CN109765381A - A kind of platelet derived growth factor PDGF-BB test strips and detection method based on the amplification of aptamer signal - Google Patents
A kind of platelet derived growth factor PDGF-BB test strips and detection method based on the amplification of aptamer signal Download PDFInfo
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Abstract
The invention discloses a kind of platelet derived growth factor PDGF-BB test strips and detection method based on the amplification of aptamer signal.The platelet derived growth factor PDGF-BB test strips include bottom plate, sample pad, bonding pad, nitrocellulose filter and blotting paper are successively pasted on the bottom plate, it partly overlaps between two adjacent pastes, the nitrocellulose filter is equipped with detection line and nature controlling line, colloidal gold-antibody-nucleic acid compound is coated on the bonding pad, it is coated with the aptamer Apt2 of PDGF-BB in the detection line, is coated with Poly A on the nature controlling line.The present invention is interacted using the specificity of the convenient and efficient and DNA- cell factor of colloidal gold lateral chromatography detection, realizes the quick detection of cytokine activity.Quick, easy, accurate, the inexpensive advantage of platelet derived growth factor PDGF-BB test strips of the invention will promote the popularization and application of this kind of detection method, provide a kind of new method and thinking for drug research and screening.
Description
Technical field:
The invention belongs to detection technique fields, and in particular to a kind of based on the platelet-derived of aptamer signal amplification
Growth factor PDGF-BB test strips and detection method.
Background technique:
Platelet derived growth factor (PDGF) is the growth factor protein in a kind of human blood platelets, have PDGF-AA,
Five kinds of PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD various forms of dimer types.Wherein, PDGF-BB is in serum
Important cell factor is the protein markers for for cancer diagnosis and directly participating in many cell conversion process.PDGF-BB
It is related to many pernicious diseases, e.g., atherosclerosis, fibrosis.It is often over-expressed in human malignancies cell.
Therefore, the qualitative and quantitative detection of PDGF-BB is most important in biological and medical field.Existing frequently-used method is used for PDGF
Detection method have: enzyme-linked immunosorbent assay, fluoroimmunoassay and chemiluminescence immune assay etc..
Currently used method has for the detection method of PDGF: enzyme-linked immunosorbent assay, fluoroimmunoassay and
Chemiluminescence immune assay etc..With apply in the colour developing enzyme or fluorescein, radioimmunoassay technique applied in Enzyme-multiplied immune technique
The fluorescein applied in isotope, serology is compared, and colloidal gold, which is gathered around, has the advantage that result naked eyes as it can be seen that without complicated, expensive
Instrument, quick diagnosis on site can be used；It is environmentally friendly, it is nontoxic without any radiocontamination；It as a result can long-term preservation；
It takes short, can be used for quick diagnosis.
Summary of the invention:
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of blood based on the amplification of aptamer signal
Platelet derivative growth factor PDGF-BB test strips and detection method.
The first purpose of the invention is to provide it is a kind of based on aptamer signal amplification platelet derived growth because
Sub- PDGF-BB test strips, including bottom plate are successively pasted with sample pad, bonding pad, nitrocellulose filter and suction on the bottom plate
Water paper partly overlaps between two adjacent pastes, and the nitrocellulose filter is equipped with detection line and nature controlling line, the knot
It closes and is coated with colloidal gold-antibody-nucleic acid compound on pad, the aptamer of PDGF-BB is coated in the detection line
Apt2 is coated with Poly A on the nature controlling line；The colloidal gold-antibody-nucleic acid compound is to make by the following method
Standby: colloid gold label PDGF-BB monoclonal antibody is used, colloidal gold-antibody complex is prepared, then passes through PDGF-BB
The terminal modified Apt for having carboxyl of amino and 3 ', Apt-Poly T2, Apt-Poly T3, Apt-Poly T4 in monoclonal antibody and
Condensation reaction occurs for the carboxyl of band on Apt-Poly T5, and colloidal gold-antibody-nucleic acid compound is prepared；The Apt is
The aptamer of PDGF-BB, the Apt-Poly T2, Apt-Poly T3, Apt-Poly T4 and Apt-Poly T5 are
Apt of the 3 ' ends added with different length Poly T.
