CN107192817B - A kind of quick detection kit and its detection method for being used to detect PD-L1 - Google Patents
A kind of quick detection kit and its detection method for being used to detect PD-L1 Download PDFInfo
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Abstract
The invention belongs to technical field of medical examination, and in particular to a kind of quick detection kit and its detection method for being used to detect PD L1.Include luminous substrate solution I, luminous substrate solution II, concentrated cleaning solution, PD L1 standard items, PD L1 quality-control products, antibody acridinium ester label and magnetic bead immune complex provided by the present invention for the quick detection kit for detecting PD L1.Have the advantages that detection time is short, dosage is few, the repeatability and reliability of efficient, high sensitivity, high specificity, stabilization provided by the present invention for the quick detection kit for detecting PD L1.
Description
Technical field
The invention belongs to technical field of medical examination, and in particular to a kind of quick detection kit for being used to detect PD-L1
And its detection method.
Background technology
PD-1 full name programmed deaths acceptor 1, english name are programmed death 1, are a kind of important are immunized
Suppress molecule, be CD28 superfamily members.It is the immunological regulation of target spot in antitumor, anti-infective, anti-autoimmune using PD-1
Disease and organ transplant survival etc. have important meaning.Its ligand PD-L1 can also be used as target spot, and corresponding antibody also may be used
To serve the same role.PD-L1 full name programmed death-ligand 1s, english name programmed cell death-
Ligand 1, is the first type transmembrane protein that size is 40kDa.Immune system can be to being gathered in lymph node or spleen under normal conditions
Dirty exotic antigen produces reaction, promotes the T cell hyperplasia with antigentic specificity.And programmed death acceptor 1 (PD-1) with
Programmed death-ligand 1 (PD-L1) combines, and can conduct the signal of inhibition, lowers the hyperplasia of T cell.Tumour cell is escaped
A kind of approach that T cell is destroyed is by producing PD-L1 on its surface, when the PD-1 on immunocyte T cell surface identifies PD-L1
Afterwards, it can conduct and suppress row signal, T cell cannot find tumour cell and send signal to attack to tumour cell.
Chinese patent application CN105579471A provides the separated antibody of specific binding human PD-L 1 and such anti-
The antigen-binding fragment of body, and be used to detect with reference to human PD-L 1 comprising the anti-PD-L1 antibody or binding fragment and one group
The kit of the reagent of the compound of the antibody or its antigen-binding fragment.Chinese patent application CN105274104A is disclosed
A kind of detection kit for predicting anti-PD-L1 antibody drugs drug effect, DNA is corrected including detection POLD albumen and POLE albumen
The primer and probe of the corresponding gene loci of domain of resultant fault.
Cancer includes disease in extensive range, and the seriousness of the negative effect of cancer is deep, has generally influenced doctor
Treat insurance and society.Since polytype cancer mark is the quick of malignant cell and not modulated propagation, change
One decisive problem of the kind method for cancer is the demand to early detection and diagnosis.PD-L1 is a kind of a variety of swollen
Dynamic, the marker of wide expression in oncocyte, antigen presenting cell and other immunocytes.Clinical and experimental study shows, hinders
Disconnected PD-L1 can effectively treat or suppress tumour growth.Therefore, clinically to need easy-to-use detection means to filter out swollen
Tumor tissue or cell altimeter reach the tumor patient of PD-L1, for personalized carry out blocking PD-L1 treatment provide instruct and according to
According to.And it is existing be used to detecting the kit of PD-L1 there are sensitivity is relatively low, and the shortcomings of specificity is not high.
The content of the invention
In view of the deficiencies of the prior art, it is an object of the invention to provide a kind of quick detection examination for being used to detect PD-L1
Agent box and its detection method.Quick detection kit provided by the present invention for detecting PD-L1 has that detection time is short, dosage
Less, efficiently, the repeatability and reliability of high sensitivity, high specificity, stabilization the advantages that.
The technical scheme is that：
A kind of quick detection kit for being used to detect PD-L1, the kit include luminous substrate solution I, and shine bottom
Thing solution II, concentrated cleaning solution, PD-L1 standard items, PD-L1 quality-control products, antibody acridinium ester label and magnetic bead immune complex.
The preparation method of the luminous substrate liquid I is：It is molten that the dust technology that concentration is 0.1M is configured to distilled water and nitric acid
Liquid, adds hydrogen peroxide, the concentration for making hydrogen peroxide is 0.08M.
The preparation method of the luminous substrate liquid II is：The hydrogen-oxygen that concentration is 0.2M is configured to distilled water and sodium hydroxide
Change sodium solution, add TritonX-100, the mass concentration for making TritonX-100 is 2%.
