CN109541215A - A kind of lesion detection object and the preparation method and application thereof - Google Patents
A kind of lesion detection object and the preparation method and application thereof Download PDFInfo
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- CN109541215A CN109541215A CN201811578161.3A CN201811578161A CN109541215A CN 109541215 A CN109541215 A CN 109541215A CN 201811578161 A CN201811578161 A CN 201811578161A CN 109541215 A CN109541215 A CN 109541215A
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- antigen
- capture antibody
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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
- G01N33/53—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
- G01N33/54326—Magnetic particles
Abstract
The present invention relates to molecular biology field, a kind of lesion detection object and the preparation method and application thereof is specifically disclosed, the detectable substance is " antigen of magnetic bead-two-capture antibody-microspheres " 5-linked complex.A kind of preparation method of lesion detection object, the preparation including " two anti-magnetic beads " compound, by the coupling of secondary antibody and superparamagnetic nanosphere, it is corresponding that secondary antibody can capture antibody with antigen respectively, and with capture antibody respectively in connection in the different epitopes of the antigen.A kind of lesion detection object of the present invention is mainly used in quantitative detection tumor markers and simultaneous quantitative detection not synantigen.It solves fluorescent reporter molecule in the prior art to be easy to be quenched, photosensitivity is low；There is no the accurate foundation of quantitative analysis；There is certain limitation in detection flux.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to a kind of lesion detection object and the preparation method and application thereof.
Background technique
With the continuous development of the various new technologies of field of biotechnology, high-throughput, accurate and quick detection method becomes
The important research means of bioscience and medical domain.Traditional high-throughput biomolecule detection technology is mostly based on biochip
Technology, the biochip technology for being usually used in biomacromolecule detection at present is genetic chip and protein-chip.But due to biology
The preparation cost of chip is high, the period is long, using being difficult to popularize, so that there are limitations for the application of biochip technology.
In recent years, the xMAP (Flexible after genetic chip and protein chip developed by Luminex company, the U.S.
Multi-Analyte Profiling) technology, for solid phase chip deficiency and develop design, improve to a certain extent
The accuracy and specificity of detection.XMAP (Flexible Multi-Analyte Profiling) technology is also known as liquid phase gene core
Chip technology or microballoon suspension dot matrix technology are that the method for the polystyrene sphere fluorescent staining for being 5.6 μm diameter is compiled
Code, the different ratio by adjusting two kinds of fluorescent dyes obtain at most up to 100 kinds of microballoons with different characteristic fluorescence Spectra, so
The capture molecules such as antigen, antibody or the nucleic acid probe of particular detection object will be directed on every kind of coding microball covalent cross-linking afterwards.It is to be added
After entering sample to be examined, under liquid-phase condition, the capture molecule that target molecule is crosslinked with microsphere surface is specifically bound, at one
Up to 100 kinds different detection reactions can be completed at the same time in reacting hole.Finally, microballoon is sent to liquid phase with liquid stream defiled
Chip detector automatically analyzes, and analyzer passes through red, green two beams laser, the classification fluorescence in red diode laser excitation microballoon
Dyestuff determines the specificity (qualitative) of measured object；Yag laser (YAC) excites the reporter fluorescence for being incorporated in microsphere surface
Plain (such as phycoerythrin, Alexa532, Cy3) determines the amount (quantitative) of measured object, completes the real-time qualitative to reaction and quantifies
Analysis.
Although liquid phase genetic chip technology enhances reaction efficiency, shortens the reaction time.But there is also one for xMAP technology
It is a little significant insufficient, such as: the problems such as fluorescent reporter molecule of the technology is easy to be quenched, and photosensitivity is low.Therefore, it is badly in need of exploitation one
The novel detectable substance of kind is used to make up the defect of the prior art.
Summary of the invention
The invention is intended to provide a kind of lesion detection object and the preparation method and application thereof, to solve fluorescence report in the prior art
It accuses molecule to be easy to be quenched, the low problem of photosensitivity.
In order to achieve the above objectives, technical scheme is as follows: a kind of lesion detection object, and the detectable substance is " magnetic bead-
Two antigens-capture antibody-microspheres " 5-linked complex.