The nucleotide sequence of the Apt is preferred are as follows: 5 '-AGGGCGCGTTCTTCGTGGTTACTTTTAGTCCCG-3 '
(as shown in SEQ ID NO.1)；The nucleotide sequence of the Apt-Poly T2 is preferred are as follows: 5 '-AGGGCGCGTTCTTCGTG
GTTACTTTTAGTCCCGTTTTTTTTTTTTTTT-3 ' (as shown in SEQ ID NO.2)；The nucleosides of the Apt-Poly T3
Acid sequence is preferred are as follows: 5 '-AGGGCGCGTTCTTCGTGGTTACTTTTAGTCCCGTTTTTTTTTTTTTTTTTTTTTTT TTTT
TTT-3 ' (as shown in SEQ ID NO.3)；The nucleotide sequence of the Apt-Poly T4 is preferred are as follows: 5 '-AGGGCGCGTTC
TTCGTGGTTACTTTTAGTCCCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTT-3 ') such as SEQ
Shown in ID NO.4)；The nucleotide sequence of the Apt-Poly T5 is preferred are as follows: 5 '-AGGGCGCGTTCTTCGTGGTTACTT
TTAGTCCCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTT-3 ') such as
Shown in SEQ ID NO.5).
The nucleotide sequence of the Poly A is preferred are as follows: 5 '-AAAAAAAAAAAAAAAAAAAAA-3 ' (such as SEQ ID
Shown in NO.6).
The nucleotide sequence of the aptamer Apt2 of the PDGF-BB is preferred are as follows: 5 '-CAGGCTACGGCACGTAG
AGCATCACCATGATCCTG-3 ' (as shown in SEQ ID NO.7).
The bottom plate is preferably PVC bottom plate.
The distance between described detection line and nature controlling line are preferably 3mm.
A second object of the present invention is to provide described in one kind based on the platelet-derived of aptamer signal amplification
The preparation method of growth factor PDGF-BB test strips, comprising the following steps:
1) preparation of sample pad: glass fibre membrane is immersed in sample pad treatment fluid, takes out drying, sample is prepared
Pad；
2) preparation of bonding pad: colloid gold label PDGF-BB monoclonal antibody is used, it is compound that colloidal gold-antibody is prepared
Then object passes through the amino and 3 ' terminal modified Apt, Apt-Poly T2, the Apt- for having carboxyl in PDGF-BB monoclonal antibody
Condensation reaction occurs for the carboxyl of band on Poly T3, Apt-Poly T4 and Apt-Poly T5, and colloidal gold-antibody-core is prepared
Sour compound is sprayed on glass fibre membrane after closing, aging, bonding pad is prepared；
3) aptamer Apt2 and the Poly A of PDGF-BB is drawn respectively on nitrocellulose filter, with PDGF-BB's
Aptamer Apt2 is as detection line, using Poly A as nature controlling line；
4) sample pad, bonding pad, nitrocellulose filter and blotting paper are successively pasted on bottom plate, adjacent two paste it
Between partly overlap, obtain based on aptamer signal amplify platelet derived growth factor PDGF-BB test strips.
It is preferred that the sample pad treatment fluid includes 4%Triton X-100,1%BSA, 2% sucrose, 2%PEG-
4000,100mM boric acid, 0.1%SDS and 0.5 μ g/mL salmon sperm dna, surplus are water, pH 9.0.
The nucleotide sequence of the competitor dna probe is preferred are as follows: 5 '-TGGTCTTGTGTGGCTGTGGCTATGTCTGA
TCTTAATCCACGAAGTCACC-3 ' (as shown in SEQ ID NO.7), the 5 ' of the competitor dna are terminal modified sulfydryl.
Third object of the present invention is to provide it is a kind of based on aptamer signal amplification platelet derived growth because
The detection method of sub- PDGF-BB, comprising the following steps: sample to be tested is added drop-wise to the platelet derived growth factor
In the sample pad of PDGF-BB test strips, then toward sample-loading buffer is added dropwise in sample pad, observed after standing 5~10min as a result,
If detection line and nature controlling line are all displayed in red band, it is judged as positive, contains PDGF-BB in sample to be tested, if detection line is not
It is displayed in red band, nature controlling line is displayed in red band, then is judged as negative, PDGF-BB is not contained in sample to be tested.
The present invention is interacted using the specificity of the convenient and efficient and DNA- cell factor of colloidal gold lateral chromatography detection,
Realize the quick detection of cytokine activity.Platelet derived growth factor PDGF-BB test strips of the invention it is quick, simple
Just, advantage accurately, inexpensive will promote the popularization and application of this kind of detection method, provide one kind newly for drug research and screening
Method and thinking.
The platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal of the invention have with
It is lower the utility model has the advantages that
(1) it greatly improves cytokines measurement to limit, in clinical serum sample, the abundance of cell factor is not high, this reality
It tests the amino using monoclonal antibody band itself and is added to the oligonucleotide condensation reaction of carboxyl, and is different by length
Oligonucleotide increases multiple cell factor binding sites, dexterously will test limit and improves 10 times or more；
(2) easy to use, the processes such as electrophoresis and enzymatic treatment are not needed, nor the use of specific apparatus is needed,
As long as blood serum sample and sample-loading buffer are added drop-wise in sample pad, each operating process be not necessarily to special training, be suitble to
Common lab uses；
(3) detection is time-consuming short, and testing result can be obtained in whole process 10min or so；
(4) detection is cytokine activity, and the maximum difference with the double sandwich methods of common antibody is that this method is guaranteeing
What is detected while specific is the DNA binding ability of cell factor, i.e., captured by aptamer specific cell because
Son.
Detailed description of the invention:
Fig. 1 is that the structure of the platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal is shown
It is intended to；Wherein, 1, bottom plate；2, sample pad；3, bonding pad；4, nitrocellulose filter；5, detection line；6, nature controlling line；7, blotting paper.
Fig. 2 is the platelet derived growth factor PDGF-BB test strips testing principle based on the amplification of aptamer signal
Figure；(A) after colloidal gold is in conjunction with PDGF-BB monoclonal antibody, colloidal gold-antibody complex (AuNP- is obtained
AntibodyComplex), pass through the amino and 3 ' the terminal modified Apt and Apt-Poly for having carboxyl in PDGF-BB monoclonal antibody
Condensation reaction occurs for T2-5, obtains colloidal gold-antibody-nucleic acid compound (AuNP-Antibody-PolyT-Apt Complex)；
When sample is the positive, the aptamer Apt of the PDGF-BB on PDGF-BB albumen and colloidal gold-antibody-nucleic acid compound and
PDGF-BB monoclonal antibody specific bond；(B) the platelet derived growth factor PDGF- based on the amplification of aptamer signal
The assembling of BB test strips and each section composition；SP is sample pad, and CP is bonding pad, and NM is nitrocellulose filter, and TL is detection line,
CL is nature controlling line, and AP is water absorption pad (blotting paper)；(C) colloidal gold by PDGF-BB albumen and Poly T respectively with T line and C line
In conjunction with.
Fig. 3 is the detection knot of the platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal
Fruit；T is detection line, and C is nature controlling line.
Specific embodiment:
The following examples are further illustrations of the invention, rather than limiting the invention.
The present invention establishes and a kind of perfect platelet derived growth factor PDGF- based on the amplification of aptamer signal
BB test strips, colloidal gold labeled monoclonal antibody and aptamer solution formula are screened, the preparation of detection line and nature controlling line with
And the committed steps such as selection, detection formula of liquid selection of nitrocellulose filter are investigated, and to test strips, specific and steady
Qualitative equal quality performance indicators are tested.
As shown in Figure 1, the platelet derived growth factor PDGF-BB examination of the invention based on the amplification of aptamer signal
Paper slip includes bottom plate 1, sample pad 2, bonding pad 3, nitrocellulose filter 4 and blotting paper 7 is successively pasted on bottom plate 1, two is adjacent
2mm is overlapped between paste, nitrocellulose filter 4 is equipped with detection line 5 and nature controlling line 6, is coated with colloidal gold-on bonding pad 3
Antibody-nucleic acid compound is coated with aptamer Apt2 (its nucleotide sequence such as SEQ ID of PDGF-BB in detection line 5
Shown in NO.7), it is coated with Poly A on nature controlling line 6 (its nucleotide sequence is as shown in SEQ ID NO.6).
The testing principle of platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal is as schemed
Shown in 2: a certain amount of colloidal gold that tiles on bonding pad-antibody-nucleic acid compound (AuNP-Antibody-PolyT-Apt
Complex), aptamer Apt2 and oligonucleotide the Poly A of PDGF-BB is covered on detection line and nature controlling line respectively.When
When sample is positive, when passing through bonding pad containing determinand (PDGF-BB) solution, dissolve and with colloidal gold-antibody-nucleic acid compound
On PDGF-BB aptamer Apt and PDGF-BB monoclonal antibody reactive, formed colloidal gold-antibody-PolyT- nucleic acid
The nucleic acid of aptamers-PDGF-BB polymer (AuNP-Antibody-PolyT-Apt-PDGF-BB Complex), PDGF-BB is suitable
Ligand Apt2 is in conjunction with the PDGF-BB in colloidal gold-antibody-Poly T- aptamer-PDGF-BB polymer, in detection zone
Aggregation forms red stripes.When sample is negative, the aptamer Apt2 and colloidal gold-antibody-nucleic acid of PDGF-BB is compound
Object can not combine, and detection line does not develop the color.The Poly A in nature controlling line region being capable of specificity and colloidal gold-antibody-nucleic acid compound
Or whether colloidal gold-antibody-Poly T- aptamer-PDGF-BB polymer Poly T is combined, no matter contain in sample
Determinand, can be poly- with colloidal gold-antibody-nucleic acid compound or colloidal gold-antibody-Poly T- aptamer-PDGF-BB
It closes object combination and forms red stripes.When nature controlling line does not develop the color, then it is considered as invalid test.
