-control product, standard substance, calibration object) reacts, generally uses Criterion curve between concentration and absorbance
Method measure, if proving whether this standard curve exists linear relationship, way is that " clinical chemistry is external according to WS/T124
Diagnostic kit quality inspection general provisions " about the regulation of the range of linearity, the solution concentration scope chosen is taken 6 points, including concentration
Lower limit (or 0), intermediate value and the upper limit, 3 absorbances of every some replication.Linear equation y=a+bx is calculated according to formula
And regression coefficient.
The relation of linear equation y=a+bx has been had, it is possible to efficiently by the suction of solution between concentration and absorbance
Shading value calculates the concentration of correspondence.Utilize the calculating of range of linearity formula, push away and demonstrate,prove out perfect calibration curve, thus for detecting
The sensitivity of reagent, accuracy, precision are laid a solid foundation.
1) reagent Linear Experiment 1
By the using method of above-mentioned detection kit, taking the sample of 5 kinds of known low concentrations, each sample measures 3 times, makes even
Average, result is as shown in Figure 1.
2) reagent Linear Experiment 2
By the using method of above-mentioned detection kit, taking the sample of 5 kinds of known high concentrations, each sample measures 3 times, makes even
Average, result is as shown in Figure 2.
2, precision
Can precision obtain the finger of the ability of identical experiment result when being and investigate test kit to same sample replication
Mark, generally represents by the coefficient of variation (CV%) of repeatedly sample measurement, precision evaluation is the important of quality control
Content.Precision assessed information is the important evidence evaluated and intend commercialized product effectiveness, is also the important Shen needed for equipment registration
One of report data.
Withinrun precision is most basic in numerous kinds precision one, and it is under strict condition of similarity, gained
The optimal precision arrived.
By the using method of above-mentioned detection kit, the sample taking two different cell number in detection range is surveyed
Fixed, each sample measures 7 times, and 7 testing results are calculated meansigma methods, and standard deviation and the coefficient of variation, result is as shown in table 2 below.
Table 2
3, accuracy
A lot of clinical experiment chamber interior, it will usually have plural detecting system, should determine between multiple detecting systems
Phase compares.For open detecting system, also should verify system, most important of which one is accuracy
Evaluating, accuracy estimating can be realized by methodology comparison, utilizes the comparison of two kinds of methods to contrast the accurate of corollary system
Degree is estimated being one of method assessing accuracy.Accuracy evaluation data is to evaluate plan the important of commercialized product effectiveness to depend on
According to, also it is one of important declaration material needed for equipment registration.
By the using method of above-mentioned detection kit, take the standard substance with traceability, detect with reagent, detect 7
Secondary, calculate meansigma methods and compare with target value, result is as shown in table 3 below.
Table 3
4, sensitivity
By the using method of above-mentioned detection kit, measuring the sample of 7 kinds of different cell concentrations, each sample is surveyed 7 times, logical
Crossing the calculating coefficient of variation (CV%), the definition point started more than 20% is the sensitivity of this test kit, the detection examination of the present invention
The sensitivity of agent box is 3/7.5mL, and result is as shown in table 4 below.
Table 4
5, stability
Stability test
Under 2～8 DEG C of storage requirement, respectively at 0 month, in April, in August, December and 14 months cells to same concentration are surveyed
Fixed, each sample is surveyed 5 times, takes average.Result shows, April, August, December, and within 14 months, the difference recorded is the least, says compared with 0 month
The detection kit of the bright present invention can be stablized 1 year under 2～8 DEG C of storage requirement, and result is as shown in table 5 below.
Table 5
Behind reagent Kaifeng, in 30 days, measure the absorbance average of antibody sample of same concentration, maximum, for 30 times
Little value, standard deviation and the absorbance coefficient of variation, it is seen that this kit reagent, behind Kaifeng, can be stablized more than one month, and result is such as
Shown in table 6.