The preparation method of the concentrated cleaning solution is：It is 7.4 to pH, concentration is to be added in the phosphate buffer of 1M
Tween-20, the volume fraction for making Tween-20 are 5%.
The preparation of the PD-L1 standard items comprises the following steps：Be 1%BSA containing volume fraction, volume fraction be
The pH of 0.05% thimerosal is 7.4, and concentration is the phosphate buffer of 0.05M, and it is respectively 100pg/ that PD-L1 is configured to concentration
The titer of mL, 250pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, 3000pg/mL, 5000pg/mL, to obtain the final product.
The preparation of the PD-L1 quality-control products comprises the following steps：Be 1%BSA containing volume fraction, volume fraction be
The pH of 0.05% thimerosal is 7.4, and concentration is the phosphate buffer of 0.05M, and PD-L1 is configured to concentration is respectively
The solution of 1500pg/mL, 4000pg/mL, to obtain the final product.
The preparation of the antibody acridinium ester label comprises the following steps：
(1) PD-L1 monoclonal antibodies are taken, are 0.05mol/L with concentration, the carbonate buffer solution that pH is 9.5 adjusts PD-
The concentration of L1 monoclonal antibodies is 1mg/mL, obtains solution A；
(2) acridinium ester is taken, adding dimethylformamide makes the concentration of acridinium ester obtain solution B to 2mg/mL；
(3) step (2) resulting solution B is added in step (1) resulting solution A, acridinium ester and PD-L1 monoclonal antibodies
Molar ratio be 14~22: 1, room temperature reaction, the reaction time is 25~50min, obtains reaction solution；
(4) reaction solution obtained by step (3) is moved in the bag filter that molecular cut off is 7500~95000, is with concentration
The carbonate buffer solution dialysis 24h that 0.05mol/L, pH are 9.5, addition and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, to obtain the final product.
The preparation of the magnetic bead immune complex comprises the following steps：
The Streptavidin MagneSphere for taking 100 μ L concentration to be 10mg/mL, is diluted with 250 μ L concentrated cleaning solutions according to 1: 100
The solution arrived washs 4 times, adds 50 μ L biotinylation PD-L1 monoclonal antibodies, is placed in 37 DEG C of constant temperature gas bath shaking tables and vibrates, shake
It is 100r/min to swing speed, is taken out after 12~16min, is washed with 200 μ L concentrated cleaning solutions according to the solution that 1: 100 dilution obtains
4 times, then, it is replaced in above-mentioned constant temperature gas bath shaking table, it is 7.4 to add 1mL containing the pH that volume fraction is 2%BSA, and concentration is
The phosphate buffer of 1M carries out capping, and the capping time is 15~22min, takes out, and Magnetic Isolation, removes supernatant, uses
200 μ L concentrated cleaning solutions wash 4 times according to the solution that 1: 100 dilution obtains, and it is 7.4 to add pH, and concentration is that the phosphate of 1M delays
Fliud flushing is made into the magnetic bead immune complex of 1mg/mL, is placed in 4 DEG C and is kept in dark place, to obtain the final product；
Wherein, the preparation method of the concentrated cleaning solution is：It is 7.4 to pH, adds in the phosphate buffer that concentration is 1M
Enter Tween-20, the volume fraction for making Tween-20 is 5%.
In the preparation of above-mentioned magnetic bead immune complex, the preparation of the Streptavidin MagneSphere comprises the following steps：
A. it is 7.0 to prepare pH, and concentration is the phosphate buffer of 0.1M, and sterilizing, sterilising temp is 120 DEG C, sterilization time
For 18min, the Streptavidin of 1mg is dissolved in the above-mentioned phosphate buffers of 1mL, obtains solution of streptavidin, take 400 μ L chains
Mould avidin solution is loaded in EP pipes；
B. the chitosan magnetic microsphere for taking 5mL to modify, adds 4.5mL glutaraldehydes, room temperature concussion reaction, the reaction time is
4h, carries out Magneto separate, washes with water after removing unnecessary glutaraldehyde, and it is 7.0 that magnetic microsphere is resuspended in 5mLpH, concentration
In the phosphate buffer of 0.1M, to obtain magnetic microsphere liquid；
C. magnetic microsphere liquid obtained by step b is added in EP pipes of the step a equipped with 400 μ L solution of streptavidin, room
Warm shake culture 4h, Magneto separate, obtains solids, and it is 7.0 that obtained solid thing is dispersed in 10mLpH, and concentration is the phosphate of 0.1M
In buffer solution, the concentration for making solids is 10mg/mL, obtains Streptavidin MagneSphere.