Embodiment 1: the preparation of the platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal
1.1 materials and methods
1.1.1 main material
| Title | Producer |
| PDGF-BB | Wuhan is excellent and gives birth to commerce and trade Co., Ltd |
| PDGF-BB monoclonal antibody | Wuhan is excellent and gives birth to commerce and trade Co., Ltd |
| PDGF-BB is mostly anti- | Wuhan is excellent and gives birth to commerce and trade Co., Ltd |
| Various anion salts | Sigma company |
| Bovine serum albumin(BSA) | Sigma company |
| Gold chloride | Sigma company |
| PBS | Gibco company |
| Nitrocellulose filter | PALL company |
| PVC bottom plate | Shanghai Han Gan trade Co., Ltd |
| Glass fibre membrane | Shanghai Jinbiao Bio-Tech Co., Ltd. |
| SDS | Guangzhou Chemical Reagent Factory |
| Two citric acid monohydrate trisodiums | Shanghai crystalline substance pure reagent Co., Ltd |
| Boric acid | Beijing Ding Guo Bioisystech Co., Ltd |
| Potassium carbonate | Tianjin Yong great chemical reagent Co., Ltd |
| Sodium chloride | Guangzhou Chemical Reagent Factory |
| PEG-4000 | MBchem company |
| Sucrose | Guangzhou Chemical Reagent Factory |
| Triton X-100 | Amresco company |
| Blotting paper | Shanghai Jinbiao Bio-Tech Co., Ltd. |
| PDGF-BB ELISA detection kit | Wuhan is excellent and gives birth to commerce and trade Co., Ltd |
| DNA | Shanghai bio-engineering corporation |
1.1.2 DNA sequence dna
1.1.3 key instrument
1.2 experimental method
1.2.1 the preparation of colloidal gold
(1) 250mL conical flask is taken, the magnetic stir bar cleaned up is put into 100mL ultrapure water is accurately added, by taper
Bottle is put into heating stirring in hot type magnetic stirring apparatus and extremely boils；
The configuration of (2) 1% gold chloride working solutions: accurately weighing 1.0g gold chloride, is dissolved with 90mL ultrapure water, waits it sufficiently molten
Xie Hou, constant volume to 100mL are made into the gold chloride working solution of l%, which saves backup in 4 DEG C of brown reagent bottles；
(3) 1% citric acid three sodium solutions configuration (ready-to-use): 0.1g two citric acid monohydrate trisodiums are accurately weighed
(Na3C6H5O7·2H2O), dissolved with 10mL ultrapure water, be made into 1% citric acid three sodium solution, then with 0.22 μM of membrane filtration；
(4) 1% gold chloride working solution of 100mL is heated, when gold chloride working solution comes to life, stirring while is fast
1% citric acid three sodium solution of the above-mentioned preparation of 4.0mL is added in speed；
(5) continue heating stirring, keep solution boiling, when solution colour from it is yellowish switch to black and become brick-red again when,
Timing continues heating 10 minutes, turns off heating knob thereafter, but is kept stirring simultaneously cooled to room temperature, and colloidal gold is prepared
Solution, 4 DEG C of the colloidal gold solution prepared preservations.
1.2.2 colloidal gold-antibody-nucleic acid compound preparation
1.2.2.1 the determination of optimum antibody label pH
(1) 8 1.5mL test tubes are taken, the above-mentioned unconcentrated colloidal gold solution of 1mL is separately added into；
(2) every pipe sequentially adds the K of 0.1M2CO30 μ L of solution, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 6 μ L, 7 μ L and 8 μ L are mixed, quiet
Set 5min；
(3) every pipe is separately added into the 1mg/mLPDGF-BB monoclonal antibody mixing of 5 μ L, shakes 30min at room temperature；
(4) every pipe is separately added into the NaCl solution that 100 μ L concentration are 10%, is uniformly mixed, stands 10min at room temperature；
(5) observing colloid gold color change, record keep brick-red minimum pH (K of 0.1M to be added2CO3When 4 μ L of solution
PH8.0)；
(6) for observing colloid gold color change until placing at room temperature 2 hours, it is most that record, which still keeps brick-red minimum pH,
(K of 0.1M is added in good antibody label pH2CO3PH8.0 when 4 μ L of solution).