Table 6
Claims (3)
1. the method for a microsphere enrichment enhancement techniques detection circulating tumor cell antigen, it is characterised in that step is as follows:
1) synthesis of HRP and HER2 antibody labeling latex beads:
Take 100ul solid content 10% w/v 80nm latex beads and add the piping and druming of MES buffer solution uniformly, add 0.614ug
HER2 rabbit antibody mab, 90ug BSA, 9ug HRP, 5mg EDC also blows and beats uniformly, and total system is 1mL；45 DEG C of oscillating reactionss
1h；It is centrifuged and washs with cleaning mixture, remove supernatant；Being settled to original volume 1mL with confining liquid, 30min is closed in 45 DEG C of vibrations, centrifugal
And washed once with confining liquid；Finally it is settled to 1mL with the confining liquid containing 0.5% sucrose；4 DEG C store for future use；
2) detection of circulating tumor cell antigen in peripheral blood:
Take 10-20uL cell suspension in micropore reaction batten in, be settled to 200uL with the PBS that pH is 2, add 1uL 0.04mg/mL
Biotin labeling EpCAM antibody and 50uL step 1) in the latex beads of antibody labeling, shaken at room temperature reaction 1h；Add 2 uL
1% Streptavidin MagneSphere, oscillating reactions 1h under room temperature；Magneto separate, washs and removes supernatant；Added for 100 microlitre TMB and the ends
Thing mixed liquor and 50 microlitre 1% carbamide peroxide solution, lucifuge 15min under room temperature condition, lucifuge adds 50 microlitre sulphuric acid after terminating
Stop buffer；Magneto separate, takes 150 microlitre supernatants and measures OD450 in microplate reader.
2. the test kit using method as claimed in claim 1, it is characterised in that include HRP and HER2 antibody labeling
80nm latex beads.
3. the test kit using method as claimed in claim 1, it is characterised in that include 80nm latex beads, for marking
The HER2 rabbit antibody mab of note latex beads and HRP.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610327849.9A CN105891492A (en) | 2016-05-17 | 2016-05-17 | Method for detecting antigens of circulating tumor cells by virtue of microsphere enrichment enhancement technique and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610327849.9A CN105891492A (en) | 2016-05-17 | 2016-05-17 | Method for detecting antigens of circulating tumor cells by virtue of microsphere enrichment enhancement technique and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105891492A true CN105891492A (en) | 2016-08-24 |
Family
ID=56716300
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610327849.9A Pending CN105891492A (en) | 2016-05-17 | 2016-05-17 | Method for detecting antigens of circulating tumor cells by virtue of microsphere enrichment enhancement technique and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105891492A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107918013A (en) * | 2017-09-25 | 2018-04-17 | 浙江天科高新技术发展有限公司 | The method and kit of K Ras albumen in chemiluminescence Enzyme immunoassay circulating tumor cell |
| CN109917121A (en) * | 2019-03-01 | 2019-06-21 | 洛阳恒恩生物科技有限公司 | Bladder chalone C determining reagent kit and preparation method thereof |
| CN112326958A (en) * | 2020-10-28 | 2021-02-05 | 厦门宝太生物科技有限公司 | Microsphere sealing method and microsphere marking method thereof |
| CN112858671A (en) * | 2019-11-27 | 2021-05-28 | 菲鹏生物股份有限公司 | Method for preparing rheumatoid factor detection reagent, kit and detection method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101256191A (en) * | 2008-03-07 | 2008-09-03 | 中国科学院上海微系统与信息技术研究所 | Method for determining minim proteins based on magnetic pearl and nano gold probe |
| CN101498719A (en) * | 2009-03-09 | 2009-08-05 | 东南大学 | Production method for enzyme functionalized nano immunity marker and use thereof |
| CN102183657A (en) * | 2011-02-24 | 2011-09-14 | 南京基蛋生物科技有限公司 | Method for greatly improving chemiluminescence magnetic enzyme immunization sensitivity by depending on polystyrene nanoparticles |
| CN102527353A (en) * | 2011-12-21 | 2012-07-04 | 深圳市老年医学研究所 | Preparation for nanometer magnetic immunomicrosphere for enriching circulating cancer cells |
| CN104076140A (en) * | 2014-07-09 | 2014-10-01 | 国家纳米科学中心 | Construction and application of chemiluminiscence-colloidal gold immunochromatography test paper |
| CN104568923A (en) * | 2014-12-01 | 2015-04-29 | 浙江省肿瘤医院 | Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection |
-
2016
- 2016-05-17 CN CN201610327849.9A patent/CN105891492A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101256191A (en) * | 2008-03-07 | 2008-09-03 | 中国科学院上海微系统与信息技术研究所 | Method for determining minim proteins based on magnetic pearl and nano gold probe |
