CN109613245A - A method of detection albumen-allinase conjugate targeting - Google Patents
A method of detection albumen-allinase conjugate targeting Download PDFInfo
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- CN109613245A CN109613245A CN201910020677.4A CN201910020677A CN109613245A CN 109613245 A CN109613245 A CN 109613245A CN 201910020677 A CN201910020677 A CN 201910020677A CN 109613245 A CN109613245 A CN 109613245A
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Abstract
The present invention provides a kind of methods for detecting albumen-allinase conjugate targeting, belong to immunochromatography technique field, it the described method comprises the following steps: protein specific antibody is fixed on cellulose membrane, protein antibodies capture has albumen-allinase conjugate of fluorescence, last test strip fluorescence intensity after chromatographing；When maximum fluorescence intensity >=350, illustrate that albumen-allinase conjugate has targeting；As maximum fluorescence intensity < 350, illustrate that albumen-allinase conjugate does not have targeting.Method of the invention detects albumen-allinase conjugate targeting using immunochromatography technique, has time-consuming short, at low cost and simple operation and other advantages.
Description
Technical field
The present invention relates to immunochromatography technique field more particularly to a kind of detection albumen-allinase conjugate targetings
Method.
Background technique
Antibody coupling drug (Antibody-Drug Conjugate, ADC) is realized as a kind of novel biological missile
The combination among the strong ones of monoclonal antibody targeting and small-molecule drug cytotoxicity, has become neoplasm targeted therapy neck with fastest developing speed
One of domain.
Garlic is the underground bulb of perennial Liliaceae allium garlic (Allium sativum L.), is with a long history
Medical and edible dual purpose plant.Garlic chemical component containing there are many, and organic compounds containing sulfur is its important active material, it is main to wrap
Include alliin and allicin.Allicin is that one generated after garlic is pulverized contains oxysulfide, alliin and allinase (garlic
Propylhomoserin lyases, Alliinase) respectively in the different parts of Garlic cell, after crushed, alliin meets garlic with allinase, fastly
Speed reaction generates allicin.In recent years, many scholars at home and abroad are dedicated to the research of garlic, it was demonstrated that garlic has various
Bioactivity.Wherein allicin plays an important role in the antibacterial activity of garlic, the anti-tumor activity of allicin, machine
Reason is to influence the cell cycle, inducing apoptosis of tumour cell has enhanced sensitivity work to anticancer medical instrument by inhibiting the proliferation of tumour cell
With etc., it induces the apoptosis of tumour cell and inhibits or kill tumour cell.But since allicin is unstable, patent medicine is difficult, institute
It is combined with existing research using alliin and allinase, in tumour cell original position release allicin, so that it is thin to generate killing tumour
The effect of born of the same parents.
For antibody drug conjugates, internal animal experiment is generally used, carries out modeling, living imaging skill is used after administration
Art studies the targeting and activity of conjugate the research in terms of animal tissue's progress cellular elements, this process is not only
Time-consuming, and at high cost, used expensive equipment, and there are animal experiment ethnics Problems；Test cell line sieves in vitro
When selecting antibody drug conjugates, height is required to experimental enviroment, it is complicated for operation and at high cost.So finding a kind of simple and fast method
Conjugate targeting primarily determine very necessary.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for detecting albumen-allinase conjugate targeting, and this method can
Quickly detection albumen-allinase conjugate targeting.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of methods for detecting albumen-allinase conjugate targeting, comprising the following steps:
1) specific antibody of albumen in albumen-allinase conjugate is fixed on nitrocellulose filter, obtains immune layer
Analyse test strips；
2) it is loaded, carries out on the step 1) immuno-chromatographic test paper strip using albumen-allinase conjugate as sample to be tested
Immunochromatography measures test strips fluorescence intensity, when maximum fluorescence intensity >=350, illustrates that albumen-allinase conjugate has targeting
Property；As maximum fluorescence intensity < 350, illustrate that albumen-allinase conjugate does not have targeting.
Preferably, the albumen is bovine serum albumin(BSA).
