CN107328939A - A kind of enzyme-linked immunosorbent assay for measuring spent based on biotinylated antigen or antibody inorganic salts hybridized nanometer - Google Patents
A kind of enzyme-linked immunosorbent assay for measuring spent based on biotinylated antigen or antibody inorganic salts hybridized nanometer Download PDFInfo
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Abstract
The invention discloses a kind of enzyme-linked immunosorbent assay for measuring spent based on biotinylated antigen or antibody inorganic salts hybridized nanometer.Belong to field of immunological detection.This method utilizes Streptavidin and the rapidly and efficiently specific effect of biotin, biotinylated antigen or antibody inorganic salts hybridized nanometer are spent and are quickly coupled on the ELISA Plate of Streptavidin modification, the problem of traditional ELISA antibody coating needs reaction overnight is solved；Secondly as biotinylated antigen or antibody inorganic salts hybridized nanometer flower have higher stability, it is set still to have preferable activity under being placed under normal temperature condition；Finally, synthesized biotinylated antigen or antibody inorganic salts hybridized nanometer flower have three-layer laminated structure, and the capture to follow-up antibody or antigen is highly efficient, are conducive to improving detection sensitivity.The method that the present invention is provided only needs to provide biotinylated not synantigen or antibody, the detection of different antibodies or antigen is can be achieved with, with high applicability.
Description
Technical field
The present invention relates to a kind of immunological detection method, more particularly to it is a kind of based on biotinylated antigen or antibody-inorganic
The enzyme-linked immunosorbent assay for measuring of salt hybridized nanometer flower.
Background technology
Enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), refers to solubility
Antigen or antibody be adsorbed onto on solid phase carrier, using Ag-Ab binding specificity, carry out the qualitative of immune response and quantitative
Detection method.Since Engvall and Perlmann, this method has obtained swift and violent development since invention in 1971, extensively should
Detection for various antigen-antibodies.
Sandwich ELISA method is presently the most the detection means of conventional antigen and antibody, this method have it is simple to operate,
Quickly, sensitive the advantages of.Its general principle is, during detection antibody, and corresponding antigen molecule, which is coated on polystyrene solid phase, to be carried
On body, add and be detected sample, be incubated, washing adds enzyme mark antiantibody, then is incubated, washs, and then adds chromogenic enzyme substrate,
Data are read on ELIASA, the content of antibody in test sample is judged according to the power of substrate chromogenic reaction；Detect antigen
When, the antibody molecule for the antigen is coated on solid phase carrier, test sample is added, is incubated, washing is added tested anti-
Former another antibody (secondary antibody), is incubated, and washing adds the antibody of the anti-secondary antibody of enzyme mark, then is incubated, washs, and is eventually adding enzyme bottom
Thing develops the color, reading, according to the power of color reaction come the content of antigen in judgement sample.
But traditional enzyme-linked immunosorbent assay comes with some shortcomings, time-consuming for such as antibody or antigen coat process,
Difference is big between costly, batch, and antibody or antigen need strict Cord blood condition；Further, while sandwich ELISA side
Method sensitiveness is high, but with the development of life science, medical science and field of optical detection, the sensitivity of the ELISA method of simple and fast
Property is still urgently.Therefore, antibody or antigen coat overlong time, antibody how to be solved or Antigen Stability is poor, antibody or antigen
The problems such as capture rate is relatively low, realizes rapidly and efficiently emphasis of the detection as current enzyme linked immunosorbent assay research.
The content of the invention
The problem of existing for traditional enzyme linked immunosorbent assay (hereinafter referred to as " 2D ELISA "), the present invention uses bionical conjunction
Into strategy be prepared for biotinylated antigen or antibody-inorganic salts hybridized nanometer flower, construct immune point of three-layer laminated structure
Analysis method (hereinafter referred to as " 3D ELISA ").This method utilizes the rapidly and efficiently specific effect of Streptavidin and biotin,
Biotinylated antigen or antibody-inorganic salts hybridized nanometer are spent and are quickly coupled on the ELISA Plate of Streptavidin modification, is solved
The problem of traditional ELISA antibody coating needs reaction overnight；Secondly as biotinylated antigen or antibody-inorganic salts hydridization are received
Popped rice has higher stability, it is still had preferable activity under being placed under normal temperature condition；Finally, synthesized biotin
Changing antigen or antibody-inorganic salts hybridized nanometer flower has three-layer laminated structure, and the capture to follow-up antibody or antigen is more high
Effect, is conducive to improving detection sensitivity (Fig. 1).
