CN107328939B - A kind of enzyme-linked immunosorbent assay for measuring based on biotinylated antigen or antibody-inorganic salts hybridized nanometer flower - Google Patents
A kind of enzyme-linked immunosorbent assay for measuring based on biotinylated antigen or antibody-inorganic salts hybridized nanometer flower Download PDFInfo
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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 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, solves the problems, such as that traditional ELISA antibody coating needs reaction overnight；Secondly as biotinylated antigen or antibody-inorganic salts hybridized nanometer flower have higher stability, still there is preferable activity under so that it is placed under normal temperature condition；Finally, synthesized biotinylated antigen or antibody-inorganic salts hybridized nanometer flower have three-layer laminated structure, highly efficient to the capture of follow-up antibody or antigen, are conducive to improve detection sensitivity.Method provided by the invention only needs to provide biotinylated not synantigen or antibody, can be achieved with the detection of different antibodies or antigen, has high applicability.
Description
Technical field
The present invention relates to a kind of immunological detection method, more particularly to one kind are inorganic based on biotinylated antigen or antibody-
The enzyme-linked immunosorbent assay for measuring of salt hybridized nanometer flower.
Background technology
Enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA), referring to will be soluble
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 this method has obtained swift and violent development to Engvall and Perlmann since invention in 1971, answer extensively
Detection for various antigen-antibodies.
Sandwich ELISA method is presently the most the detection means of common antigen and antibody, this method have it is easy to operate,
Quickly, the advantages that sensitive.Its basic principle is, when detecting antibody, corresponding antigen molecule is coated on polystyrene solid phase and is carried
It on body, being added and is detected sample, be incubated, washing is added enzyme mark antiantibody, then is incubated, washs, and chromogenic enzyme substrate is then added,
Data are read in microplate reader, and 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 adds tested anti-
Former another antibody (secondary antibody) is incubated, and the antibody of the anti-secondary antibody of enzyme mark is added in washing, then is incubated, washs, and is eventually adding enzyme bottom
Object 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, such as antibody or antigen coat process time-consuming,
It is costly, difference is big between batch and antibody or antigen need stringent Cord blood condition；Further, while sandwich ELISA side
Method sensibility is high, but with the development of life science, medicine and field of optical detection, the sensitivity of simple and fast ELISA method
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 that rapidly and efficiently detection is as the emphasis of current enzyme linked immunosorbent assay research.
Invention content
For traditional enzyme linked immunosorbent assay (hereinafter referred to as " 2D ELISA ") there are the problem of, the present invention use bionical conjunction
At 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, still has preferable activity under so that it is placed under normal temperature condition；Finally, synthesized biotin
Changing antigen or antibody-inorganic salts hybridized nanometer flower has three-layer laminated structure, more high to the capture of follow-up antibody or antigen
Effect is conducive to improve detection sensitivity (Fig. 1).
Technical solution is used by realizing above-mentioned purpose of the present invention：
In a first aspect, providing a kind of enzyme linked immunological suction based on biotinylated antigen or antibody-inorganic salts hybridized nanometer flower
Attached assay method, which is characterized 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 is incubated at 37 DEG C, will be incubated the enzyme for having biotinylated antigen or antibody-inorganic salts hybridized nanometer flower
Target is used for the enzyme linked immunosorbent assay (ELISA) of sample to be tested.