Preferably, the albumen-allinase conjugate preparation method the following steps are included:
A) allinase and carbonate buffer solution are mixed, centrifugation obtains allinase supernatant；
B) fluorescein isothiocynate and carbonate buffer solution are mixed, obtains fluorescein isothiocynate solution；
C) allinase supernatant described in step a) is mixed with fluorescein isothiocynate solution described in step b), carries out garlic
The fluorescent marker of enzyme reacts, and obtains allinase fluorescent marker；
D) albumen and phosphate buffer are mixed, obtains protein solution；
E) by the step d) protein solution, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxyl
Base thiosuccimide mixing, ultrafiltration centrifugation, obtains ultrafiltration solution；
F) the step e) ultrafiltration solution is mixed with step c) the allinase fluorescent marker, carries out coupling reaction, obtains
To albumen-allinase conjugate；
The step a) and b) between without time sequencing limit.
Preferably, the reaction of fluorescent marker described in step c) is carried out in the dark.
Preferably, the temperature of the reaction of fluorescent marker described in step c) is 1~5 DEG C；The time of the fluorescent marker reaction
For 10~14h.
Preferably, the volume ratio of allinase supernatant described in step c) and fluorescein isothiocynate solution be 3~5:0.5~
1.5。
Preferably, protein solution described in step e), 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and
The ratio of fluorescein isothiocynate solution is 0.5~1.5mL:190~210mg:14~16mg.
Preferably, the volume ratio of ultrafiltration solution described in step f) and allinase fluorescent marker be 0.5~1.5:0.5~
1.5。
Preferably, the molecular cut off of the centrifugation of ultrafiltration described in step e) is 9~11KDa；The revolving speed of the ultrafiltration centrifugation
Time for 4000~6000rpm, the ultrafiltration centrifugation is 15~25min.
Preferably, the temperature of coupling reaction described in step f) is 16~25 DEG C；The time of the coupling reaction be 2~
4h。
Beneficial effects of the present invention: the present invention provides a kind of methods for detecting albumen-allinase conjugate targeting, should
Protein specific antibody is fixed on cellulose membrane by method, and protein antibodies capture has albumen-allinase of fluorescence after chromatographing
Conjugate, last test strip fluorescence intensity；When maximum fluorescence intensity >=350, illustrate that albumen-allinase conjugate has target
Tropism；As maximum fluorescence intensity < 350, illustrate that albumen-allinase conjugate does not have targeting.Method of the invention, which utilizes, to be exempted from
Epidemic disease chromatographic technique detects albumen-allinase conjugate targeting, has time-consuming short, at low cost and easy to operate etc. excellent
Point.
Detailed description of the invention:
Fig. 1 is fluorescence immune chromatography detection schematic diagram, wherein being 1. fluorescein isothiocynate, is 2. albumen, is 3. non-mesh
Object is marked, is 4. allinase fluorescent marker, is 5. albumen-allinase conjugate, is 6. the specific antibody of albumen；
Fig. 2 is the fluorescence detection schematic diagram of test strips, wherein 1 indicates unstressed configuration bottom plate, 2 indicate nitrocellulose filter, 3 tables
Show light source, 4 indicate detector, and 5 indicate fluorescence cuvette；
Fig. 3 is the fluorescence spectrum scanning figure of fluorescein isothiocynate in embodiment 1；
Fig. 4 is the fluorescence spectrum scanning figure of immuno-chromatographic test paper strip in embodiment 1, wherein 1 indicates conjugate, 2 indicate different
Thiocyanic acid fluorescein；
Fig. 5 is the fluorescence spectrum scanning figure of the hollow white board of embodiment 1 and blank nitrocellulose filter item, wherein 1 indicates
Blank unstressed configuration cardboard, 2 indicate blank nitrocellulose filter.
Specific embodiment
The present invention provides a kind of methods for detecting albumen-allinase conjugate targeting, comprising the following steps:
1) specific antibody of albumen in albumen-allinase conjugate is fixed on nitrocellulose filter, obtains immune layer
Analyse test strips；
2) it is loaded, carries out on the step 1) immuno-chromatographic test paper strip using albumen-allinase conjugate as sample to be tested
Immunochromatography measures test strips fluorescence intensity, when maximum fluorescence intensity >=350, illustrates that albumen-allinase conjugate has targeting
Property；As maximum fluorescence intensity < 350, illustrate that albumen-allinase conjugate does not have targeting.
Testing principle of the invention: the sample to be tested with fluorescence adds in the sample pad of test strips, and solution is through sample pad
Capillarity, chromatography is to nitrocellulose filter.Object makes to fix in conjunction with antibody on nitrocellulose filter in sample to be tested
There is the position on the tunica fibrosa of antibody that there is fluorescence, fluorescence immune chromatography detection schematic diagram of the invention is referring to Fig. 1, wherein being 1.