Realize technical scheme that above-mentioned purpose of the present invention used for：
There is provided a kind of enzyme linked immunological suction based on biotinylated antigen or antibody-inorganic salts hybridized nanometer flower for first aspect
Attached assay method, it is characterised in that by biotinylated antigen or antibody, it is 6.8 that pH value is added to together with copper-bath
In~7.4 phosphate buffer solution, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound are obtained, with strepto-
The ELISA Plate of Avidin modification has incubation the enzyme of biotinylated antigen or antibody-inorganic salts hybridized nanometer flower in 37 DEG C of incubations
Target is used for the enzyme linked immunosorbent assay (ELISA) of testing sample.
Specifically, enzyme-linked immunosorbent assay for measuring of the invention comprises the following steps：
(a) by disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride solution in deionized water, adjust mixed solution pH value to
6.8~7.4, phosphate buffer solution is obtained, the antigen or antibody of copper-bath and biotin modification is added, shakes up rear room temperature
18h is placed, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound is obtained；
(b) gained biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound are centrifuged and must precipitated, be washed with water
2~4 times, biotinylated antigen or antibody after must washing-inorganic salts hybridized nanometer flower compound；
(c) biotinylated antigen after washing or antibody-inorganic salts hybridized nanometer flower compound are dispersed in PBST
In cushioning liquid, in the orifice plate for being placed in Streptavidin modification, 37 DEG C are incubated after 30~120min, with wash liquid five times, then
2%BSA solution is added, 45min are closed in 37 DEG C, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower must be coated with
ELISA Plate (referred to as " 3D Substrate "), it is placed in 4 DEG C and saves backup；Described washing lotion is PBST cushioning liquid, wherein phosphoric acid
Salt buffer solution concentration is 10mM, and NaCl concentration is 150mM, and Tween 20 concentration is that 0.05%, pH is 7.2；
(d) testing sample is added and is coated with the ELISA Plate of biotinylated antigen or antibody-inorganic salts hybridized nanometer flower,
Mixing is gently rocked, ELISA Plate is plus lid or overlay film, 37 DEG C of reaction 45min；
(e) abandoning supernatant, is washed five times, pats dry, and the anti-object antibody of 50 μ L HRP marks is added per hole, and 37 DEG C anti-
Answer 45min；
(f) after incubating, liquid in hole is discarded, board-washing 5 times is patted dry, the TMB colour developing μ L of work night 50 is added per hole, 37 DEG C are kept away
Light develops the color 10min, sequentially adds the μ L of sulfuric acid terminate liquid 50 per hole, terminating reaction, and now solution colour switchs to yellow immediately by blueness；
(g) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with ELIASA, or solution is transferred to cuvette,
It is positioned in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.Pass through shade or absorption intensity
Visualization half-quantitative detection or quantitative determination can be carried out to determinand.
Wherein, the phosphate buffer solution concentration described in step (a) is 0.1M, and the concentration of described copper-bath is 10
~200mM, the antigen or antibody of described biotin modification are alpha-fetoprotein (AFP) monoclonal antibody of biotin modification, dense
Degree scope is 0.001~0.5mg/mL；
Rotating speed during each centrifugation described in step (b) is 10000r/min, and centrifugation time is 10min；
Anti- object antibody described in step (e) is AntiAFP antibody；
Sulfuric acid terminate liquid concentration is 2M H in step (f)2SO4。
The inventive method compared with the conventional method, with advantages below and effect：
1st, antigen or antibody have been had concurrently using bionic method synthesizing biotinylated antigen or antibody-inorganic salts hybridized nanometer flower
High activity and nano material high-specific surface area three-dimensional structure, three-dimensional ELISA detection pattern is realized, with traditional two dimension
ELISA is compared, and improves the capture rate to antibody or antigen, therefore, and the present invention has higher sensitivity and wider inspection
Survey scope.