In the preparation of above-mentioned Streptavidin MagneSphere, the preparation of the chitosan magnetic microsphere modified described in step b include with
Lower step：
A weighs 4.02g anhydrous sodium acetates, adds 2.9mL glacial acetic acid, and stirring adds deionized water dilution to being completely dissolved
To 500mL, the sodium acetate buffer that concentration is 0.1M is obtained；
B weighs 2.5g chitosans, adds 1.5mL glacial acetic acid, stirring to being completely dissolved, add 250mL deionized waters and
Concentration is the sodium acetate buffer of 0.1M obtained by 250mL steps A, shakes up, obtains chitosan acetate buffer；
C weighs 4.7g iron ammonium sulfates and 9.67g ammonium ferric sulfates, is ground, and adds 250mL steps A gained concentration and is
Chitosan acetate buffer obtained by the sodium acetate buffer and 100mL steps B of 0.1M, is uniformly mixed, obtains mixed liquor；
It is molten to add the sodium hydroxide that 100mL concentration is 6M into three-necked bottle under the enclosed system environment that nitrogen is protected by D
Liquid, stirring, is heated to 60 DEG C, average to add mixed liquor obtained by step C in three times, and the time interval added every time is 30 points
Clock, maintains reaction 45min, products therefrom is washed repeatedly with deionized water, until supernatant liquid becomes clarification, use reagent after adding
AgNO3Sulfate ion is checked untill no precipitation, Magneto separate, obtains solid product, and obtained solid product is freeze-dried to obtain shell
Glycan magnetic nano-particle；
E takes chitosan magnetic nano particle obtained by 0.5g steps D, adds the polyvinyl alcohol that 10mL mass concentrations are 0.1%
Solution, ultrasonic disperse, obtains inner aqueous phase, weighs 0.6g methoxy polyethylene glycol-polylactic acid and is dissolved in 15mL dichloromethane, obtains oily
Phase, above-mentioned inner aqueous phase and oil phase is mixed, ultrasonic emulsification, phaco time 16min, obtains emulsion；
Emulsion obtained by step E is added in the poly-vinyl alcohol solution that 20mL mass concentrations are 0.1% by F, emulsifying,
Matter emulsifying rate is 6000r/min, and the emulsifying time is 18min, dichloromethane is volatilized complete, obtains product, gained is produced
Thing is washed repeatedly with deionized water, and Magneto separate, obtains solid product, and it is 7.0 that pH is added into obtained solid thing, and concentration is 0.1M's
Phosphate buffer, the concentration for making solid product is 20mg/mL, obtains the chitosan magnetic microsphere of modification.
In the preparation of above-mentioned magnetic bead immune complex, the preparation of the biotinylation PD-L1 monoclonal antibodies is including following
Step：
I takes PD-L1 monoclonal antibodies, is 0.05mol/L with concentration, and the carbonate buffer solution that pH is 9.5 adjusts PD-L1
The concentration of monoclonal antibody is 1mg/mL, obtains solution A；
II is dissolved in biotin, dicyclohexylcarbodiimide and n-hydroxysuccinimide in dimethylformamide, biology
The molar ratio of element, dicyclohexylcarbodiimide and n-hydroxysuccinimide is 1: 1: 1.2, and magnetic agitation is anti-in 50 DEG C of water-baths
Should, reaction time 12h, filters off the white precipitate dicyclohexylurea (DCU) generated in reaction solution, collects filtrate, adds ether and extremely precipitates
It is not further added by, is placed in ice bath, standing time 1h, is collected by filtration precipitation, obtains active bio element ester crude product, and above-mentioned activity is raw
Thing element ester crude product is dissolved with hot isopropanol, is placed in ice bath, standing time 3h, collected by filtration, uses dimethylformamide
Ether is added after dissolving, is placed in ice bath, standing time 1h, is collected by filtration precipitation, and vacuum drying, obtains active bio element ester；
III adds active bio element ester obtained by step II into step I resulting solution A, and active bio element ester and PD-L1 are mono-
The molar ratio of clonal antibody is 11~14: 1, and room temperature reaction, the reaction time is 35~50min, obtains reaction solution；
IV moves to step III gained reaction solution in the bag filter that molecular cut off is 7500~95000, is with concentration
The carbonate buffer solution dialysis 24h that 0.05mol/L, pH are 9.5, addition and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, to obtain the final product.