2. fluorescein isothiocynate is albumen, be 3. non-targeted object, is 4. allinase fluorescent marker, is 5. albumen-allinase conjugate,
6. being the specific antibody of albumen.
In the present invention, the specific antibody of albumen in albumen-allinase conjugate is fixed on nitrocellulose filter, is obtained
To immuno-chromatographic test paper strip；The present invention is not particularly limited the sample pad and water absorption pad of immuno-chromatographic test paper strip, and this field is normal
Rule setting；The albumen is preferably bovine serum albumin(BSA)；The bovine serum albumin(BSA) is purchased from Shanghai source leaf biotechnology
Co., Ltd, lot number L16M6S2；The specific antibody of the bovine serum albumin(BSA) is bovine serum albumin(BSA) antibody；The ox blood
Pure protein antibodies are purchased from Beijing Bo Aosen Bioisystech Co., Ltd, lot number AG03031588.
In the present invention, the mode of the fixation preferably sprays the scribing line of fixed or point sample and fixes；The specificity of the albumen
The concentration of antibody is preferably 1mg/mL；The fixed amount of the specific antibody of the albumen is preferably 0.5 μ L.
In the present invention, add on the immuno-chromatographic test paper strip using the albumen-allinase conjugate as sample to be tested
Sample carries out immunochromatography, measures test strips fluorescence intensity, when maximum fluorescence intensity >=350, illustrates albumen-allinase conjugate
With targeting；As maximum fluorescence intensity < 350, illustrate that albumen-allinase conjugate does not have targeting；Wherein 350 be empty
White fluorescein isothiocynate maximum fluorescence intensity.
In the present invention, the tool of the point sample is preferably liquid-transfering gun；The albumen-allinase conjugate point sample amount is preferably
60μL；The chromatographic solution is albumen-allinase conjugate solution.
The present invention after carrying out immunochromatography, preferably further include washed away using carbonate buffer solution it is extra more in test strips
The remaining free fluorescein isothiocynate not synthesized；The pH of the carbonate buffer solution is preferably 9~10, and more preferably 9.5；This hair
In bright specific implementation process, the carbonate buffer solution preferably includes sodium bicarbonate, sodium carbonate and water；The bicarbonate
The ratio of sodium, sodium carbonate and water is preferably 3.5~3.9g:0.4~0.8g:500~700mL, more preferably 3.7g:0.6g:
600mL。
The present invention measures test strips fluorescence intensity after carrying out immunochromatography, and the measurement test strips fluorescence intensity is set
Standby preferably sepectrophotofluorometer；The sepectrophotofluorometer is preferably LS-55 sepectrophotofluorometer；The LS-55
Sepectrophotofluorometer is purchased from U.S. Perkin Elmer company.
The method of heretofore described measurement test strips fluorescence intensity, the specific steps are as follows: take the cardboard without fluorescence
It is cut into and is suitable for fluorescence cuvette size, be placed in fluorescence cuvette in diagonal line, then by the nitrocellulose test paper after chromatography
Item is fixed on unstressed configuration cardboard, carries out fluorescence spectrum sweep measuring test strips fluorescence to test strips using sepectrophotofluorometer
Intensity illustrates albumen-allinase coupling when maximum fluorescence intensity >=350 (blank fluorescein isothiocynate maximum fluorescence intensity)
Object has targeting；When maximum fluorescence intensity < 350 (blank fluorescein isothiocynate maximum fluorescence intensity), illustrate albumen-
Allinase conjugate does not have targeting.In the present invention, the excitation wavelength of the sepectrophotofluorometer is preferably 490nm；It is described
The launch wavelength of sepectrophotofluorometer is preferably 505~600nm；The slit width of the launch wavelength be preferably (2.5,
2.5)；Referring to fig. 2, wherein 1 indicates unstressed configuration bottom plate, 2 indicate nitrocellulose filter to the fluorescence detection schematic diagram of the test strips,
3 indicate light source, and 4 indicate detector, and 5 indicate fluorescence cuvette.