| CN101498719A (en) * | 2009-03-09 | 2009-08-05 | 东南大学 | Production method for enzyme functionalized nano immunity marker and use thereof |
| CN102183657A (en) * | 2011-02-24 | 2011-09-14 | 南京基蛋生物科技有限公司 | Method for greatly improving chemiluminescence magnetic enzyme immunization sensitivity by depending on polystyrene nanoparticles |
| CN102527353A (en) * | 2011-12-21 | 2012-07-04 | 深圳市老年医学研究所 | Preparation for nanometer magnetic immunomicrosphere for enriching circulating cancer cells |
| CN104076140A (en) * | 2014-07-09 | 2014-10-01 | 国家纳米科学中心 | Construction and application of chemiluminiscence-colloidal gold immunochromatography test paper |
| CN104568923A (en) * | 2014-12-01 | 2015-04-29 | 浙江省肿瘤医院 | Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection |
Non-Patent Citations (4)
| Title |
|---|
| ELIZABETH A.PUNNOOSE: "Molecular Biomarker Analyses Using Circulating Tumor Cells", 《PLOS ONE》 * |
| HELEN SCHNECK ET AL: "EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast cancer", 《PLOS ONE》 * |
| 梁容瑞 等: "Vimentin联合CK、EpCAM用于中晚期乳腺癌患者循环肿瘤细胞的检测", 《广西医科大学学报》 * |
| 黄云艳 等: "两种纳米金标记p53抗体探针的制备、表征和性质研究", 《中国医药生物技术》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107918013A (en) * | 2017-09-25 | 2018-04-17 | 浙江天科高新技术发展有限公司 | The method and kit of K Ras albumen in chemiluminescence Enzyme immunoassay circulating tumor cell |
| CN109917121A (en) * | 2019-03-01 | 2019-06-21 | 洛阳恒恩生物科技有限公司 | Bladder chalone C determining reagent kit and preparation method thereof |
| CN112858671A (en) * | 2019-11-27 | 2021-05-28 | 菲鹏生物股份有限公司 | Method for preparing rheumatoid factor detection reagent, kit and detection method |
| CN112326958A (en) * | 2020-10-28 | 2021-02-05 | 厦门宝太生物科技有限公司 | Microsphere sealing method and microsphere marking method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2177614A2 (en) | Composition and method for diagnosing esophageal cancer and metastasis of esophageal cancer | |
| CN105891492A (en) | Method for detecting antigens of circulating tumor cells by virtue of microsphere enrichment enhancement technique and kit | |
| TWI698639B (en) | Prostate antigen standards and uses thereof | |
| CN104471402A (en) | Biomarkers for triple negative breast cancer | |
| CN104568923A (en) | Method and kit for detecting circulating tumor cell antigens in peripheral blood through electrochemical luminescence detection | |
| CN111044724B (en) | Thymidine kinase 1 magnetic particle chemiluminescence assay kit and preparation method thereof | |
| CN101231288B (en) | Novel method for analyzing human thymidine kinase fluorescence immune based on magnetic nanometer particular | |
| CN110187109B (en) | Autoantibody joint detection ELISA kit for early screening of cardia adenocarcinoma | |
| CN106248940B (en) | The system of multi objective Combining diagnosis oophoroma and/or the malignant tumour of non-ovary origin | |
| CN101424661B (en) | Serodiagnosis model establishing method for active tuberculosis disease | |
| EP3796001A1 (en) | Method for determining prostate carcinoma | |
| CN101539576A (en) | Hepatitis B virus pre S1 antigen chemiluminscence immunoassay kit and preparation method thereof | |
| CN103048453B (en) | Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof | |
| Liu et al. | Profiling of single-vesicle surface proteins via droplet digital immuno-PCR for multi-subpopulation extracellular vesicles counting towards cancer diagnostics | |
| WO2016082445A1 (en) | Application of peptidylarginine deiminase 2 in preparation of reagent for clinical diagnosis of tumors | |
| CN101373188A (en) | Tumor-associated antigen 19-9 chemical luminescence immune analytic determination reagent kit and preparation method thereof | |
| JP6857185B2 (en) | Protein biomarker panel for non-small cell lung cancer diagnosis and non-small cell lung cancer diagnostic method using this | |
| CN101726601A (en) | Double antibody identification based quantitative detection method of MG7-Ag contained in serum | |
| CN106319069A (en) | Kit for accurately determining pathogen cells and application of kit | |
| CN110244058A (en) | ENPP1 is preparing the application in high-level serous ovarian cancer diagnosis and prognosis kit | |
| CN106680512B (en) | A kind of quick detection kit of NMP-22 and its application | |
| WO2016082444A1 (en) | Application of peptidylarginine deiminase 1 in preparation of reagent for clinical diagnosis of tumors | |
| CN115372616A (en) | Gastric cancer related biomarker and application thereof | |
| CN104880424B (en) | A kind of dual-functional nanometer probe, kit and method for being used to detect AMACR | |
| CN113702464A (en) | P-tau detection immunosensor and preparation and application methods thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160824 |