Furthermore the technical program also provides a kind of preparation method of lesion detection object, includes the following steps,
Step 1: capture antibody and a plurality of types of carboxyl microballoons being coupled, it is compound to form " capture antibody-microspheres " bigeminy
Object；
Step 2: sample to be tested is mixed to form with " capture antibody-microspheres " bigeminy compound to " antigen-capture antibody-is micro-
Three compound of ball "；
Step 3: by the coupling of secondary antibody and superparamagnetic nanosphere, forming " two anti-magnetic beads " compound；The secondary antibody is to plant
Object hemagglutinin, secondary antibody is the secondary antibody respectively in conjunction with antigen and capture antibody specificity, and secondary antibody and capture antibody are anti-
Binding site in original is different；
Step 4: by " antigen-capture antibody-microspheres " three compounds in step 2 and " the two anti-magnetic beads " in step 3
Compound mixing, forms " antigen of magnetic bead-two-capture antibody-microspheres " 5-linked complex, as detection marker；
Step 5: the 5-linked complex in step 4 is as on magnetic frame, and after supernatant is got rid of after standing, dissolution is heavy again
It forms sediment；
Step 6: the label letter of different microballoons in detection " antigen of magnetic bead-two-capture antibody-microspheres " 5-linked complex
Number, determine the type and content of antigen in sample to be tested.
The principle and beneficial effect of the technical program are: in the technical program capture antibody be used for in sample to be tested
Antigen binding, carboxyl microballoon is used for marking signal so that in late detection, after capture antibody combines antigen, with carboxyl
When microballoon combines, surface markers have detection fluorescence, convenient for screening.Secondary antibody is used for and the antigen knot in capture antibody and sample to be tested
It closes, superparamagnetic nanosphere is for separating combination, so that capture antigen that superparamagnetic nanosphere combines and to be checked
After antibody combines, it can be adsorbed under the magnetic fields of magnetic frame and be deposited in reaction bottom of the tube；And extra carboxyl microballoon
The compounds such as the capture antibody not in conjunction with determined antigen then float in reaction tube supernatant liquor, realize the compound of successfully combination
The separation of body (molecule to be checked) and the complex (non-molecule to be checked) combined not successfully.And then make in late detection, theoretically
It is molecule to be checked, because non-molecule to be checked has been separated, without also detecting non-molecule to be checked, therefore detects
Time opposite shortening, avoids the problem of fluorophor is quenched under the irradiation of prolonged exciting light, so that fluorescence intensity is whole
Body improves.In addition, select phytohemagglutinin as secondary antibody in the technical program, it is existing that phytohemagglutinin is answered
In, phytohemagglutinin is usually to treat disease separately as drug, due to the therapeutic effect of phytohemagglutinin
Preferably, the motivation of its biological nature and binding site is not studied at all.And inventor is after touching phytohemagglutinin,
During doing promotion applied research after understanding its function, initiative has studied biologic specificity.Due in this programme to
Sample product are whole blood sample, and antigen to be detected is usually on the surface of red blood cell, and phytohemagglutin phytolectin is in combination with red blood cell table
Face, but surface portion of the binding site in conjunction with general antigen, antibody is different will not occur to combine conflict, it can be achieved that three
Stable bond.
Further, carboxyl microballoon is marked with the fluorescent dye of multiple proportions.
The fluorescent dye of different ratio is marked on carboxyl microballoon, the different ratio of fluorescent dye can make the glimmering of fluorescent dye
Light color changes, so that being integrated with the marking signal of different colours on carboxyl microballoon, when detecting, can pass through different fluorescence
The quantity of marking signal determines the content of variety classes antigen, increases detection range.
Further, carboxyl microballoon is using preceding through overactivation.
Carboxyl microballoon after overactivation can with capture antibody covalent coupling so that preparation process is carried out.
Further, the activation of carboxyl microballoon uses 1- ethyl-(3- dimethylaminopropyl) -3- ethyl carbodiimide hydrochloride
Salting liquid is activated with N- hydroxy thiosuccinimide solution.
Using 1- ethyl-(3- dimethylaminopropyl) -3- ethyl-carbodiimide hydrochloride solution and N- hydroxy amber
Amber imide solution progress activation act is simple, and can guarantee the activation effect of carboxyl microballoon.
Further, in step 6, the marking signal for detecting different microballoons in 5-linked complex uses Luminex.