Heretofore described albumen-allinase conjugate preparation method, comprising the following steps:
A) allinase and carbonate buffer solution are mixed, centrifugation obtains allinase supernatant；
B) fluorescein isothiocynate and carbonate buffer solution are mixed, obtains fluorescein isothiocynate solution；
C) allinase supernatant described in step a) is mixed with fluorescein isothiocynate solution described in step b), carries out garlic
The fluorescent marker of enzyme reacts, and obtains allinase fluorescent marker；
D) albumen and phosphate buffer are mixed, obtains protein solution；
E) by the step d) protein solution, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxyl
Base thiosuccimide mixing, ultrafiltration centrifugation, obtains ultrafiltration solution；
F) the step e) ultrafiltration solution is mixed with step c) the allinase fluorescent marker, carries out coupling reaction, obtains
To albumen-allinase conjugate；
The step a) and b) between without time sequencing limit.
In the present invention, allinase and carbonate buffer solution are mixed, centrifugation obtains allinase supernatant；The carbonate buffer
The pH of liquid is preferably 9~10, and more preferably 9.5；The mass volume ratio of the allinase and carbonate is preferably 30~50mg:0.5
~1.5mL, more preferably 40mg:1L；The mixed temperature is preferably 18~26 DEG C, and preferably 20 DEG C；The present invention is to mixing
Time be not particularly limited, be subject to be uniformly mixed.In specific implementation process of the invention, the allinase is purchased from Xinjiang angstrom
Le Xin pharmaceutcal corporation, Ltd；The carbonate buffer solution preferably includes sodium bicarbonate, sodium carbonate and water；The sodium bicarbonate,
The ratio of sodium carbonate and water is preferably 3.5~3.9g:0.4~0.8g:500~700mL, more preferably 3.7g:0.6g:600mL；
The revolving speed of the centrifugation preferably 4000~6000rpm, more preferably 5000rpm；The time of the centrifugation is preferably 8~12min,
More preferably 10min.
In the present invention, fluorescein isothiocynate and carbonate buffer solution are mixed, obtains fluorescein isothiocynate solution；
The pH of the carbonate buffer solution is preferably 9~10, and more preferably 9.5；The quality of the fluorescein isothiocynate and carbonate
Volume ratio is preferably 0.5~1.5mg:0.5~1.5mL, more preferably 1mg:1L；The mixed temperature is preferably 18~26
DEG C, preferably 20 DEG C；The present invention is not particularly limited the mixed time, is subject to and is uniformly mixed.Specific implementation of the invention
In the process, the fluorescein isothiocynate is purchased from Suo Laibao Biotechnology Co., Ltd, lot number No.915C011；The carbonic acid
Salt buffer preferably includes sodium bicarbonate, sodium carbonate and water；The ratio of the sodium bicarbonate, sodium carbonate and water is preferably 3.5
~3.9g:0.4~0.8g:500~700mL, more preferably 3.7g:0.6g:600mL.
The present invention after obtaining allinase supernatant and fluorescein isothiocynate solution, by the allinase supernatant with it is described different
The mixing of thiocyanic acid luciferin solution carries out the fluorescent marker reaction of allinase, obtains allinase fluorescent marker；The allinase supernatant
Volume ratio with fluorescein isothiocynate solution is preferably 3~5:0.5~1.5, more preferably 4:1；The mixed mode is excellent
It is selected as being stirred, more preferably magnetic agitation mixes；The revolving speed of the magnetic agitation mixing is preferably 100~150rpm, more
Preferably 120rpm；The fluorescent marker reaction is preferably carried out in the dark；The temperature of the fluorescent marker reaction is preferably 1~5
DEG C, more preferably 4 DEG C；The time of the fluorescent marker reaction is preferably 10~14h, more preferably 12h.
It in the present invention, is that the time of fluorescent marker reaction is 10~14h using the reason of magnetic agitation, when reaction
Between it is longer, and sample total amount is smaller, so carrying out mixing reaction using magnetic agitation.
In the present invention, the reason of temperature setting of fluorescent marker reaction is 1~5 DEG C, is, in fluorescent marker reaction,
The reaction temperature that isothiocyanic acid calculates fluorescein is 1~5 DEG C, and under conditions of 1~5 DEG C, allinase can preferably keep enzyme activity
Power.
It preferably further include that fluorescence spectrum scanning is carried out to fluorescein isothiocynate in the present invention；The purpose of the step is
Determine the individual scanning curve of fluorescein isothiocynate.