In addition, present invention also offers the detection method of the quick detection kit for being used to detect PD-L1, including with
Lower step：
S1 is pre-processed sample to be tested, takes supernatant, and 25 DEG C balance 10 minutes；
S2 adds 25 μ LPD-L1 calibration objects, PD-L1 quality-control products or sample to be tested, 37 DEG C of incubations in each flat based tubes
10 minutes, 25 μ L magnetic bead immune complexs are added, after mixing, 37 DEG C incubate 10 minutes；
S3 stands flat based tubes on magnetic separator, and time of repose is 3 minutes, supernatant is removed, by flat based tubes and magnetic
Separator is together upside down on blotting paper and pats 3 times, the liquid in hole is removed completely, 200 μ L are added in each flat based tubes
Concentrated cleaning solution, after mixing, flat based tubes are stood on magnetic separator, and time of repose is 3 minutes, supernatant is removed, by test tube
And magnetic separator is together upside down on blotting paper and pats dry beating 3 times, is repeated twice；
S4 adds antibody acridinium ester label into flat based tubes, and the addition of antibody acridinium ester label is per flat examination
50 μ L of pipe, mix 37 DEG C of postposition and are incubated 15 minutes；
The operation of S5 repeat steps S3；
S6 adds luminous substrate solution I into flat based tubes, and the addition of luminous substrate solution I is per 50 μ of flat based tubes
Luminous substrate solution II is added after L, 1S, the addition of luminous substrate solution II is per 50 μ L of flat based tubes；
S7 detects the luminous value of often pipe with Chemiluminescence Apparatus, and standard curve is drawn according to each standard solution luminous value, you can
Draw the concentration of PD-L1 in sample to be tested.
It is provided by the present invention for the principle of quick detection kit for detecting PD-L1：Using double antibody sandwich method.This
Invention is using Streptavidin MagneSphere as solid phase carrier, by the affinity interaction of streptavidin-biotin, by biotin labeling
PD-L1 monoclonal antibodies are coupled at Streptavidin MagneSphere surface, and magnetic bead immune complex is prepared.Using being connected to solid phase
The PD-L1 monoclonal antibodies of biotin labeling on carrier Streptavidin MagneSphere, and antibody acridinium ester label respectively with
Two antigenic determinants on antigen molecule are detected in sample to combine, and form solid matrix antibody-antigen-antibody sandwich compound.By
The amount of the antibody of insolubilized antibody and acridinium ester label relative to determined antigen is excessive in reaction system, therefore compound
Forming amount is directly proportional to the content of determined antigen.Acridinium ester in measure compound acts on the luminous value after the substrate of addition,
It can determine that determined antigen content.
Compared with prior art, had the advantage that provided by the present invention for detecting the quick detection kit of PD-L1：
(1) there is stable repeatability and reliability provided by the present invention for the quick detection kit for detecting PD-L1.
(2) there is efficient, high sensitivity, specificity provided by the present invention for the quick detection kit for detecting PD-L1
The characteristics of strong, not with other soluble structure analogue cross reactions.
(3) provided by the present invention for detect PD-L1 quick detection kit be applicable in serum, blood plasma, tissue homogenate,
The a variety of specimen types of cell culture supernatant, urine etc..
(4) provided by the present invention for detecting, the quick detection kit detection time of PD-L1 is short, dosage is few.
(5) when being detected using kit of the present invention, it is not necessary to the mode of centrifugation to unnecessary in detection architecture
Impurity is separated, and only need to can reach quick separating impurity under the action of externally-applied magnetic field and solid matrix antibody-antigen-antibody is multiple
The purpose of compound, makes compound efficiently separate out from complex environment, achievees the purpose that to separate specific antigen, in addition,
Disturbing factor of the environment to detection can also be reduced.
Embodiment
Below by way of the description of embodiment, the invention will be further described, but this is not the limit to the present invention
System, those skilled in the art's basic thought according to the present invention, various modifications may be made or improves, but without departing from this
The basic thought of invention, within the scope of the present invention.
Present procedure death-ligand 1 antibody is purchased from the cold bio tech ltd in Haicheng, article No. CL-
1103T, chitosan are purchased from Shanghai Lian Shuo bio tech ltd, article No. 448877, and Streptavidin is purchased from Shanghai Rong
River bend and reach Industrial Co., Ltd., article No. 21122, methoxy polyethylene glycol-polylactic acid is purchased from the auspicious auspiciousness limited public affairs of biotechnology in Xi'an
Department, article No. 32423, polyvinyl alcohol are purchased from the good Chemical Industry Science Co., Ltd of Shanghai fond dream, article No. 2099, programmed death-match somebody with somebody
Body 1 (PD-L1) is purchased from Hangzhou Lian Ke Biotechnology Ltd., article No. 70-EK1261S.