The determination of 1 optimum antibody of table label pH
1.2.2.2 the determination of most suitable antibody labelled amount
(1) 8 1.5mL test tubes are taken, the above-mentioned unconcentrated colloidal gold solution of 1mL is separately added into；
(2) according to step 1.2.2.1's as a result, every pipe is separately added into the K of the 0.1M of Optimal content2CO34 μ L of solution is mixed
It is even, stand 5min；
(3) the 1mg/mL PDGF-BB monoclonal antibody that every pipe is separately added into 0 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 6 μ L, 7 μ L and 8 μ L mixes
It closes, shakes 30min at room temperature；
(4) every pipe is separately added into the NaCl solution that 100 μ L concentration are 10%, is uniformly mixed, stands 10min at room temperature；
(5) observing colloid gold color change, record keep the brick-red minimum antibody labelled amount (PDGF-BB of 1mg/mL
6 μ L of monoclonal antibody)；
(6) for observing colloid gold color change until placing at room temperature 2 hours, record still keeps brick-red minimum antibody mark
Note amount is most suitable antibody labelled amount (the 6 μ L of PDGF-BB monoclonal antibody of 1mg/mL).
The determination of the most suitable antibody labelled amount of table 2
1.2.2.3 the coupling and closing of antibody and colloidal gold
(1) colloidal gold solution (the 10* concentration, by taking 10mL as an example: 10mL of 1mL 10* concentration is added in 1.5mL EP pipe
Unconcentrated colloidal gold solution, 10000rpm are centrifuged 35min, remove supernatant, take precipitating, add PBS buffer solution fixed to 1mL), add 4 μ
The K of L0.1M2CO3Solution (pH 8.0)；
(2) it takes the PDGF-BB monoclonal antibody of 6 μ L 1mg/mL to be slowly added in the colloidal gold solution that above-mentioned 1mL has been concentrated, adds
Vortex is kept in the process；
(3) 4min, 10000rpm, 10 DEG C are reacted, 35min is centrifuged；
(4) supernatant is removed, precipitating (i.e. colloidal gold-antibody complex) is taken, is resuspended with 5 times of diluted PBS, obtains colloidal gold-
Antibody complex solution.
1.2.2.4 colloidal gold-antibody-nucleic acid compound coupling
(1) 1mL colloidal gold-antibody complex solution is taken, DNA6 (i.e. Apt-COOH, the Apt-COOH of 100mM are gradually added
For 3 ' the terminal modified Apt for having carboxyl, the nucleotide sequence of Apt is as shown in SEQ ID NO.1), DNA7 (i.e. Apt-Poly T2-
COOH, Apt-Poly T2-COOH be 3 ' terminal modified Apt-Poly T2, the Apt-Poly T2 for having carboxyl nucleotide sequence such as
Shown in SEQ ID NO.2), (i.e. Apt-Poly T3-COOH, Apt-Poly T3-COOH are 3 ' terminal modified to have carboxyl to DNA8
The nucleotide sequence of Apt-Poly T3, Apt-Poly T3 are as shown in SEQ ID NO.3), DNA9 (i.e. Apt-Poly T4-
COOH, Apt-Poly T4-COOH be 3 ' terminal modified Apt-Poly T4, the Apt-Poly T4 for having carboxyl nucleotide sequence such as
Shown in SEQ ID NO.4) and DNA10 (i.e. Apt-Poly T5-COOH, Apt-Poly T5-COOH are 3 ' terminal modified to have carboxyl
The nucleotide sequence of Apt-Poly T5, Apt-Poly T5 are as shown in SEQ ID NO.5) each 50 μ L of solution, jog at room temperature
30min makes DNA6-10 be sufficiently dispersed in colloidal gold-antibody complex surface；
(2) EDC solution of 120 μM of 50 μ L and the NHS solution of 120 μM of 50 μ L is added, activation modification is on DNA6-10
Carboxyl, jog 40min in room temperature；
(3) 4 DEG C of centrifugations, 11500rpm, 20 minutes；
(4) EDC solution of 120 μM of 50 μ L and the NHS solution of 120 μM of 50 μ L is added, activation modification is on DNA6-10
Carboxyl, jog 40min in room temperature；
(5) 4 DEG C of centrifugations, 11500rpm, 20 minutes；
(6) configuration of re-suspension liquid: 1g BSA adds 3g sucrose, adds 50mL PBS, is dissolved to 100mL with water, filtering, 4 DEG C of guarantors
It deposits spare；
(7) with 100 μ L re-suspension liquids be resuspended, obtain colloidal gold-antibody-nucleic acid complex solution, be placed in 4 DEG C save it is standby
With.