The marking signal that different microballoons are detected using Luminex determines that this is micro- by measuring the fluorescent marker of microballoon
The type of ball and counting, can measure the quantity of antigen to be checked, easy to operate, high sensitivity simultaneously.
Further, " two anti-magnetic beads " compound is using being preceding stored under 4 DEG C of environment.
4 DEG C for " two anti-magnetic beads " compound store when optimum temperature, it is ensured that the chemistry of " two anti-magnetic beads " compound
Stability avoids " two anti-magnetic beads " compound from failing using preceding due to storing improper.
Further, a kind of application preparing lesion detection drug using detectable substance described in claim 1.
Specific embodiment
It is further described below by specific embodiment:
3 kinds of blood group antibody A antibody, B antibody, Rh (D) antibody sources are in abcam company used in the present invention；Secondary antibody used
Sigma company is derived from for phytohemagglutinin；Superparamagnetic nanosphere derives from NEB company；Different types of carboxyl is micro-
Ball is purchased in Shanghai Tou Jing scientific & technical corporation；1- ethyl-(3- dimethylaminopropyl) -3- ethyl-carbodiimide hydrochloride (EDC),
N- hydroxy thiosuccinimide (NHS) is purchased in the raw work biology Co., Ltd in Shanghai, selects to number the commercialization for No. 9
Microballoon.
Buffer:
Activation buffer (Activation buffer): 100mM NaH2PO4, pH 6.3；
Coupling buffer (Coupling buffer): 50mM HEPES, pH 7.4；
Phosphate buffer (PBS): 10mM NaH2PO4,150mM NaCl, pH 7.4；
2-morpholine ethane sulfonic acid buffer (MES): 0.01M, pH 6.0；
2-morpholine ethane sulfonic acid tween solution (MEST): 0.01M, 0.05%Tween-20, pH 6.0；
Human cancer embryoantigen detection method, the lesion detection object used in detection process is " antigen of magnetic bead-two-capture is anti-
Body-microballoon " 5-linked complex, the preparation method of " antigen of magnetic bead-two-capture antibody-microspheres " 5-linked complex include as follows
Step:
Step 1: the activation of required microballoon
1.1 full speed vortex carboxyl microballoon storing liquids, form uniform carboxyl microsphere suspensions；
1.2 prepare the EDC and NHS of 5mg/ml with deionized water respectively；
1.3 take the microsphere suspensions 10000g of 1ml to be centrifuged 3min, carefully remove supernatant；
The activation buffer of 1.4 80 μ l of addition suspends microballoon again；
1.5 are separately added into 100 μ l, 5mg/ml NHS solution of 5mg/ml EDC solution, 100 μ l, are uniformly mixed, in room temperature
(20-25 DEG C) is protected from light, oscillation incubation 30min.
Step 2: the preparation of " capture antibody-microspheres " bigeminy compound
2.1, which will capture antibody with coupling buffer, is diluted to volume as 500 μ l, and concentration is the solution of 0.2mg/ml；
Carboxyl microballoon is centrifuged 3min in 10000g by 2.2, carefully removes supernatant；
The antibody-solutions diluted in 2.3 addition steps 2.1；
2.4, by the carboxyl microballoon and antibody-solutions of activation, are protected from light, oscillation incubation 2h in room temperature (20-25 DEG C), and formation " is caught
Obtain antibody-microspheres " bigeminy compound；
Microballoon is centrifuged 3min in 10000g by 2.5, carefully removes supernatant；
2.6 500 μ l PBS of addition suspend microballoon again, and 10000g is centrifuged 3min, carefully removes supernatant；
2.7 are added 1ml PBS/1%BSA (BSA: bovine serum albumin(BSA)) suspends microballoon again；
2.8 count microballoon by thrombocytometer；
Step 3: the preparation of " two anti-magnetic beads " compound
3.1 take 1mg carboxylated superparamagnetic nanosphere, are placed in clean centrifuge tube；
3.2 are washed 2 times with 150 μ l of MEST solution, and supernatant is abandoned after Magneto separate；
3.3 take 100 μ l 5mg/ml EDC and NHS solution to mix well with magnetic bead respectively, are placed in (37 in constant temperature shaking case
DEG C, 150rpm) activation 30min, form " two anti-magnetic beads " compound；
3.4 are washed the superparamagnetic nanosphere of activation 3 times with MEST solution, Magneto separate, abandon supernatant, suspend 1mL again；
3.5 take 100 μ g magnetic beads, mix (being dissolved in MEST) with 40 μ l of phytohemagglutinin (0.25mg/ml), room temperature concussion
Coupling is overnight.