In specific implementation process of the invention, take fluorescein isothiocynate solution point sample on chromatograph test strip, utilization is glimmering
Light spectrophotometer carries out the fluorescence spectrum scanning of fluorescein isothiocynate；The solvent of the fluorescein isothiocynate solution is preferred
For carbonate buffer solution；The concentration of the fluorescein isothiocynate solution is preferably 9~11 μ g/mL, more preferably 10 μ g/mL；
The point sample amount of the fluorescein isothiocynate solution is preferably 4~6 μ L, more preferably 5 μ L；The sepectrophotofluorometer swashs
Sending out wavelength is preferably 490nm；The launch wavelength of the sepectrophotofluorometer is preferably 505~600nm, more preferably 525nm；
The slit width is preferably 10nm, 10nm；The sepectrophotofluorometer is preferably LS-55 sepectrophotofluorometer；It is described
LS-55 sepectrophotofluorometer is purchased from U.S. Perkin Elmer company.
In the present invention, albumen and phosphate buffer are mixed, obtains protein solution；The pH of the phosphate buffer
Value preferably 5~7, more preferably 6；The mass volume ratio of the albumen and phosphate buffer be preferably 3~5mg:0.5~
1.5mL, more preferably 4mg:1mL；The mixed temperature is preferably 18~26 DEG C, and preferably 20 DEG C；The present invention is to mixing
Time is not particularly limited, and is subject to and is uniformly mixed.In specific implementation process of the invention, the phosphate buffer is preferred
Including disodium hydrogen phosphate aqueous solution and biphosphate sodium water solution；The concentration of the disodium hydrogen phosphate aqueous solution is preferably 0.1~
0.3mol/L, more preferably 0.2mol/L；The concentration of the disodium hydrogen phosphate aqueous solution is preferably 0.1~0.3mol/L, more excellent
It is selected as 0.2mol/L；The volume ratio of the disodium hydrogen phosphate aqueous solution and biphosphate sodium water solution is preferably 12.1~12.5:
87.5~87.9, more preferably 12.3:87.7.It is a kind of normal that it is bovine serum albumin that the present invention, which uses the reason of bovine serum albumin(BSA),
High molecular weight protein substance, it is representative and cheap.
In the present invention, bovine serum albumin(BSA)-allinase conjugate preparation method is according to the carboxyl on bovine serum albumin(BSA)
Synthetic reaction is carried out with the amino on allinase, the polypeptide chain that protein is made of amino acid has by what tortuous folding was formed
The substance of certain space structure.Amino acid is the organic compound containing basic amine group and acidic carboxypolymer, so, this reaction is also fitted
For other protein.
It is after the present invention obtains protein solution, the protein solution, 1- (3- dimethylamino-propyl) -3- ethyl carbon two is sub-
Amine hydrochlorate and the mixing of N- hydroxy thiosuccinimide, ultrafiltration centrifugation obtain ultrafiltration solution.
In the present invention, the protein solution, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and different sulphur cyanogen
The ratio of sour luciferin solution is preferably 0.5~1.5mL:190~210mg:14~16mg, more preferably 1mL:200mg:
15mg；1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride is preferably purchased from Shanghai source leaf biotechnology
Co., Ltd, lot number Z20J8N40263, specification 25g；The N- hydroxy thiosuccinimide is preferably purchased from Beijing Suo Lai
Precious Science and Technology Ltd., lot number No.608A031, specification 1g.
In the present invention, 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxy amber
Acid imide is provided commonly for the carboxyl on activated protein, forms reactive intermediate；Reactive intermediate is carried out with the amino on allinase again
Reaction.
In the present invention, the mixed mode is preferably stirred, and more preferably magnetic agitation mixes；The magnetic force stirs
The revolving speed for mixing mixing is preferably 100~150rpm, more preferably 120rpm；The time of magnetic agitation mixing is preferably 30~
40min, more preferably 35min；The temperature of the magnetic agitation mixing is preferably 18~26 DEG C, and more preferably 20 DEG C；It is described super
The molecular cut off of filter centrifugation is preferably 9~11KDa, more preferably 10KDa；The revolving speed of ultrafiltration centrifugation is preferably 4000~
The time of 6000rpm, more preferably 5000rpm, the ultrafiltration centrifugation are preferably 15~25min, more preferably 20min.
In the present invention, the effect of the ultrafiltration centrifugation is to remove unreacted small-molecule substance.