Specifically, enzyme-linked immunosorbent assay for measuring of the invention includes the following steps：
(a) by disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride solution in deionized water, adjust 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, shakes up rear room temperature
18h is placed, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound are obtained；
(b) gained biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound are centrifuged into obtain precipitation, are washed with water
2~4 times, biotinylated antigen or antibody-inorganic salts hybridized nanometer after must washing spend compound；
(c) by after washing biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound be dispersed in PBST
In buffer solution, it is placed in the orifice plate of Streptavidin modification, after 37 DEG C are incubated 30~120min, with wash liquid five times, then
2%BSA solution is added, 45min are closed in 37 DEG C, must be coated with what biotinylated antigen or antibody-inorganic salts hybridized nanometer were spent
ELISA Plate (referred to as " 3D Substrate "), is placed in 4 DEG C and saves backup；The washing lotion is PBST buffer solutions, wherein phosphoric acid
A concentration of 150mM of salt buffer solution a concentration of 10mM, NaCl, a concentration of 0.05%, the pH 7.2 of Tween 20；
(d) sample to be tested is added in the ELISA Plate for being coated with biotinylated antigen or antibody-inorganic salts hybridized nanometer flower,
Mixing is gently shaken, ELISA Plate is plus lid or overlay film, 37 DEG C of reaction 45min；
(e) liquid is discarded supernatant, washs five times, pats dry, the anti-object antibody of 50 μ L HRP labels is added per hole, 37 DEG C anti-
Answer 45min；
(f) after incubating, liquid in hole is discarded, board-washing 5 times pats dry, and TMB colour developing 50 μ L of work night is added per hole, 37 DEG C are kept away
Light colour developing 10min, 50 μ L of sulfuric acid terminate liquid are sequentially added per hole, terminate reaction, solution colour switchs to yellow immediately by blue at this time；
(g) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with microplate reader, 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, a concentration of 0.1M of phosphate buffer solution described in step (a), a concentration of the 10 of the copper-bath
~200mM, the antigen or antibody of the biotin modification are alpha-fetoprotein (AFP) monoclonal antibody of biotin modification, dense
Spend ranging from 0.001~0.5mg/mL；
Rotating speed when centrifuging every time described in step (b) is 10000r/min, centrifugation time 10min；
Anti- object antibody described in step (e) is AntiAFP antibody；
A concentration of 2M H of sulfuric acid terminate liquid in step (f)2SO4。
The method of the present invention compared with the conventional method, has the following advantages and effect：
1, antigen or antibody have been had both 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, the detection pattern of three-dimensional ELISA is realized, with traditional two dimension
ELISA is compared, and improves the capture rate to antibody or antigen, and therefore, the present invention has higher sensitivity and wider inspection
Survey range.
2, 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.
3, 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 the problems, such as traditional antigen or antibody coating overlong time, experiment only need it is several
Hour can be completed.
4. in the present invention, it is only necessary to provide biotinylated not synantigen or antibody, prepare corresponding antigen or anti-
Body-inorganic salts hybridized nanometer flower, can utilize the ELISA method to realize the detection of different antibodies or antigen, have high applicability.
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
The schematic diagram of AFP
Fig. 2 is the scanning electron microscope diagram (SEM for biotinylated antibody-inorganic salts hybridized nanometer flower that embodiment 1 synthesizes
Figure).
Fig. 3 is the X-ray powder diffraction for biotinylated antibody-inorganic salts hybridized nanometer floral material that embodiment 1 synthesizes
(XRD)。
Fig. 4 is the working curve that 3D ELISA quantitatively detect AFP in embodiment 1.
Fig. 5 is the working curve that 2D ELISA quantitatively detect AFP in embodiment 2.
Fig. 6 is that the 3D ELISA in 2D ELISA and embodiment 1 in embodiment 2 visualize half-quantitative detection AFP comparisons
Figure
It is that the 2D ELISA in embodiment 2 visualize half-quantitative detection AFP design sketch 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 synthesizes.
Fig. 8 is the different incubation times of the 3D Substrate and traditional 2D Substrate and antigen in embodiment 5
Capture the effect contrast figure of antigen.
Fig. 9 is the 3D Substrate that embodiment 5 synthesizes and stores antibody under traditional 2D Substrate normal temperature conditions
Activity change comparison diagram.
Figure 10 is that the biotinylated antibody-inorganic salts hybridized nanometer for the various concentration that embodiment 7 synthesizes is spent and caught to antigen
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 is to calculate biotinylated antibody-inorganic salts hybridized nanometer that embodiment 1 synthesizes to spend middle antibody fixed efficiency
Canonical plotting.
Specific implementation mode
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
The ELISA Plate of Streptavidin modification and the ELISA Plate of traditional 2D ELISA 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
It is purchased from Beijing Bo Aosen Bioisystech Co., Ltd, the mouse source antibody of HRP labels is purchased from Sino Biological Inc..