2nd, compared with free state antigen or antibody, the antigen or antibody-inorganic salts hybridized nanometer flower stability are good, make its
Still there is preferable activity under room temperature.
3rd, the antigen or antibody coating technique are due to having introduced biotin-Streptavidin system, so as to utilize him
Between fast and efficiently specifically bind, solve traditional antigen or antibody coating overlong time the problem of, experiment only need it is several
Hour can complete.
4. in the present invention, it is only necessary to which biotinylated not synantigen or antibody are provided, prepare corresponding antigen or anti-
Body-inorganic salts hybridized nanometer flower, can just realize the detection of different antibodies or antigen, with high applicability using the ELISA method.
Brief description of the drawings
Fig. 1 is that 2D Substrate are tested with 3D Substrate acquisition performances and 2D ELISA and 3D ELISA are detected
AFP schematic diagram
Fig. 2 is the scanning electron microscope diagram (SEM for biotinylated antibody-inorganic salts hybridized nanometer flower that embodiment 1 is synthesized
Figure).
Fig. 3 is the X-ray powder diffraction for biotinylated antibody-inorganic salts hybridized nanometer floral material that embodiment 1 is synthesized
(XRD)。
Fig. 4 quantitatively detects AFP working curve for 3D ELISA in embodiment 1.
Fig. 5 quantitatively detects AFP working curve for 2D ELISA in embodiment 2.
Fig. 6 is the 3D ELISA visualization half-quantitative detection AFP contrasts in the 2D ELISA and embodiment 1 in embodiment 2
Figure
It is the 2D ELISA visualization half-quantitative detection AFP design sketch in embodiment 2 to scheme a；It is the 3D in embodiment 1 to scheme b
ELISA visualizes half-quantitative detection AFP design sketch.
Fig. 7 is the time-optimized figure of coating for biotinylated antibody-inorganic salts hybridized nanometer flower that embodiment 1 is synthesized.
Fig. 8 is different incubation times of the 3D Substrate in embodiment 5 from traditional 2D Substrate and antigen
Capture the effect contrast figure of antigen.
Fig. 9 is to store antibody under the 3D Substrate and traditional 2D Substrate normal temperature conditions that embodiment 5 is synthesized
Activity change comparison diagram.
Figure 10 is that the biotinylated antibody-inorganic salts hybridized nanometer for the various concentrations that embodiment 7 is synthesized is spent and antigen is caught
Obtain effect contrast figure
Wherein a, b, c, d, e representative contain 0.005,0.01,0.016,0.02,0.05mg/mL biotin modification respectively
Sheep anti mouse secondary antibody sample.
Figure 11 spends middle antibody fixed efficiency to calculate biotinylated antibody-inorganic salts hybridized nanometer that embodiment 1 is synthesized
Canonical plotting.
Embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only the explanation to the inventive method, without limiting remaining content that the present invention is disclosed in any way.
The ELISA Plate of Streptavidin modification and traditional 2D ELISA ELISA Plate are purchased from Thermo Fisher
Scientific Inc., alpha-fetoprotein (AFP) monoclonal antibody of biotin modification, alpha-fetoprotein (AFP), HRP modifications
Alpha-fetoprotein (AFP) monoclonal antibody is purchased from Shanghai Linc-Bio Science Co., Ltd., the sheep anti mouse secondary antibody of biotin modification
Beijing Bo Aosen Bioisystech Co., Ltd is purchased from, the mouse source antibody of HRP marks is purchased from Sino Biological Inc..
【Embodiment 1】Biotinylated antibody-inorganic salts hybridized nanometer of the present invention is spent for rapidly and efficiently detecting cancer mark
Will thing alpha-fetoprotein (AFP) tests (3D ELISA)
1) 4 μ L copper-baths are added into the phosphate that 600 μ L contain biotinylation alpha-fetoprotein (AFP) monoclonal antibody
In cushioning liquid, room temperature places 18h, obtains biotinylated antibody-inorganic salts hybridized nanometer flower；
Described phosphate buffer solution concentration is 0.1M, pH 7.4；The concentration of described copper-bath is 120mM；
Described antibody is that the concentration of biotinylation alpha-fetoprotein monoclonal antibody is 0.016mg/mL.