The present invention is after obtaining ultrafiltration solution and allinase fluorescent marker, by the ultrafiltration solution and the allinase fluorescence mark
Remember object mixing, carries out coupling reaction, obtain albumen-allinase conjugate；The volume of the ultrafiltration solution and allinase fluorescent marker
Than being preferably 0.5~1.5:0.5~1.5, more preferably 1:1；The mixed mode is preferably stirred, more preferably magnetic
Power is stirred；The revolving speed of the magnetic agitation mixing is preferably 100~150rpm, more preferably 120rpm；The coupling is anti-
The temperature answered is preferably 16~25 DEG C, and more preferably 20 DEG C；The time of the coupling reaction is preferably 2~4h, more preferably 3h.
Below with reference to embodiment to a kind of method progress for detecting albumen-allinase conjugate targeting provided by the invention
Detailed description, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
1. instrument and reagent
1) instrument LS-55 sepectrophotofluorometer (Perkin Elmer company, the U.S.)
2) reagent allinase Xinjiang Ailexin Pharmacy Co., Ltd. provides；Bovine serum albumin(BSA) Shanghai source leaf biotechnology is limited
Company provides, lot number L16M6S2；Fluorescein isothiocynate Suo Laibao Biotechnology Co., Ltd provides, lot number No.915C011；
Bovine serum albumin(BSA) antibody Beijing Bo Aosen Bioisystech Co., Ltd provides, lot number AG03031588.
2. solution is prepared:
1) 9.5 carbonate buffer solution of pH: weighing sodium bicarbonate 3.70g, and sodium carbonate 0.60g is dissolved in 600mL distilled water i.e.
?.
2) 6.0 phosphate buffer of pH: 0.2mol/L disodium hydrogen phosphate 12.3mL, 0.2mol/L sodium dihydrogen phosphate
87.7mL mixing to obtain the final product.
3. the fluorescent marker of allinase
Allinase is configured to 40mg/ml allinase solution through the carbonate buffer solution dissolution of pH 9.5, be centrifuged (5000rpm,
Supernatant solution and the fluorescein isothiocynate (1mg/ml) of the carbonate buffer liquor dissolution through pH 9.5 10min) is taken to mix
The volume ratio of reaction, allinase supernatant solution and fluorescein isothiocynate is 4:1.Magnetic agitation reaction is protected from light at 4 DEG C
(120rpm/min, 12h) obtains allinase fluorescent marker.
4. fluorescein isothiocynate fluorescence spectrum scans
Take the 5 μ l point sample of fluorescein isothiocynate of 10 μ g/mL on chromatograph test strip, excitation wavelength 490nm, launch wavelength
505~600nm, slit width 10nm, 10nm are taken, the fluorescence spectrum scanning of fluorescein isothiocynate, isosulfocyanic acid fluorescence are carried out
The fluorescence spectrum scanning figure of element is referring to Fig. 3, and scanning result is shown using 490nm as excitation wavelength, in 505-600nm wave-length coverage
It is scanned, maximum emission wavelength 528nm.
5. bovine serum albumin-allinase conjugate synthesis
Bovine serum albumin is configured to the bovine serum albumen solution of 4mg/ml through the phosphate buffer dissolution of pH 6.0, respectively
200mg 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDCHCl), 15mg N- hydroxy amber is added
Amber acid imide (Sulfo-NHS) magnetic agitation (room temperature, 120rpm/min, 35min) ultrafiltration centrifugation (10KDa, 5000rpm,
20min), it adds the allinase solution 1:1 (v:v) with fluorescence to mix, triethylamine adjusting pH 8-9, magnetic agitation (room temperature,
120rpm/min, 3h).
6. bovine serum albumin-allinase conjugate targeting verifying
Bovine serum albumin antibody is fixed on nitrocellulose filter, the conjugate point sample with fluorescence is in the sample of cellulose membrane
On product pad, after chromatography immune response, the carbonate buffer solution of pH 9.5 washes away extra fluorescein isothiocynate in test strips, will
Test strips are placed in fluorescence cuvette with excitation wavelength 490nm in sepectrophotofluorometer, and launch wavelength takes 505~600nm,
Slit width (2.5nm, 2.5nm) carries out fluorescence spectrum scanning to test strips.As a result referring to fig. 4, wherein 1 indicate conjugate, 2
Indicate fluorescein isothiocynate.