【Embodiment 1】The biotinylated antibody of the present invention-inorganic salts hybridized nanometer flower is for rapidly and efficiently detecting cancer mark
Will object alpha-fetoprotein (AFP) tests (3D ELISA)
1) phosphate that 600 μ L contain biotinylation alpha-fetoprotein (AFP) monoclonal antibody is added in 4 μ L copper-baths
In buffer solution, it is placed at room temperature for 18h, obtains biotinylated antibody-inorganic salts hybridized nanometer flower；
The phosphate buffer solution a concentration of 0.1M, pH 7.4；A concentration of 120mM of the copper-bath；
The antibody is a concentration of 0.016mg/mL of biotinylation alpha-fetoprotein monoclonal antibody.
2) said mixture is centrifuged into obtain precipitation, be washed with deionized 3 times, biotinylated antibody-nothing after must washing
Machine salt hybridized nanometer spends compound；Centrifugal rotational speed is 10000r/min, centrifugation time 10min.
3) above-mentioned biotinylated antibody-inorganic salts hybridized nanometer flower is dispersed in PBST buffer solutions, is placed in chain
In the ELISA Plate (Thermo Scientific Nunc Immobilizer Streptavidin) of mould Avidin modification, in 37
After DEG C being incubated 1h, with wash liquid five times, 300 μ L 2%BSA solution are added, 45min is closed 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 be PBST buffer solutions, wherein a concentration of 10mM of phosphate buffer solution, a concentration of 150mM of NaCl, Tween's 20
A concentration of 0.05%, pH 7.2.
4) addition of the antigen A FP standard solution of various concentration biotinylated antibody-inorganic salts hybridized nanometer is coated with to spend
ELISA Plate, gently shake 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.A concentration of 20 μ g/ μ L HRP labels of 50 μ L are added per hole
AntiAFP antibody, 37 DEG C reaction 45min.
6) after incubating, liquid in hole is discarded, board-washing 5 times pats dry.TMB colour developing 50 μ L of working solution are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add 50 μ L of 2M sulfuric acid terminate liquid per hole, terminate reaction, solution colour switchs to Huang immediately by blue at this time
Color.
7) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with microplate reader, 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.It is mapped to the concentration of AFP according to the absorption value at 450nm wavelength, quantitative detection can be obtained
The working curve of AFP.
Electron microscope is scanned to the biotinylated antibody synthesized by the present embodiment-inorganic salts hybridized nanometer floral material
(SEM) it characterizes, the SEM of gained is as shown in Figure 2.SEM shows that product morphology is the spherical nano flower of 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
Characterization, gained XRD such as Fig. 3.It is 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
Object AFP concentration is marked 0.1 between 100ng/mL, ultraviolet-ray visible absorbing intensity is linear related to target concentration, by right
Test result carries out linear regression, and obtaining regression equation is：
Y=0.0613x+0.222 (R2=0.990) detection, is calculated and is limited to 0.029ng/mL, has higher sensitive
Degree.
【Embodiment 2】Traditional 2D ELISA are for detecting cancer markers alpha-fetoprotein (AFP) experiment
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, 300 μ L BSA solution (2%) is added, 2h is closed in 37 DEG C.
3) the AFP standard solution of various concentration is added to the ELISA Plate for being coated with antibody, gently shakes mixing, ELISA Plate adds
Upper cover or overlay film, 37 DEG C of reaction 45min.
4) liquid is discarded, washs five times, pats dry.The anti-AFP that the HRP labels of a concentration of 20 μ g/ μ L of 50 μ L are added per hole is anti-
Body, 37 DEG C of reaction 45min.
5) after incubating, liquid in hole is discarded, board-washing 5 times pats dry.TMB colour developing 50 μ L of working solution are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add 50 μ L of 2M sulfuric acid terminate liquid per hole, terminate reaction, solution colour switchs to Huang immediately by blue at this time
Color.
6) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with microplate reader, or solution is transferred in cuvette,
It is positioned in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.By shade to determinand into
Row half-quantitative detection.It is mapped to the concentration of AFP according to the absorption value at 450nm wavelength, the work of quantitative detection AFP can be obtained
Curve.