Coupled bead is carried out Magneto separate by 3.6, takes supernatant in clean dried centrifuge tube, and 1% is added in magnetic bead
BSA, oscillation closing 30min.
3.7 are washed 4 times with PBS solution, Magneto separate, abandon supernatant, are suspended magnetic bead, are stored under 4 DEG C of environment again with PBS.
Step 4: the preparation of the microspheres solution of coupling capture antibody
The microballoon for being coupled the antibody of anti-human carcinomebryonic antigen is taken, making the final concentration of microballoon is respectively 400/μ l, and 4 DEG C are protected from light
It saves.
Step 5: " antigen of magnetic bead-two-capture antibody-microspheres " preparation of 5-linked complex and the inspection of human cancer embryoantigen
It surveys
5.1 20 parts of colorectal cancer patients whole blood samples and 20 parts of normal person's whole blood sample；
5.2 are separately added into the microspheres solution of the microspheres solution of coupled antibody and non-coupled antibody in 96 hole elisa Plates, 20 μ
The hole l/；
5.3 are added patient, normal person's whole blood sample, 20 holes μ l/；
5.4 mix mixture with the volley of rifle fire above and below, pay attention to control action amplitude, then close the lid, are protected from light incubate at room temperature
Educate 30min；
5.5 are added the superparamagnetic nanosphere containing phytohemagglutinin, 20 holes μ l/；Upper and lower with volley of rifle fire tenderness mixes
It is even, it closes the lid, is protected from light is incubated for 30min at room temperature, it is compound to form " antigen of magnetic bead-two-capture antibody-microspheres " 5-linked
Body；
5.6 are placed in 96 hole elisa Plates on magnetic board, stand 3min；
5.7 on liquid-phase chip instrument (Luminex 200), analyze the mixture of reaction, read fluorescent marker, often
Group sampling test in triplicate, is averaged.
Testing result and analysis
The count results and comparison (average value) of table 1CEA antibody microballoon
CUTOFF value: 2.375
CUTOFF value is the threshold value of detection method in the present embodiment, is colon cancer when the ratio of detection is higher than CUTOFF value
Patient, the above results show can from the ratio of each microballoon number and internal reference in conjunction with CUTOFF value with method detection CEA of the invention
To find out, 1-20 sample is colorectal cancer patients sample, and ratio is above CUTOFF value；21-40 sample is normal person's mark
This, ratio is below CUTOFF value, is analyzed by SPSS software it is found that the sensitivity of this method and specificity are 100%.
Comparative experiments
20 parts and 20 parts of normal person's whole blood sample of above-mentioned identical colorectal cancer patients whole blood sample are chosen, use is clinically normal
People CEA ELISA detection kit (producer: Sigma company, model 96T) detection, all operations step are said by kit
The operating procedure provided in bright book carries out, testing result such as following table.
Testing result and analysis
1 CEA ELISA testing result (average value) of table
| Sample number | OD value | Sample number | OD value |
| 1 | 1.42 | 21 | 0.20 |
| 2 | 0.84 | 22 | 0.41 |
| 3 | 0.75 | 23 | 0.21 |
| 4 | 1.07 | 24 | 0.62 |
| 5 | 0.81 | 25 | 0.22 |
| 6 | 1.19 | 26 | 0.38 |
| 7 | 0.65 | 27 | 0.35 |
| 8 | 1.00 | 28 | 0.47 |
| 9 | 1.36 | 29 | 0.35 |
| 10 | 1.05 | 30 | 0.37 |
| 11 | 0.79 | 31 | 0.75 |
| 12 | 1.14 | 32 | 0.40 |
| 13 | 1.25 | 33 | 0.34 |
| 14 | 0.83 | 34 | 0.43 |
| 15 | 1.22 | 35 | 0.46 |
| 16 | 1.18 | 36 | 0.48 |
| 17 | 1.32 | 37 | 0.62 |
| 18 | 1.04 | 38 | 0.20 |
| 19 | 1.37 | 39 | 0.45 |
| 20 | 0.61 | 40 | 0.41 |
CUTOFF value: 0.545
Testing result with ELISA detection CEA is as shown in table 2, the results showed that, 1-20 sample is colorectal cancer patients sample
Value is above CUTOF value, is to be detected as normal person's sample in addition to 24,31, No. 37 in 21-40 sample, but 24,31 and 37
Number reality is normal person's sample, occurs detecting wrong phenomenon.It is analyzed by SPSS software it is found that the sensitivity of this method is
100%, specificity is 85%.