1.2.2.5 colloidal gold-antibody-nucleic acid compound closing and aging
(1) configuration (10%BSA) of confining liquid: 10 μ g BSA are weighed and are dissolved in 100 μ L ddH2In O, 0.22 μm of filter membrane mistake
Filter, 4 DEG C save backup；
(2) colloidal gold-antibody-nucleic acid complex solution for taking above-mentioned 4 DEG C of preservations, is slowly added to 10% BSA solution, makes
BSA final concentration of 1% keeps vortex in adding procedure；
(3) it is stored at room temperature closing 30 minutes；
(4) configuration of aged solution: taking the sodium chloride solution of 1.5 μM of 700 μ L, and the 1% SDS solution of 7 μ L is added to mix；
(5) aged solution is slowly added in colloidal gold-antibody-nucleic acid complex solution that step (3) BSA has been closed,
Vortex is kept in adding procedure；
(6) 4 DEG C of age overnights；
(7) 4 DEG C of centrifugations, 9500rpm 20 minutes, abandon supernatant；
(8) bottom colloidal gold-antibody-nucleic acid composite particles are resuspended with 1mL PBS buffer solution (pH7.4), repeat step
(7) and (8) twice；
(9) 4 DEG C of centrifugations, 11500rpm 20 minutes, abandon supernatant；
(10) with 100 μ L PBS buffer solution (pH7.4) be resuspended through closing, aging, be collected by centrifugation after colloidal gold-antibody-
Nucleic acid complexes particle obtains being closed, the colloidal gold of aging-antibody-nucleic acid complex solution, and 4 DEG C save backup.
1.2.3 the preparation of detection line (T line) and nature controlling line (C line)
1.2.3.1 prepared by the area T
(1) aptamer Apt2 (its nucleotide sequence such as SEQ ID NO.7 institute of the PDGF-BB of 1 pipe 1.0OD is taken
Show), high speed centrifugation 5min slowly opens lid, adds water, and oscillation dissolution makes its final concentration of 100 μM, obtains the core of PDGF-BB
Sour aptamers Apt2 solution；
(2) 4 DEG C of preservations prepare for making the detection zone on nitrocellulose membrane.
1.2.3.2 prepared by the area C
(1) the Poly A (DNA11, nucleotide sequence is as shown in SEQ ID NO.6) of 1 pipe 1.0OD, high speed centrifugation are taken
5min slowly opens lid, adds water, and oscillation dissolution makes its final concentration of 100 μM, obtains Poly solution A；
(2) 4 DEG C of preservations prepare for making the quality control region on nitrocellulose membrane.
1.2.4 the preparation of the platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal
1.2.4.1 the pretreatment of sample pad
(1) formula of sample pad treatment fluid: pH 9.0, including 4%Triton X-100,1%BSA, 2% sucrose, 2%
PEG-4000,100mM boric acid, 0.1%SDS, 0.5 μ g/mL salmon sperm dna, surplus is water；
(2) glass fibre membrane of size needed for cutting, is immersed in sample pad treatment fluid；
(3) glass fibre membrane is immersed in sample pad treatment fluid to take out after half an hour and is dried, then 37 DEG C of drying, be prepared into
To sample pad；
(4) it is saved under drying at room temperature.
1.2.4.2 the preparation of bonding pad
Colloidal gold-antibody-nucleic acid complex solution through closing, aging is sprayed on glass fibre membrane, metal spraying amount is
Overnight, bonding pad is prepared in 0.8uL/CM, 37 DEG C of drying.
1.2.4.3 nitrocellulose filter is crossed
(1) nitrocellulose filter of size needed for cutting is careful not to the integrality of damage film；
(2) by point film instrument, scribing line amount is adjusted to 1.0 μ L/cm, the distance of nature controlling line and detection line is 3.0mm；
(3) the aptamer Apt2 solution of ready PDGF-BB in advance and Poly solution A are respectively taken into 30 μ L successively
It draws on nitrocellulose filter；
(4) 37 DEG C of drying overnight, using the aptamer Apt2 of PDGF-BB as detection line, using Poly A as Quality Control
Line, kept dry, the group of the platelet derived growth factor PDGF-BB test strips for being amplified based on aptamer signal
Dress.
1.2.4.4 the assembling of the platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal
(1) the platelet derived growth factor PDGF-BB test strips one based on the amplification of aptamer signal are divided into four
A part, is followed successively by sample pad, bonding pad, nitrocellulose filter and blotting paper, successively pastes it on a PVC bottom plate,
2mm is overlapped between two adjacent pastes；
(2) with cutting machine by it is assembled based on aptamer signal amplification platelet derived growth factor PDGF-
BB test strips are cut into the strip of 4.0mm wide, dry to be sealed for use.