Quantitatively AFP effects are detected as shown in figure 5, the detection method range of linearity is 5-50ng/mL, by test result
Linear regression is carried out, obtaining regression equation is：Y=0.0301x+0.336 (R2=0.994) detection, is calculated to be limited to
0.92ng/mL。
The present embodiment detects AFP antigens, visualization sxemiquantitative inspection using traditional enzyme linked immunosorbent assay (2D ELISA)
Survey AFP effects are as shown in Figure 6 a, and 2D ELISA Visual retrieval AFP detection ranges are narrow, only have more in 5-50ng/mL ranges
Apparent color change can not differentiate the AFP of low concentration, reach platform, Wu Fafen for the AFP detection signals of high concentration
It distinguishes；Fig. 6 b are the visualization half-quantitative detection AFP design sketch of 1 3D ELISA of embodiment, Visual retrieval AFP detection ranges
Extensively, it can see apparent color change in the AFP concentration ranges of 0.1-100ng/mL.
Sensitivity and detection range in 3D ELISA are substantially better than 2D ELISA.It is analyzed in terms of signal, due to antibody
Inorganic salts hybridized nanometer spend it is middle loaded a large amount of capture antibody and its special three-dimensional structure, keep it anti-to the AFP of low concentration
Body also can be captured effectively；The a large amount of AFP capture antibody of its load simultaneously can also meet the capture of the AFP of high concentration.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, to reduce
Background.
【Embodiment 3】The coating of the biotinylated antibody that embodiment 1 synthesizes-inorganic salts hybridized nanometer flower is time-optimized
1) biotinylated antibody-inorganic salts hybridized nanometer flower that embodiment 1 obtains is dispersed in PBST buffer solutions
In, it is placed in the orifice plate of Streptavidin modification, after 37 DEG C are incubated and are incubated 5,10,30,60,90,120min respectively, with washing
Liquid washs five times, adds 300 μ L 2%BSA solution, closes 45min in 37 DEG C, it is inorganic must to be coated with biotinylated antibody-
The ELISA Plate (referred to as " 3D Substrate ") of salt hybridized nanometer flower, is placed in 4 DEG C and saves backup.The washing lotion is PBST
(10mM, 150mM NaCl, 0.05%Tween 20, pH 7.2).
2) 50 microlitres of 25ng/mL AFP standard solution additions biotinylated antibody-inorganic salts hybridized nanometer is coated with to spend
ELISA Plate, gently shake mixing, ELISA Plate is plus lid or overlay film, 37 DEG C of reaction 45min.
3) liquid is discarded, washs five times, pats dry.The anti-AFP that the HRP labels of a concentration of 20 μ g/ μ L of 50 μ L are added per hole is anti-
Body, 37 DEG C of reaction 45min.
4) after incubating, liquid in hole is discarded, board-washing 5 times pats dry.TMB colour developing 50 μ L of working solution are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add 50 μ L of sulfuric acid terminate liquid per hole, terminate reaction, solution colour switchs to yellow immediately by blue at this time.
5) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with microplate reader, 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 of biotinylated antibody-inorganic salts hybridized nanometer flower and Streptavidin modification
Soon, being incubated 5min has more apparent detection signal, and when incubation time is more than 60min, detection signal reaches platform.In order to protect
It demonstrate,proves higher detection signal while realizing the purpose quickly detected again, in this experiment, biotinylated antibody-inorganic salts hybridized nanometer
The incubation time of flower and the ELISA Plate of Streptavidin modification is selected as 60min.
【Embodiment 4】Verify reliability and accuracy of this method in actual sample detection
The AFP standard items of various concentration 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 measures between 93.0% to 109.3%
As a result it can coincide well with spiked levels, illustrate that this method has preferable accuracy.
1 recovery of standard addition of table
【Embodiment 5】Three-dimensional enzyme linked immunosorbent assay (3D is spent based on biotinylated antibody-inorganic salts hybridized nanometer
ELISA antibody) and in traditional 2D ELISA is respectively captured to compare in different incubation time experiment effects
One, 3D ELISA
1) copper-bath is added in the phosphate buffer solution containing biotinylated antibody, is placed at room temperature for 18h, obtains
Biotinylated antibody-inorganic salts hybridized nanometer flower；
The phosphate buffer solution a concentration of 0.1M, pH 7.4；A concentration of 120mM of the copper-bath；
The antibody is the sheep anti mouse secondary antibody of biotin modification, a concentration of 0.016mg/mL.