Embodiment 1, the present invention are used for composition and the preparation for detecting the quick detection kit of PD-L1
Kit of the present invention includes following components：
(1) luminous substrate solution I；(2) luminous substrate solution II；(3) concentrated cleaning solution；(4) PD-L1 standard items；(5)
PD-L1 quality-control products；(6) antibody acridinium ester label and (7) magnetic bead immune complex.
The preparation of each reagent comprises the following steps in kit of the present invention：
(1) preparation of luminous substrate solution I：
Preparation method：The dilute nitric acid solution that concentration is 0.1M is configured to distilled water and nitric acid, hydrogen peroxide is added, made
The concentration of hydrogen oxide is 0.08M.
(2) preparation of luminous substrate solution II：
Preparation method：The sodium hydroxide solution that concentration is 0.2M is configured to distilled water and sodium hydroxide, is added
TritonX-100, the mass concentration for making TritonX-100 are 2%.
(3) preparation of concentrated cleaning solution：
Preparation method：It is 7.4 to pH, concentration is to add Tween-20 in the phosphate buffer of 1M, makes Tween-20's
Volume fraction is 5%.
(4) preparation of PD-L1 standard items：
Preparation method：Be 1%BSA containing volume fraction, the pH that volume fraction is 0.05% thimerosal be 7.4, concentration
For the phosphate buffer of 0.05M, by PD-L1 be configured to concentration be respectively 100pg/mL, 250pg/mL, 500pg/mL,
The titer of 1000pg/mL, 2000pg/mL, 3000pg/mL, 5000pg/mL, to obtain the final product.
(5) preparation of PD-L1 quality-control products：
Preparation method：Be 1%BSA containing volume fraction, the pH that volume fraction is 0.05% thimerosal be 7.4, concentration
For the phosphate buffer of 0.05M, PD-L1 is configured to the solution that concentration is respectively 1500pg/mL, 4000pg/mL, to obtain the final product.
(6) preparation of antibody acridinium ester label：
Preparation method：
(1) PD-L1 monoclonal antibodies are taken, are 0.05mol/L with concentration, the carbonate buffer solution that pH is 9.5 adjusts PD-
The concentration of L1 monoclonal antibodies is 1mg/mL, obtains solution A；
(2) acridinium ester is taken, adding dimethylformamide makes the concentration of acridinium ester obtain solution B to 2mg/mL；
(3) step (2) resulting solution B is added in step (1) resulting solution A, acridinium ester and PD-L1 monoclonal antibodies
Molar ratio be 14~22: 1, room temperature reaction, the reaction time is 25~50min, obtains reaction solution；
(4) reaction solution obtained by step (3) is moved in the bag filter that molecular cut off is 7500~95000, is with concentration
The carbonate buffer solution dialysis 24h that 0.05mol/L, pH are 9.5, addition and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, to obtain the final product.
(7) preparation of magnetic bead immune complex：
1. the preparation for the chitosan magnetic microsphere modified：
A weighs 4.02g anhydrous sodium acetates, adds 2.9mL glacial acetic acid, and stirring adds deionized water dilution to being completely dissolved
To 500mL, the sodium acetate buffer that concentration is 0.1M is obtained；
B weighs 2.5g chitosans, adds 1.5mL glacial acetic acid, stirring to being completely dissolved, add 250mL deionized waters and
Concentration is the sodium acetate buffer of 0.1M obtained by 250mL steps A, shakes up, obtains chitosan acetate buffer；
C weighs 4.7g iron ammonium sulfates and 9.67g ammonium ferric sulfates, is ground, and adds 250mL steps A gained concentration and is
Chitosan acetate buffer obtained by the sodium acetate buffer and 100mL steps B of 0.1M, is uniformly mixed, obtains mixed liquor；
It is molten to add the sodium hydroxide that 100mL concentration is 6M into three-necked bottle under the enclosed system environment that nitrogen is protected by D
Liquid, stirring, is heated to 60 DEG C, average to add mixed liquor obtained by step C in three times, and the time interval added every time is 30 points
Clock, maintains reaction 45min, products therefrom is washed repeatedly with deionized water, until supernatant liquid becomes clarification, use reagent after adding
AgNO3Sulfate ion is checked untill no precipitation, Magneto separate, obtains solid product, and obtained solid product is freeze-dried to obtain shell
Glycan magnetic nano-particle；
E takes chitosan magnetic nano particle obtained by 0.5g steps D, adds the polyvinyl alcohol that 10mL mass concentrations are 0.1%
Solution, ultrasonic disperse, obtains inner aqueous phase, weighs 0.6g methoxy polyethylene glycol-polylactic acid and is dissolved in 15mL dichloromethane, obtains oily
Phase, above-mentioned inner aqueous phase and oil phase is mixed, ultrasonic emulsification, phaco time 16min, obtains emulsion；
Emulsion obtained by step E is added in the poly-vinyl alcohol solution that 20mL mass concentrations are 0.1% by F, emulsifying,
Matter emulsifying rate is 6000r/min, and the emulsifying time is 18min, dichloromethane is volatilized complete, obtains product, gained is produced
Thing is washed repeatedly with deionized water, and Magneto separate, obtains solid product, and it is 7.0 that pH is added into obtained solid thing, and concentration is 0.1M's
Phosphate buffer, the concentration for making solid product is 20mg/mL, obtains the chitosan magnetic microsphere of modification.