2) said mixture is centrifuged and must precipitated, be washed with deionized 3 times, biotinylated antibody-nothing after must washing
Machine salt hybridized nanometer spends compound；Centrifugal rotational speed is 10000r/min, and centrifugation time is 10min.
3) above-mentioned biotinylated antibody-inorganic salts hybridized nanometer flower is dispersed in PBST cushioning liquid, is placed in chain
In the ELISA Plate (Thermo Scientific Nunc Immobilizer Streptavidin) of mould Avidin modification, in 37
DEG C it is incubated after 1h, with wash liquid five times, adds 300 μ L 2%BSA solution, close 45min in 37 DEG C, biology must be coated with
The ELISA Plate (referred to as " 3D Substrate ") of elementization antibody-inorganic salts hybridized nanometer flower, is placed in 4 DEG C and saves backup.Described
Washing lotion is PBST cushioning liquid, and wherein phosphate buffer solution concentration is 10mM, and NaCl concentration is 150mM, Tween's 20
Concentration is that 0.05%, pH is 7.2.
4) the antigen A FP standard liquids of various concentrations are added and is coated with biotinylated antibody-inorganic salts hybridized nanometer flower
ELISA Plate, gently rock mixing, ELISA Plate is plus lid or overlay film, 37 DEG C of reaction 45min.
5) liquid is discarded, PBST buffer solutions are washed five times, patted dry.50 μ L concentration are added per hole to mark for 20 μ g/ μ L HRP
AntiAFP antibody, 37 DEG C reaction 45min.
6) after incubating, liquid in hole is discarded, board-washing 5 times is patted dry.The TMB colour developing μ L of working solution 50 are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add the μ L of 2M sulfuric acid terminate liquid 50 per hole, terminating reaction, now solution colour Huang is switched to by blueness immediately
Color.
7) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with ELIASA, or solution is transferred to cuvette, is put
It is placed in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.Pass through shade or absorption intensity pair
Determinand carries out half-quantitative detection.AFP concentration is mapped according to the absorption value at 450nm wavelength, quantitative detection can be obtained
AFP working curve.
Electron microscope is scanned to the biotinylated antibody synthesized by the present embodiment-inorganic salts hybridized nanometer floral material
(SEM) characterize, the SEM of gained is as shown in Figure 2.SEM shows the spherical nano flower that product morphology is about 10 μm of diameter.
X-ray powder diffraction (XRD) is carried out to the miscellaneous floral material of biotinylated antibody-inorganic salts synthesized by the present embodiment
Characterize, gained XRD such as Fig. 3.Identified through X-ray powder diffraction, the inorganic component of the material is Copper phosphate (Cu3(PO4)2) trihydrate.
Figure 4, it is seen that with the increase of object AFP concentration, ultraviolet-ray visible absorbing intensity increases therewith.Mesh
Thing AFP concentration is marked 0.1 between 100ng/mL, ultraviolet-ray visible absorbing intensity is linear related to target concentration, by right
Result of the test carries out linear regression, and obtaining regression equation is：
Y=0.0613x+0.222 (R2=0.990), calculating obtains detection limit for 0.029ng/mL, with higher sensitive
Degree.
【Embodiment 2】Traditional 2D ELISA are used to detect that cancer markers alpha-fetoprotein (AFP) is tested
1) in Thermo Fisher Scientific Inc. (article No.s：446469) 50 μ L first tires are added in ELISA Plate hole
Albumen (AFP) monoclonal antibody (20 μ g/mL), in 4 DEG C of overnight incubations.
2) solution in the ELISA Plate being coated with is inclined, adds 300 μ L BSA solution (2%), 2h is closed in 37 DEG C.
3) the AFP standard liquids of various concentrations are added to the ELISA Plate for being coated with antibody, mixing are gently rocked, ELISA Plate adds
Upper lid or overlay film, 37 DEG C of reaction 45min.
4) liquid is discarded, washs five times, pats dry.50 μ L concentration are added per hole for the 20 μ g/ μ L HRP anti-AFP marked to resist
Body, 37 DEG C of reaction 45min.