2) said mixture is centrifuged into obtain precipitation, be washed with deionized 3 times, the antibody after must washing-inorganic salts hydridization
Nano flower compound；Rotating speed when centrifuging every time is 10000r/min, and centrifugation time is 10 min.
3) above-mentioned biotinylated antibody-inorganic salts hybridized nanometer flower is dispersed in PBST buffer solutions, is placed in chain
In the orifice plate of mould Avidin modification, after 37 DEG C are incubated 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) mouse source antibody (100ng/mL) addition of 50 μ L HRP labels is coated with biotinylated antibody-inorganic salts hydridization
The ELISA Plate of nano flower gently shakes mixing, and ELISA Plate is plus lid or overlay film, and 37 DEG C of reactions, control time is respectively 5,30,
60,90,120min.
5) after incubating, liquid in hole is discarded, board-washing 5 times pats dry.TMB colour developing 50 μ L of working solution are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add 50 μ L of sulfuric acid terminate liquid per hole, terminate reaction, solution colour switchs to yellow immediately by blue at this time.
6) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with microplate reader, or solution is transferred to cuvette, is put
It is placed in uv-visible absorption spectra instrument, surveys its absorption value at 450nm wavelength.
Two, traditional 2D ELISA
1) secondary antibody (20 μ g/mL) of the sheep anti mouse of 50 μ L biotin modifications is added dissolved in PBST buffer solutions, is placed in
In the orifice plate of Streptavidin modification, after 37 DEG C are incubated 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) of 50 μ L HRP labels is added to the enzyme mark for the antibody for being coated with biotin modification
Plate gently shakes mixing, and ELISA Plate is plus lid or overlay film, and 37 DEG C of reactions, control time is respectively 5,30,60,90,120min.
3) after incubating, liquid in hole is discarded, board-washing 5 times pats dry.TMB colour developing 50 μ L of working solution are added per hole, 37 DEG C are kept away
Light colour developing 10min.Sequentially add 50 μ L of sulfuric acid terminate liquid per hole, terminate reaction, solution colour switchs to yellow immediately by blue at this time.
4) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with microplate reader, 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, captures energy
Power gradually increases, and the capture ability of 3D substrate 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) stores under room temperature, 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, the repeatedly step 3) -6 in embodiment 5 " one, 3D ELISA " respectively) or " two, traditional 2D
1 in ELISA ") it is -4), 37 DEG C of 45min with reacting for the mouse source antibody of HRP labels.
Experimental result as shown in figure 9, the storage stability for the antibody being fixed on during hybridized nanometer is spent compared with free antibodies
It is substantially improved, relative activity is still maintained at 90% or more within one week.
【Embodiment 7】Three-dimensional enzyme linked immunosorbent assay (3D is spent based on biotinylated antibody-inorganic salts hybridized nanometer
ELISA in), various concentration biotinylated antibody is coated with experiment effect test
1) copper-bath is added in the phosphate buffer solution containing various concentration biotinylated antibody, is placed at room temperature for
18h obtains antibody-inorganic salts hybridized nanometer flower；
The phosphate buffer solution a concentration of 0.1M, pH 7.4；A concentration of 120mM of the copper-bath；
The 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 into obtain precipitation, be washed with deionized 3 times, biotinylated antibody-nothing after must washing
Machine salt hybridized nanometer spends compound；Rotating speed when centrifuging every time is 10000r/min, centrifugation time 10min.
3) above-mentioned biotinylated antibody-inorganic salts hybridized nanometer flower is dispersed in PBST buffer solutions, is placed in chain
In the orifice plate of mould Avidin modification, after 37 DEG C are incubated 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.The washing lotion be PBST (10mM,
150mM NaCl, 0.05%Tween 20, pH 7.2).
4) mouse source antibody (100ng/mL) addition of 50 μ L HRP labels is coated with biotinylated antibody-inorganic salts hydridization
The ELISA Plate of nano flower gently shakes mixing, and lid or overlay film, 37 DEG C of 45 min of reaction are added on ELISA Plate.
5) optical density (OD values) in each hole is sequentially measured in 450nm wavelength with microplate reader, 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 of nano flower enhances.