2. the preparation of Streptavidin MagneSphere：
A. it is 7.0 to prepare pH, and concentration is the phosphate buffer of 0.1M, and sterilizing, sterilising temp is 120 DEG C, sterilization time
For 18min, the Streptavidin of 1mg is dissolved in the above-mentioned phosphate buffers of 1mL, obtains solution of streptavidin, take 400 μ L chains
Mould avidin solution is loaded in EP pipes；
B. the chitosan magnetic microsphere for taking 5mL to modify, adds 4.5mL glutaraldehydes, room temperature concussion reaction, the reaction time is
4h, carries out Magneto separate, washes with water after removing unnecessary glutaraldehyde, and it is 7.0 that magnetic microsphere is resuspended in 5mLpH, concentration
In the phosphate buffer of 0.1M, to obtain magnetic microsphere liquid；
C. magnetic microsphere liquid obtained by step b is added in EP pipes of the step a equipped with 400 μ L solution of streptavidin, room
Warm shake culture 4h, Magneto separate, obtains solids, and it is 7.0 that obtained solid thing is dispersed in 10mLpH, and concentration is the phosphate of 0.1M
In buffer solution, the concentration for making solids is 10mg/mL, obtains Streptavidin MagneSphere.
3. the preparation of biotinylation PD-L1 monoclonal antibodies comprises the following steps：
I takes PD-L1 monoclonal antibodies, is 0.05mol/L with concentration, and the carbonate buffer solution that pH is 9.5 adjusts PD-L1
The concentration of monoclonal antibody is 1mg/mL, obtains solution A；
II is dissolved in biotin, dicyclohexylcarbodiimide and n-hydroxysuccinimide in dimethylformamide, biology
The molar ratio of element, dicyclohexylcarbodiimide and n-hydroxysuccinimide is 1: 1: 1.2, and magnetic agitation is anti-in 50 DEG C of water-baths
Should, reaction time 12h, filters off the white precipitate dicyclohexylurea (DCU) generated in reaction solution, collects filtrate, adds ether and extremely precipitates
It is not further added by, is placed in ice bath, standing time 1h, is collected by filtration precipitation, obtains active bio element ester crude product, and above-mentioned activity is raw
Thing element ester crude product is dissolved with hot isopropanol, is placed in ice bath, standing time 3h, collected by filtration, uses dimethylformamide
Ether is added after dissolving, is placed in ice bath, standing time 1h, is collected by filtration precipitation, and vacuum drying, obtains active bio element ester；
III adds active bio element ester obtained by step II into step I resulting solution A, and active bio element ester and PD-L1 are mono-
The molar ratio of clonal antibody is 11~14: 1, and room temperature reaction, the reaction time is 35~50min, obtains reaction solution；
IV moves to step III gained reaction solution in the bag filter that molecular cut off is 7500~95000, is with concentration
The carbonate buffer solution dialysis 24h that 0.05mol/L, pH are 9.5, addition and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, to obtain the final product.
4. the preparation of magnetic bead immune complex：
The Streptavidin MagneSphere for taking 100 μ L concentration to be 10mg/mL, is diluted with 250 μ L concentrated cleaning solutions according to 1: 100
The solution arrived washs 4 times, adds 50 μ L biotinylation PD-L1 monoclonal antibodies, is placed in 37 DEG C of constant temperature gas bath shaking tables and vibrates, shake
It is 100r/min to swing speed, is taken out after 12~16min, is washed with 200 μ L concentrated cleaning solutions according to the solution that 1: 100 dilution obtains
4 times, then, it is replaced in above-mentioned constant temperature gas bath shaking table, it is 7.4 to add 1mL containing the pH that volume fraction is 2%BSA, and concentration is
The phosphate buffer of 1M carries out capping, and the capping time is 15~22min, takes out, and Magnetic Isolation, removes supernatant, uses
200 μ L concentrated cleaning solutions wash 4 times according to the solution that 1: 100 dilution obtains, and it is 7.4 to add pH, and concentration is that the phosphate of 1M delays
Fliud flushing is made into the magnetic bead immune complex of 1mg/mL, is placed in 4 DEG C and is kept in dark place, to obtain the final product；
Wherein, the preparation method of the concentrated cleaning solution is：It is 7.4 to pH, adds in the phosphate buffer that concentration is 1M
Enter Tween-20, the volume fraction for making Tween-20 is 5%.