1.2.5 the detection of PDGF-BB albumen
(1) preparation of buffer
1. the preparation of DNA- protein hybridization liquid: weighing a certain amount of Tris-HCl, KCl, MgCl2, BSA, be added to certain
In the sterilizing ultrapure water of volume, it is placed in stirring and dissolving in magnetic stirring apparatus and mixes, adjust pH to 7.5, and make the end of Tris-HCl
Concentration is 10mM, the final concentration of 150mM, MgCl of KCl2Concentration be 1mM, the concentration of BSA is 5%.After mixing, it is added
A certain amount of glycerol, make its final concentration of 10%, and mix well.DTT is added before the use, is allowed to final concentration of 1mM.
2. the preparation of sample-loading buffer: weighing a certain amount of Tris-HCl, NaCl, the sterilizing for being added to certain volume is ultrapure
In water, it is placed in stirring and dissolving in magnetic stirring apparatus and mixes, adjust pH to 8.0, and make the final concentration of 20mM, NaCl of Tris-HCl
Final concentration of 150mM.
(2) people's recombination PDGF-BB albumen (Wuhan is excellent and gives birth to commerce and trade Co., Ltd) is diluted to DNA- protein hybridization liquid
10 μ g/mL add 2 μ L to the sample for the platelet derived growth factor PDGF-BB test strips amplified based on aptamer signal
On pad, then toward 60 μ L sample-loading buffers are added dropwise in sample pad, be horizontally arranged the test strips and put be stored at room temperature 5-10min after see
Examine result.
1.2.6 testing result judgment criteria
It is positive: when detection line and nature controlling line are all displayed in red band, to be then judged as positive, illustrate to contain in sample to be tested
PDGF-BB.PDGF-BB concentration in the more deep then sample to be tested of the red stripes of detection line is higher.
Negative: when detection line is not displayed in red band, nature controlling line is displayed in red band, then is judged as negative, illustrates to be measured
PDGF-BB is not contained in sample.
Invalid: when nature controlling line is not displayed in red band, no matter detection line is aobvious is not displayed in red band, is all judged as invalid.
1.3 testing result
The people's recombinant cytokine PDGF-BB albumen for being diluted to 10 μ g/mL is added drop-wise to platelet derived growth factor
It is detected in the sample pad of PDGF-BB test strips, testing result such as Fig. 3.
As shown in figure 3, the testing result of people's recombinant cytokine PDGF-BB albumen is the positive, someone's recombinant cell is free of
The testing result of the control of factor PDGF-BB albumen is feminine gender.
Embodiment 2: specificity experiments
With DNA- protein hybridization liquid (formula is with embodiment 1) respectively by PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC
10 μ g/mL are diluted to PDGF-DD albumen.Then the blood based on the amplification of aptamer signal for taking 2 μ L to be added to embodiment 1 is small
In the sample pad of plate derivative growth factor PDGF-BB test strips, then (formula is the same as real toward 60 μ L sample-loading buffers are added dropwise in sample pad
Apply example 1), be horizontally arranged the test strips and put be stored at room temperature 5~10min after observe result.
The result shows that: only PDGF-BB albumen testing result be the positive, PDGF-AA, PDGF-AB, PDGF-CC and
The testing result of PDGF-DD albumen is feminine gender.Illustrate that platelet derived growth factor PDGF-BB test strips of the invention have
The specificity of height.
Sequence table
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Claims (9)
1. a kind of platelet derived growth factor PDGF-BB test strips based on the amplification of aptamer signal, including bottom plate, institute
It is successively pasted with sample pad, bonding pad, nitrocellulose filter and blotting paper on the bottom plate stated, is partially weighed between two adjacent pastes
Folded, the nitrocellulose filter is equipped with detection line and nature controlling line, which is characterized in that is coated with colloid on the bonding pad
Gold-antibody-nucleic acid compound, is coated with the aptamer Apt2 of PDGF-BB in the detection line, on the nature controlling line
It is coated with Poly A；The colloidal gold-antibody-nucleic acid compound is prepared by the following method: using colloid gold label
Colloidal gold-antibody complex is prepared in PDGF-BB monoclonal antibody, then passes through the amino in PDGF-BB monoclonal antibody
With band on 3 ' the terminal modified Apt for having carboxyl, Apt-Poly T2, Apt-Poly T3, Apt-Poly T4 and Apt-Poly T5
Condensation reaction occurs for carboxyl, and colloidal gold-antibody-nucleic acid compound is prepared；The nucleic acid that the Apt is PDGF-BB is adapted to
Body, the Apt-Poly T2, Apt-Poly T3, Apt-Poly T4 and Apt-Poly T5 are 3 ' ends added with different length
The Apt of Poly T.