It takes the cardboard without fluorescence to be cut into and is suitable for fluorescence cuvette size, then by nitrocellulose filter from test strips
It removes, sticks on cardboard, with excitation wavelength 490nm, launch wavelength takes 505~600nm, and slit width 2.5,2.5 carries out fluorescence
Spectral scan.As a result referring to Fig. 5, wherein 1 indicates blank unstressed configuration cardboard, 2 indicate blank nitrocellulose filter, as the result is shown institute
The blank cardboard and blank nitrocellulose filter item of choosing do not have fluorescence, do not have blank to the targeting Journal of Sex Research of subsequent conjugate
Interference.
The above results show that the blank film item of fluorescein isothiocynate is washed till colourless after fluorescent scanning using buffer
Afterwards, still there is fluorescence on film item, but intensity is lower；Conjugate after immunochromatography, using buffer be washed till it is colourless after, it is different with blank
Thiocyanic acid fluorescein film item compared to after conjugate chromatographs film item have stronger fluorescence, maximum emission wavelength be 525nm with
The maximum emission wavelength of the fluorescence spectrum scanning figure of fluorescein isothiocynate is identical.In conclusion allinase-bovine serum albumin coupling
Object can be in conjunction with bovine serum albumin antibody specificity, and has fluorescence.So allinase-bovine serum albumin conjugate is to ox blood
Albumin antibody has targeting.
As seen from the above embodiment, the present invention uses immunochromatography technique can be with preliminary screening antibody-drug conjugates
Targeting.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method for detecting albumen-allinase conjugate targeting, comprising the following steps:
1) specific antibody of albumen in albumen-allinase conjugate is fixed on nitrocellulose filter, obtains immunity-chromatography test
Paper slip；
2) it is loaded, is immunized on the step 1) immuno-chromatographic test paper strip using albumen-allinase conjugate as sample to be tested
Chromatography measures test strips fluorescence intensity, when maximum fluorescence intensity >=350, illustrates that albumen-allinase conjugate has targeting；
As fluorescence intensity < 350, illustrate that albumen-allinase conjugate does not have targeting.
2. detection method according to claim 1, which is characterized in that the albumen is bovine serum albumin(BSA).
3. detection method according to claim 1 or 2, which is characterized in that the albumen-allinase conjugate preparation side
Method, comprising the following steps:
A) allinase and carbonate buffer solution are mixed, centrifugation obtains allinase supernatant；
B) fluorescein isothiocynate and carbonate buffer solution are mixed, obtains fluorescein isothiocynate solution；
C) allinase supernatant described in step a) is mixed with fluorescein isothiocynate solution described in step b), carries out allinase
Fluorescent marker reaction, obtains allinase fluorescent marker；
D) albumen and phosphate buffer are mixed, obtains protein solution；
E) by the step d) protein solution, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxyl sulphur
It is mixed for succinimide, ultrafiltration centrifugation obtains ultrafiltration solution；
F) the step e) ultrafiltration solution is mixed with step c) the allinase fluorescent marker, carries out coupling reaction, obtains egg
White-allinase conjugate；
The step a) and b) between without time sequencing limit.
4. detection method according to claim 3, which is characterized in that fluorescent marker described in step c) reaction be protected from light into
Row.
5. according to the method described in claim 4, it is characterized in that, the temperature of the reaction of fluorescent marker described in step c) is 1~5
℃；The time of the fluorescent marker reaction is 10~14h.
6. according to the method described in claim 5, it is characterized in that, allinase supernatant and isosulfocyanic acid fluorescence described in step c)
The volume ratio of plain solution is 3~5:0.5~1.5.
7. according to the method described in claim 3, it is characterized in that, protein solution described in step e), 1- (3- dimethylamino third
Base) ratio of -3- ethyl-carbodiimide hydrochloride and fluorescein isothiocynate solution is 0.5~1.5mL:190~210mg:14
~16mg.
8. according to the method described in claim 3, it is characterized in that, ultrafiltration solution described in step f) and allinase fluorescent marker
Volume ratio be 0.5~1.5:0.5~1.5.
9. according to the method described in claim 3, it is characterized in that, the molecular cut off of the centrifugation of ultrafiltration described in step e) is 9
~11KDa；The revolving speed of the ultrafiltration centrifugation is 4000~6000rpm, and the time of the ultrafiltration centrifugation is 15~25min.