【Embodiment 8】Biotinylated antibody-inorganic salts hybridized nanometer of the present invention spends the fixed efficiency of middle antibody to calculate
In the BSA protein standards solution to the test tube of respective markers for respectively taking 50 μ L various concentrations.It is bright that 50 μ L coomassies are added dropwise
It in blue enhanced reagent to every pipe and mixes well, is placed in black out at room temperature and is incubated 10min.Suction of the determination sample at 595nm
Receipts value (A595), and map to the concentration of BSA, draw standard curve, such as Figure 11.Go out unknown sample with this standard curve interpretation again
Albumen 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 object element modification, detailed process are placed at room temperature for 18h referring to embodiment 1, obtain biotinylated antibody-nothing
Machine salt hybridized nanometer is spent；Centrifugation, takes supernatant liquor to be placed in sample to be tested pipe.Take 50 μ L samples to be tested that isometric coomassie is added
The enhanced reagent of brilliant blue, mixes well, and measures absorption value of the sample to be tested 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 sample to be tested at 595nm is 0.013.Bringing standard curve into, to obtain albumen in sample to be tested a concentration of
10.8μg/mL。
It is a concentration of that albumen is added before reaction：
After the completion of reaction, the albumen of supernatant liquor is a concentration of：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, special
Sign is, by biotinylated antigen or antibody, it is molten that the phosphoric acid buffer that pH value is 6.8 ~ 7.4 is added to together with copper-bath
In liquid, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound, the ELISA Plate with Streptavidin modification are obtained
It is incubated at 37 DEG C, will be incubated has the ELISA Plate of biotinylated antigen or antibody-inorganic salts hybridized nanometer flower for sample to be tested
Enzyme linked immunosorbent assay (ELISA).
2. according to the method described in claim 1, it is characterized in that, specifically comprising the 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 are added, rear room temperature is shaken up and puts
18 h are set, biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound are obtained；
（b）It centrifuges gained biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound to obtain precipitation, it is washed with water 2 ~
4 times, biotinylated antigen or antibody-inorganic salts hybridized nanometer after must washing spend compound；
（c）By after washing biotinylated antigen or antibody-inorganic salts hybridized nanometer flower compound be dispersed in PBST bufferings
It in solution, is placed in the orifice plate of Streptavidin modification, after 37 DEG C are incubated 30 ~ 120 min, with wash liquid five times, then adds
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；The washing lotion be PBST buffer solutions, wherein a concentration of 10mM of phosphate buffer solution,
A concentration of 0.05%, the pH 7.2 of a concentration of 150mM of NaCl, Tween 20；
（d）Sample to be tested is added in the ELISA Plate for being coated with biotinylated antigen or antibody-inorganic salts hybridized nanometer flower, gently
Mixing is shaken, ELISA Plate is plus lid or overlay film, 37 DEG C of 45 min of reaction；
（e）Liquid is discarded supernatant, PBST buffer solutions are washed five times, patted dry, and the anti-object that 50 μ L HRP labels are added per hole is anti-
Body, 37 DEG C of 45 min of reaction；
（f）After incubation, discard liquid in hole, board-washing 5 times pats dry, and is added 50 μ L of TMB colour developing work nights per hole, 37 DEG C be protected from light it is aobvious
10 min of color sequentially adds 50 μ L of sulfuric acid terminate liquid per hole, terminates reaction, and solution colour switchs to yellow immediately by blue at this time；
（g）The optical density in each hole is sequentially measured in 450 nm wavelength with microplate reader（OD values）, or solution is transferred to cuvette, it places
In uv-visible absorption spectra instrument, its absorption value at 450 nm wavelength is surveyed, it can be right by shade or absorption intensity
Determinand carries out visualization half-quantitative detection or quantitative determination.
3. according to the method described in claim 2, it is characterized in that, step（a）The phosphate buffer solution a concentration of 0.1
M, a concentration of 10 ~ 200 mM of the copper-bath, the antigen or antibody of the biotin modification are repaiied for biotin
The AFP AFP monoclonal antibody of decorations, concentration range are 0.001 ~ 0.5 mg/mL；Step（b）Rotating speed when centrifugation every time
For 10000 r/min, centrifugation time is 10 min；Step（e）The anti-object antibody is AntiAFP antibody, a concentration of 20 μ
g/μL；A concentration of 2M H of sulfuric acid terminate liquid in step (f)2SO4。
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