5) after incubating, liquid in hole is discarded, board-washing 5 times is patted dry.The TMB colour developing μ L of working solution 50 are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add the μ L of 2M sulfuric acid terminate liquid 50 per hole, terminating reaction, now solution colour Huang is switched to by blueness immediately
Color.
6) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with ELIASA, or solution is transferred in cuvette,
It is positioned in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.Determinand is entered by shade
Row half-quantitative detection.AFP concentration is mapped according to the absorption value at 450nm wavelength, quantitative detection AFP work can be obtained
Curve.
Quantitatively detection AFP effects are as shown in figure 5, the detection method range of linearity is 5-50ng/mL, by result of the test
Linear regression is carried out, obtaining regression equation is：Y=0.0301x+0.336 (R2=0.994), calculating obtains detection limit and is
0.92ng/mL。
The present embodiment is using traditional enzyme linked immunosorbent assay (2D ELISA) detection AFP antigens, and it visualizes sxemiquantitative inspection
Survey AFP effects as shown in Figure 6 a, 2D ELISA Visual retrieval AFP detection ranges are narrow, only have more in 5-50ng/mL scopes
Obvious color change, can not be differentiated for the AFP of low concentration, and platform is reached for the AFP detection signals of high concentration, it is impossible to point
Distinguish；Fig. 6 b are the 3D ELISA of embodiment 1 visualization half-quantitative detection AFP design sketch, its Visual retrieval AFP detection ranges
Extensively, obvious color change can be seen in 0.1-100ng/mL AFP concentration ranges.
Sensitivity and detection range in 3D ELISA are substantially better than 2D ELISA.Analyzed in terms of signal, due to antibody
Inorganic salts hybridized nanometer spend it is middle loaded substantial amounts of capture antibody and its special three-dimensional structure, resist its AFP to low concentration
Body also can be captured effectively；The substantial amounts of AFP capture antibody of its load simultaneously can also meet the AFP of high concentration capture.From background
Aspect is analyzed, and the inorganic component in being spent due to antibody inorganic salts hybridized nanometer can effectively reduce non-specific adsorption, so as to reduce
Background.
【Embodiment 3】The coating of the biotinylated antibody that embodiment 1 is synthesized-inorganic salts hybridized nanometer flower is time-optimized
1) biotinylated antibody for obtaining embodiment 1-inorganic salts hybridized nanometer flower is dispersed in PBST cushioning liquid
In, in the orifice plate for being placed in Streptavidin modification, after 37 DEG C are incubated and are incubated 5,10,30,60,90,120min respectively, with washing
Liquid is washed five times, adds 300 μ L 2%BSA solution, and 45min are closed in 37 DEG C, must be coated with biotinylated antibody-inorganic
The ELISA Plate (referred to as " 3D Substrate ") of salt hybridized nanometer flower, is placed in 4 DEG C and saves backup.Described washing lotion is PBST
(10mM, 150mM NaCl, 0.05%Tween 20, pH 7.2).
2) 50 microlitres of 25ng/mL AFP standard liquids are added and is coated with biotinylated antibody-inorganic salts hybridized nanometer flower
ELISA Plate, gently rock mixing, ELISA Plate is plus lid or overlay film, 37 DEG C of reaction 45min.
3) liquid is discarded, washs five times, pats dry.50 μ L concentration are added per hole for the 20 μ g/ μ L HRP anti-AFP marked to resist
Body, 37 DEG C of reaction 45min.
4) after incubating, liquid in hole is discarded, board-washing 5 times is patted dry.The TMB colour developing μ L of working solution 50 are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add the μ L of sulfuric acid terminate liquid 50 per hole, terminating reaction, now solution colour yellow is switched to by blueness immediately.
5) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with ELIASA, or solution is transferred to cuvette, is put
It is placed in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.
Shown in Fig. 7, the ELISA Plate coupling rates that biotinylated antibody-inorganic salts hybridized nanometer flower is modified with Streptavidin
It hurry up, it is to have more obvious detection signal to be incubated 5min, when incubation time is more than 60min, detection signal reaches platform.In order to protect
The higher detection signal of card realizes the purpose of quick detection, in this experiment, biotinylated antibody-inorganic salts hybridized nanometer again simultaneously
The incubation time for the ELISA Plate that flower is modified with Streptavidin is selected as 60min.