Embodiment 2, the quick detection kit progress PD-L1 detections for being used to detect PD-L1 using the present invention
S1 is pre-processed sample to be tested, takes supernatant, and 25 DEG C balance 10 minutes；
S2 adds 25 μ LPD-L1 calibration objects, PD-L1 quality-control products or sample to be tested, 37 DEG C of incubations in each flat based tubes
10 minutes, 25 μ L magnetic bead immune complexs are added, after mixing, 37 DEG C incubate 10 minutes；
S3 stands flat based tubes on magnetic separator, and time of repose is 3 minutes, supernatant is removed, by flat based tubes and magnetic
Separator is together upside down on blotting paper and pats 3 times, the liquid in hole is removed completely, 200 μ L are added in each flat based tubes
Concentrated cleaning solution, after mixing, flat based tubes are stood on magnetic separator, and time of repose is 3 minutes, supernatant is removed, by test tube
And magnetic separator is together upside down on blotting paper and pats dry beating 3 times, is repeated twice；
S4 adds antibody acridinium ester label into flat based tubes, and the addition of antibody acridinium ester label is per flat examination
50 μ L of pipe, mix 37 DEG C of postposition and are incubated 15 minutes；
The operation of S5 repeat steps S3；
S6 adds luminous substrate solution I into flat based tubes, and the addition of luminous substrate solution I is per 50 μ of flat based tubes
Luminous substrate solution II is added after L, 1S, the addition of luminous substrate solution II is per 50 μ L of flat based tubes；
S7 detects the luminous value of often pipe with Chemiluminescence Apparatus, and standard curve is drawn according to each standard solution luminous value, you can
Draw the concentration of PD-L1 in sample to be tested.
Comparative example 1, the composition of kit and preparation
The composition of 1 kit of comparative example and prepare and the composition of 1 kit of embodiment and prepare essentially identical, distinguish
In, the magnetic bead immune complex is prepared by Streptavidin MagneSphere and biotinylation PD-L1 monoclonal antibodies, wherein,
The chitosan magnetic microsphere of modification is replaced with into chitosan magnetic nano particle in the preparation of Streptavidin MagneSphere.
Test example one, the stability of quick detection kit for detecting PD-L1
1st, accelerate the failure stability
In order to examine the stability that accelerates the failure of kit, the embodiments 1 for placing 4 DEG C, 37 DEG C preservations 10 days are made respectively
The standby quick detection kit for being used to detect PD-L1 is tested for the property, the performance indicator of test kit.Accelerate the failure steady
Qualitative results are as shown in table 1.
Table 1：Kit accelerates the failure stability result
The empirical method of the kit term of validity is calculated according to the experiment that accelerates the failure, is accelerated the failure 10 days at 37 DEG C, equivalent to
The term of validity 1 year half, can meet the requirement of the kit term of validity of 1 year.As can be seen from Table 1, the present invention is used to detect PD-L1
Quick detection kit 4 DEG C, 37 DEG C preserve 10 days after, sensitivity, accuracy and accuracy of kit etc. be satisfied by will
Ask.
2nd, freeze-thaw stability
It is to simulate kit in transportational process, kit each component multigelation is to stabilization of kit under low temperature environment
Influence, be used to detect the quick detection kit of the PD-L1 freeze overnight in a low temperature of -20 DEG C by prepared by embodiment 1, so
Redissolve at room temperature afterwards, freeze overnight in a low temperature of being then placed in -20 DEG C again, such multigelation 5 times.The results are shown in Table 2.
Table 2：Kit freeze-thaw stability result
As can be seen from Table 2, the quick detection kit that the present invention is used to detect PD-L1 is after multigelation 5 times, reagent
Sensitivity, accuracy and accuracy of box etc. are satisfied by requiring.This explanation, kit of the present invention have preferable freeze-thaw stability
Property.
3rd, transportation stability is simulated
Be simulation kit in transportational process, vehicle jolt and influence of the hot environment to stabilization of kit,
The quick detection kit for being used to detect PD-L1 prepared by embodiment 1 is fixed on micro oscillator, is positioned over 37 DEG C of constant temperature
Case vibrates 7 days.The results are shown in Table 3.