10. according to the method described in claim 9, it is characterized in that, the temperature of coupling reaction described in step f) is 16~25
℃；The time of the coupling reaction is 2~4h.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538616A (en) * | 2002-04-09 | 2009-09-23 | 唐舜荣 | Improved method for detecting target molecule through granula combination |
| CN101539573A (en) * | 2009-04-30 | 2009-09-23 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | High-flux visible chip detecting method of protein-protein interaction and detecting kit for protein-protein interaction |
| CN101537185A (en) * | 2009-04-27 | 2009-09-23 | 武汉理工大学 | Tumor cell actively targeted drug delivery system, preparation method and application thereof |
| CN101539578A (en) * | 2009-02-17 | 2009-09-23 | 李红玉 | Colloidal gold test strip for testing melamine content |
| CN201429617Y (en) * | 2009-06-10 | 2010-03-24 | 北京维德维康生物技术有限公司 | Detection card for detecting melamine |
| CN102269758A (en) * | 2010-06-04 | 2011-12-07 | 国家纳米科学中心 | Method for visual detection of antigen-antibody reaction, kit and application thereof |
| CN102798708A (en) * | 2012-08-23 | 2012-11-28 | 中国科学院长春应用化学研究所 | Method for detecting binding specificity between ligand and target and drug screening method |
| CN103439506A (en) * | 2013-08-12 | 2013-12-11 | 杭州戈登生物科技有限公司 | Rapid detection device and method for interaction of biomacromolecules |
| CN107157952A (en) * | 2017-06-30 | 2017-09-15 | 广东工业大学 | A kind of fibroin albumen nanoparticle and load medicine fibroin albumen nanoparticle |
| CN107543922A (en) * | 2016-06-27 | 2018-01-05 | 北京康华源科技发展有限公司 | A kind of centrichromatography fluorescence immunoassay detection technique and application thereof |
-
2019
- 2019-01-09 CN CN201910020677.4A patent/CN109613245B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538616A (en) * | 2002-04-09 | 2009-09-23 | 唐舜荣 | Improved method for detecting target molecule through granula combination |
| CN101539578A (en) * | 2009-02-17 | 2009-09-23 | 李红玉 | Colloidal gold test strip for testing melamine content |
| CN101537185A (en) * | 2009-04-27 | 2009-09-23 | 武汉理工大学 | Tumor cell actively targeted drug delivery system, preparation method and application thereof |
| CN101539573A (en) * | 2009-04-30 | 2009-09-23 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | High-flux visible chip detecting method of protein-protein interaction and detecting kit for protein-protein interaction |
| CN201429617Y (en) * | 2009-06-10 | 2010-03-24 | 北京维德维康生物技术有限公司 | Detection card for detecting melamine |
| CN102269758A (en) * | 2010-06-04 | 2011-12-07 | 国家纳米科学中心 | Method for visual detection of antigen-antibody reaction, kit and application thereof |
| CN102798708A (en) * | 2012-08-23 | 2012-11-28 | 中国科学院长春应用化学研究所 | Method for detecting binding specificity between ligand and target and drug screening method |
| CN103439506A (en) * | 2013-08-12 | 2013-12-11 | 杭州戈登生物科技有限公司 | Rapid detection device and method for interaction of biomacromolecules |
| CN107543922A (en) * | 2016-06-27 | 2018-01-05 | 北京康华源科技发展有限公司 | A kind of centrichromatography fluorescence immunoassay detection technique and application thereof |
| CN107157952A (en) * | 2017-06-30 | 2017-09-15 | 广东工业大学 | A kind of fibroin albumen nanoparticle and load medicine fibroin albumen nanoparticle |
Non-Patent Citations (3)
| Title |
|---|
| ELENA APPEL ET AL.: "Conjugates of daidzein-alliinase as a targeted pro-drug enzyme system against ovarian carcinoma", 《JOURNAL OF DRUG TARGETING》, vol. 19, no. 5, 31 December 2011 (2011-12-31), pages 326 - 335 * |
| MIRON T等: "Inhibition of tumor growth by a novel approach: in situ allicin generation using targeted alliinase delivery", 《MOL CANCER THER》, vol. 2, no. 12, 31 December 2003 (2003-12-31), pages 1295 - 1301, XP002619682 * |
| 刘耕陶: "《当代药理学（第二版）》", 29 February 2008, 中国协和医科大学出版社, pages: 25 - 29 * |
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