【Embodiment 4】Verify reliability and accuracy of this method in actual sample detection
The AFP standard items of various concentrations are added in blood serum sample, gained recovery of standard addition is listed in the table below in 1.From table 1
As can be seen that the standard curve obtained using embodiment 1, the recovery of standard addition of detection is determined between 93.0% to 109.3%
As a result it can well be coincide with spiked levels, illustrate that this method has preferable accuracy.
The recovery of standard addition of table 1
【Embodiment 5】Three-dimensional enzyme linked immunosorbent assay (3D is spent based on biotinylated antibody-inorganic salts hybridized nanometer
ELISA) and in traditional 2D ELISA each capture antibody is contrasted in different incubation time experiment effects
First, 3D ELISA
1) copper-bath is added in the phosphate buffer solution containing biotinylated antibody, room temperature places 18h, obtains
Biotinylated antibody-inorganic salts hybridized nanometer flower；
Described phosphate buffer solution concentration is 0.1M, pH 7.4；The concentration of described copper-bath is 120mM；
Described antibody is the sheep anti mouse secondary antibody of biotin modification, and concentration is 0.016mg/mL.
2) said mixture is centrifuged and must precipitated, be washed with deionized 3 times, the antibody after must washing-inorganic salts hydridization
Nano flower compound；Rotating speed during described each centrifugation is 10000r/min, and centrifugation time is 10 min.
3) above-mentioned biotinylated antibody-inorganic salts hybridized nanometer flower is dispersed in PBST cushioning liquid, is placed in chain
In the orifice plate of mould Avidin modification, it is incubated in 37 DEG C after 1h, with wash liquid five times, 2% BSA solution is added, in 37 DEG C of envelopes
45min is closed, the ELISA Plate (referred to as " 3D Substrate ") of biotinylated antibody-inorganic salts hybridized nanometer flower must be coated with.Institute
The washing lotion stated is PBST (10mM, 150mM NaCl, 0.05% Tween 20, pH 7.2).
4) the mouse source antibody (100ng/mL) for marking 50 μ L HRP adds and is coated with biotinylated antibody-inorganic salts hydridization
The ELISA Plate of nano flower, gently rocks mixing, and ELISA Plate is plus lid or overlay film, and 37 DEG C are reacted, and control time is respectively 5,30,
60,90,120min.
5) after incubating, liquid in hole is discarded, board-washing 5 times is patted dry.The TMB colour developing μ L of working solution 50 are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add the μ L of sulfuric acid terminate liquid 50 per hole, terminating reaction, now solution colour yellow is switched to by blueness immediately.
6) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with ELIASA, or solution is transferred to cuvette, is put
It is placed in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.
2nd, traditional 2D ELISA
1) secondary antibody (20 μ g/mL) of the sheep anti mouse of 50 μ L biotin modifications is added dissolved with PBST cushioning liquid, be placed in
In the orifice plate of Streptavidin modification, it is incubated in 37 DEG C after 1h, with wash liquid five times, 1%BSA solution is added, in 37 DEG C
45min is closed, the ELISA Plate (referred to as " 2D Substrate ") of antibody must be coated with.
2) the mouse source antibody (100ng/mL) for marking 50 μ L HRP adds the enzyme mark for the antibody for being coated with biotin modification
Plate, gently rocks mixing, and ELISA Plate is plus lid or overlay film, and 37 DEG C are reacted, and control time is respectively 5,30,60,90,120min.
3) after incubating, liquid in hole is discarded, board-washing 5 times is patted dry.The TMB colour developing μ L of working solution 50 are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add the μ L of sulfuric acid terminate liquid 50 per hole, terminating reaction, now solution colour yellow is switched to by blueness immediately.
4) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with ELIASA, or solution is transferred in cuvette,
It is positioned in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.
As shown in figure 8, enhancings of the 3D ELISA and traditional 2D ELISA with the capture antibody incubation time, it captures energy
Power gradually increases, and 3D substrate capture ability is better than traditional 2D ELISA under identical incubation time.