In the case where detection sample, testing conditions are all the same, the detection method that the technical program uses detects sensitive
Degree and specificity have reached 100%, when meeting the requirement of 95% or more accuracy rate of lesion detection, and using ELISA detection, spirit
Sensitivity is 100%, but specificity is only 85%, lower than the standard requirements of lesion detection.Therefore, lesion detection object of the invention is special
Anisotropic strong, high sensitivity can provide more stable data source in medical oncology research, improve the accuracy of tumor research,
Theoretical reference is provided for the research and development of tumour medicine, it is with a high credibility.
What has been described above is only an embodiment of the present invention, the common sense such as well known specific technical solution and/or characteristic in scheme
It does not describe excessively herein.It should be pointed out that for those skilled in the art, before not departing from technical solution of the present invention
It puts, several modifications and improvements can also be made, these also should be considered as protection scope of the present invention, these all will not influence this
Invent the effect and patent practicability implemented.The scope of protection required by this application should be based on the content of the claims,
The records such as the specific embodiment in specification can be used for explaining the content of claim.
Claims (8)
1. a kind of lesion detection object, it is characterised in that: the detectable substance is " antigen of magnetic bead-two-capture antibody-microspheres " five
Join complex.
2. a kind of preparation method of lesion detection object as described in claim 1, it is characterised in that: include the following steps,
Step 1: capture antibody and a plurality of types of carboxyl microballoons being coupled, " capture antibody-microspheres " bigeminy compound is formed；
Step 2: sample to be tested and " capture antibody-microspheres " bigeminy compound are mixed to form " antigen-capture antibody-microspheres " three
Join compound；
Step 3: by the coupling of secondary antibody and superparamagnetic nanosphere, forming " two anti-magnetic beads " compound；The secondary antibody is plant blood
Ball agglutinin, secondary antibody is the secondary antibody respectively in conjunction with antigen and capture antibody specificity, and secondary antibody and capture antibody are on antigen
Binding site it is different；
Step 4: " antigen-capture antibody-microspheres " three compounds in step 2 and " the two anti-magnetic beads " in step 3 are compound
Object mixing, forms " antigen of magnetic bead-two-capture antibody-microspheres " 5-linked complex, as detection marker；
Step 5: the 5-linked complex in step 4 is as on magnetic frame, and after supernatant is got rid of after standing, dissolution is precipitated again；
Step 6: the marking signal of different microballoons in detection " antigen of magnetic bead-two-capture antibody-microspheres " 5-linked complex, really
Determine the type and content of antigen in sample to be tested.
3. a kind of preparation method of lesion detection object according to claim 2, it is characterised in that: the carboxyl microballoon label
There is the fluorescent dye of multiple proportions.
4. a kind of preparation method of lesion detection object according to claim 3, it is characterised in that: the carboxyl microballoon uses
It is preceding through overactivation.
5. a kind of preparation method of lesion detection object according to claim 3, it is characterised in that: the work of the carboxyl microballoon
Change sub- using 1- ethyl-(3- dimethylaminopropyl) -3- ethyl-carbodiimide hydrochloride solution and N- hydroxy succinyl
Amine aqueous solution is activated.
6. a kind of preparation method of lesion detection object according to claim 2, it is characterised in that: in step 6, detect 5-linked
The marking signal of different microballoons uses Luminex in complex.
7. a kind of preparation method of lesion detection object according to claim 2, it is characterised in that: " the two anti-magnetic beads "
Compound is using being preceding stored under 4 DEG C of environment.
8. a kind of application for preparing lesion detection drug using detectable substance described in claim 1.
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Cited By (1)
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| CN111830251A (en) * | 2020-06-24 | 2020-10-27 | 瑞芯智造(深圳)科技有限公司 | Biological sample detection method and detection kit |
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