2. the platelet derived growth factor PDGF-BB examination according to claim 1 based on the amplification of aptamer signal
Paper slip, it is characterised in that: the nucleotide sequence of the Apt are as follows: 5 '-AGGGCGCGTTCTTCGTGGTTACTTTTAGTCCCG-
3'；The nucleotide sequence of the Apt-Poly T2 are as follows: 5 '-AGGGCGCGTTCTTCGTGGTTACTTTTAGTCCCGTTTTT
TTTTTTTTTT-3'；The nucleotide sequence of the Apt-Poly T3 are as follows: 5 '-AGGGCGCGTTCTTCGTGGTTACTTTTA
GTCCCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3；The nucleotide sequence of the Apt-Poly T4 are as follows: 5 '-AG
GGCGCGTTCTTCGTGGTTACTTTTAGTCCCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-
3'；The nucleotide sequence of the Apt-Poly T5 are as follows: 5 '-AGGGCGCGTTCTTCGTGGTTACTTTTAGTCCCGTTTTT
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’。
3. the platelet derived growth factor PDGF-BB examination according to claim 1 based on the amplification of aptamer signal
Paper slip, which is characterized in that the nucleotide sequence of the Poly A are as follows: 5 '-AAAAAAAAAAAAAAAAAAAAA-3 '.
4. the platelet derived growth factor PDGF-BB examination according to claim 1 based on the amplification of aptamer signal
Paper slip, which is characterized in that the nucleotide sequence of the aptamer Apt2 of the PDGF-BB are as follows: 5 '-CAGGCTACGGCAC
GTAGAGCATCACCATGATCCTG-3’。
5. the platelet derived growth factor PDGF-BB examination according to claim 1 based on the amplification of aptamer signal
Paper slip, which is characterized in that the bottom plate is PVC bottom plate.
6. the platelet derived growth factor PDGF-BB examination according to claim 1 based on the amplification of aptamer signal
Paper slip, which is characterized in that the distance between described detection line and nature controlling line are 3mm.
7. a kind of platelet derived growth factor described in any one of claims 1-6 based on the amplification of aptamer signal
The preparation method of PDGF-BB test strips, which comprises the following steps:
1) preparation of sample pad: glass fibre membrane is immersed in sample pad treatment fluid, takes out drying, sample pad is prepared；
2) preparation of bonding pad: colloid gold label PDGF-BB monoclonal antibody is used, colloidal gold-antibody complex is prepared, so
Afterwards by amino in PDGF-BB monoclonal antibody and 3 ' the terminal modified Apt for having carboxyl, Apt-Poly T2, Apt-Poly T3,
Condensation reaction occurs for the carboxyl of band on Apt-Poly T4 and Apt-Poly T5, and it is compound that colloidal gold-antibody-nucleic acid is prepared
Object is sprayed on glass fibre membrane after closing, aging, dry, and bonding pad is prepared；
3) aptamer Apt2 and the Poly A of PDGF-BB is drawn respectively on nitrocellulose filter, with the nucleic acid of PDGF-BB
Aptamers Apt2 is as detection line, using Poly A as nature controlling line；
4) sample pad, bonding pad, nitrocellulose filter and blotting paper are successively pasted on bottom plate, between two adjacent pastes
Divide overlapping, obtains the platelet derived growth factor PDGF-BB test strips amplified based on aptamer signal.
8. preparation method according to claim 7, which is characterized in that the sample pad treatment fluid includes 4%Triton
X-100,1%BSA, 2% sucrose, 2%PEG-4000,100mM boric acid, 0.1%SDS and 0.5 μ g/mL salmon sperm dna, surplus are
Water, pH 9.0.
9. a kind of detection method of the platelet derived growth factor PDGF-BB based on the amplification of aptamer signal, feature
It is, comprising the following steps: sample to be tested is added drop-wise to platelet derived growth factor described in any one of claims 1-6
In the sample pad of PDGF-BB test strips, then toward sample-loading buffer is added dropwise in sample pad, observed after standing 5~10min as a result,
If detection line and nature controlling line are all displayed in red band, it is judged as positive, contains PDGF-BB in sample to be tested, if detection line is not
It is displayed in red band, nature controlling line is displayed in red band, then is judged as negative, PDGF-BB is not contained in sample to be tested.
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