【Embodiment 6】The biotinylated antibody of the present invention-inorganic salts hybridized nanometer flower and free state antibody storage stability
Compare
The biotinylated antibody synthesized with above-described embodiment 5-inorganic salts hybridized nanometer flower is carrier loaded antibody, and investigated
When phosphate buffer (pH 7.4) is stored under room temperature condition, the relative activity of immobilized antibody and free antibodies.In room temperature and
Under conditions of phosphate buffer pH 7.4, by biotinylated antibody-inorganic salts hybridized nanometer flower and free biotin antibody
After placing 1-7 days respectively, respectively repeatedly embodiment 5 " one, the step 3 in 3D ELISA ") -6) or " two, traditional 2D
1) -4 in ELISA ") it is, 37 DEG C of 45min with the reaction of the HRP mouse source antibody marked.
Experimental result is as shown in figure 9, the storage stability for the antibody being fixed on during hybridized nanometer is spent is compared with free antibodies
It is substantially improved, relative activity was still maintained at more than 90% within one week.
【Embodiment 7】Three-dimensional enzyme linked immunosorbent assay (3D is spent based on biotinylated antibody-inorganic salts hybridized nanometer
ELISA in), the coating experiment effect test of various concentrations biotinylated antibody
1) copper-bath is added in the phosphate buffer solution containing various concentrations biotinylated antibody, room temperature is placed
18h, obtains antibody-inorganic salts hybridized nanometer flower；
Described phosphate buffer solution concentration is 0.1M, pH 7.4；The concentration of described copper-bath is 120mM；
Described antibody is the sheep anti mouse secondary antibody of biotin modification, and concentration is respectively 0.005,0.01,0.016,0.02,0.05mg/
mL。
2) said mixture is centrifuged and must precipitated, be washed with deionized 3 times, biotinylated antibody-nothing after must washing
Machine salt hybridized nanometer spends compound；Rotating speed during described each centrifugation is 10000r/min, and centrifugation time is 10min.
3) above-mentioned biotinylated antibody-inorganic salts hybridized nanometer flower is dispersed in PBST cushioning liquid, is placed in chain
In the orifice plate of mould Avidin modification, it is incubated in 37 DEG C after 1h, with wash liquid five times, 2%BSA solution is added, in 37 DEG C of envelopes
45min is closed, the ELISA Plate of biotinylated antibody-inorganic salts hybridized nanometer flower must be coated with.Described washing lotion be PBST (10mM,
150mM NaCl, 0.05%Tween 20, pH 7.2).
4) the mouse source antibody (100ng/mL) for marking 50 μ L HRP adds and is coated with biotinylated antibody-inorganic salts hydridization
The ELISA Plate of nano flower, is gently rocked on mixing, ELISA Plate plus lid or overlay film, 37 DEG C of 45 min of reaction.
5) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with ELIASA, or solution is transferred in cuvette,
It is positioned in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.To the difference synthesized in the present embodiment
Biotin flower antibody-inorganic salts hybridized nanometer flower of concentration carries out acquisition performance test, the ultraviolet-visible absorption spectroscopy figure of gained
As shown in Figure 10, with the increase for the biotinylated antibody concentration being fixed on nano flower, the capture ability enhancing of nano flower.
【Embodiment 8】Biotinylated antibody-inorganic salts hybridized nanometer of the present invention spends the fixed efficiency of middle antibody to calculate
The BSA protein standards solution of 50 μ L various concentrations is respectively taken into the test tube of respective markers.50 μ L coomassies are added dropwise bright
Blue enhanced reagent is into every pipe and fully mixes, and is placed in black out at room temperature and is incubated 10min.Suction of the determination sample at 595nm
Receipts value (A595), and BSA concentration is mapped, draw standard curve, such as Figure 11.Go out unknown sample with this standard curve interpretation again
Protein concentration.
4 μ L copper-baths (120mM) and 10 μ L lifes are added in 600 μ L phosphate buffer solutions (0.1M, pH 7.4)
The AFP antibody (1mg/mL) of thing element modification, detailed process places 18h, obtains biotinylated antibody-nothing referring to embodiment 1, room temperature
Machine salt hybridized nanometer is spent；Centrifugation, takes supernatant liquor to be placed in testing sample pipe.50 μ L testing samples are taken to add isometric coomassie
The enhanced reagent of light blue, is fully mixed, and determines absorption value of the testing sample at 595nm.
Experimental result：It can be seen from fig. 11 that Abs595It is y=0.006x-0.05 (R with standard protein concentration relationship2
=0.994).Absorption value of the testing sample at 595nm is 0.013.Bring standard curve into and obtain protein concentration in testing sample and be
10.8μg/mL。
Protein concentration is added before reaction is：
After the completion of reaction, the protein concentration of supernatant liquor is：10.5μg/mL.
Obtaining biotinylated antibody-inorganic salts hybridized nanometer by experiment spends the fixed efficiency of middle antibody to be：
Claims (3)
1. a kind of enzyme-linked immunosorbent assay for measuring based on biotinylated antigen or antibody-inorganic salts hybridized nanometer flower, it is special
Levy and be, by biotinylated antigen or antibody, the phosphoric acid buffer that pH value is 6.8 ~ 7.4 is added to together with copper-bath molten
In liquid, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound, the ELISA Plate modified with Streptavidin are obtained
In 37 DEG C of incubations, will be incubated has biotinylated antigen or the ELISA Plate of antibody-inorganic salts hybridized nanometer flower to be used for testing sample
Enzyme linked immunosorbent assay (ELISA).
2. according to the method described in claim 1, it is characterised in that specifically include following steps：
（a）By disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride is dissolved in deionized water, adjusts the pH value of mixed solution to 6.8
~ 7.4, phosphate buffer solution is obtained, the antigen or antibody of copper-bath and biotin modification is added, rear room temperature is shaken up and puts
18 h are put, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound is obtained；
（b）Gained biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound are centrifuged and must precipitated, it is washed with water 2 ~
4 times, biotinylated antigen or antibody after must washing-inorganic salts hybridized nanometer flower compound；
（c）Biotinylated antigen after washing or antibody-inorganic salts hybridized nanometer flower compound are dispersed in PBST bufferings
In solution, in the orifice plate for being placed in Streptavidin modification, 37 DEG C are incubated after 30 ~ 120 min, with wash liquid five times, then add
Enter 2% BSA solution, 45min is closed in 37 DEG C, the enzyme of biotinylated antigen or antibody-inorganic salts hybridized nanometer flower must be coated with
Target, is placed in 4 DEG C and saves backup；Described washing lotion is PBST cushioning liquid, and wherein phosphate buffer solution concentration is 10mM,
NaCl concentration is 150mM, and Tween 20 concentration is that 0.05%, pH is 7.2；
（d）Testing sample is added and is coated with the ELISA Plate of biotinylated antigen or antibody-inorganic salts hybridized nanometer flower, gently
Mixing is rocked, ELISA Plate is plus lid or overlay film, 37 DEG C of 45 min of reaction；
（e）Abandoning supernatant, PBST buffer solutions are washed five times, are patted dry, and the anti-object that 50 μ L HRP marks are added per hole resists
Body, 37 DEG C of 45 min of reaction；
（f）After incubation, liquid in hole is discarded, board-washing 5 times is patted dry, the μ L of TMB colour developing work nights 50 are added per hole, 37 DEG C of lucifuges show
The min of color 10, sequentially adds the μ L of sulfuric acid terminate liquid 50 per hole, terminating reaction, and now solution colour switchs to yellow immediately by blueness；
（g）The optical density in each hole is sequentially measured in 450 nm wavelength with ELIASA（OD values）, or solution is transferred to cuvette, place
In uv-visible absorption spectra instrument, its absorption value at 450 nm wavelength is surveyed, can be right by shade or absorption intensity
Determinand carries out visualization half-quantitative detection or quantitative determination.
3. method according to claim 2, it is characterised in that step（a）Described phosphate buffer solution concentration is 0.1
M, the concentration of described copper-bath is 10 ~ 200 mM, and the antigen or antibody of described biotin modification are repaiied for biotin
The AFP monoclonal antibody of decorations, concentration range is 0.001 ~ 0.5 mg/mL；Step（b）During described each centrifugation
Rotating speed be 10000 r/min, centrifugation time be 10 min；
Step（e）Described anti-object antibody is AntiAFP antibody, and concentration is 20 μ g/ μ L；Sulfuric acid terminate liquid is dense in step (f)
Spend for 2M H2SO4。
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