Table 3：Kit simulates transportation stability result
As can be seen from Table 3, the present invention is positioned over 37 DEG C of insulating boxs vibrations for detecting the quick detection kit of PD-L1
After 7 days, sensitivity, accuracy and accuracy of kit etc. are satisfied by requiring.
Prepared by test example two, 1 kit of comparative example and the embodiment of the present invention 1 is used to detect the quick detection examination of PD-L1
The results contrast that agent box is detected PD-L1
The quick detection kit for being used to detect PD-L1 prepared by the kit and embodiment 1 prepared using comparative example 1,
Sample is detected, the specific step of the method reference implementation example 2 of detection.
The kit of the preparation of comparative example 1 and the quick detection reagent for being used to detect PD-L1 of the preparation of embodiment 1 is respectively adopted
Box, while Serum of Patients with Lung Cancer sample 100 parts (PD-L1 positive rates are 100%) is detected, the results are shown in Table 4.
Table 4：Testing result compares
As can be seen from Table 4, the sensitivity decrease detected using kit prepared by comparative example 1 to PD-L1 in serum, batch
It is interior and batch between accuracy reduce.This explanation, the quick detection kit for being used to detect PD-L1 prepared by the present invention are remarkably improved
Detection sensitivity and detection accuracy.
Claims (4)
- A kind of 1. quick detection kit for being used to detect PD-L1, it is characterised in that：The kit includes luminous substrate solution I, luminous substrate solution II, concentrated cleaning solution, PD-L1 standard items, PD-L1 quality-control products, antibody acridinium ester label and magnetic bead are exempted from Epidemic disease compound；The preparation of the antibody acridinium ester label comprises the following steps：1.1 take PD-L1 monoclonal antibodies, are 0.05mol/L with concentration, and the carbonate buffer solution that pH is 9.5 adjusts PD-L1 The concentration of monoclonal antibody is 1mg/mL, obtains solution A；1.2 take acridinium ester, and adding dimethylformamide makes the concentration of acridinium ester obtain solution B to 2mg/mL；1.3 are added to step 1.2 resulting solution B in step 1.1 resulting solution A, acridinium ester and PD-L1 monoclonal antibodies Molar ratio is 14~22: 1, and room temperature reaction, the reaction time is 25~50min, obtains reaction solution；1.4 move to step 1.3 gained reaction solution in the bag filter that molecular cut off is 7500~95000, are with concentration The carbonate buffer solution dialysis 24h that 0.05mol/L, pH are 9.5, addition and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C Preserve, to obtain the final product；The preparation of the magnetic bead immune complex comprises the following steps：The Streptavidin MagneSphere that 100 μ L concentration are 10mg/mL is taken, is obtained with 250 μ L concentrated cleaning solutions according to 1: 100 dilution Solution washs 4 times, adds 50 μ L biotinylation PD-L1 monoclonal antibodies, is placed in 37 DEG C of constant temperature gas bath shaking tables and vibrates, vibration speed Spend for 100r/min, taken out after 12~16min, washed 4 times according to the solution that 1: 100 dilution obtains with 200 μ L concentrated cleaning solutions, Then, it is replaced in above-mentioned constant temperature gas bath shaking table, it is 7.4 to add 1mL containing the pH that volume fraction is 2%BSA, and concentration is 1M's Phosphate buffer carries out capping, and the capping time is 15~22min, takes out, Magnetic Isolation, removes supernatant, with 200 μ L Concentrated cleaning solution washs 4 times according to the solution that 1: 100 dilution obtains, and it is 7.4 to add pH, and concentration is that the phosphate buffer of 1M is matched somebody with somebody Into the magnetic bead immune complex of 1mg/mL, be placed in 4 DEG C and be kept in dark place, to obtain the final product；Wherein, the preparation method of concentrated cleaning solution is：It is 7.4 to pH, concentration is to add Tween- in the phosphate buffer of 1M 20, the volume fraction for making Tween-20 is 5%；The preparation of the Streptavidin MagneSphere comprises the following steps：2.1 preparation pH are 7.0, and concentration is the phosphate buffer of 0.1M, and sterilizing, sterilising temp is 120 DEG C, and sterilization time is 18min, the Streptavidin of 1mg is dissolved in the above-mentioned phosphate buffers of 1mL, obtains solution of streptavidin, takes 400 μ L strepto-s Avidin solution is loaded in EP pipes；2.2 take 5mL modify chitosan magnetic microsphere, add 4.5mL glutaraldehydes, room temperature concussion reaction, reaction time 4h, into Row Magneto separate, washes with water after removing unnecessary glutaraldehyde, and it is 7.0 that magnetic microsphere is resuspended in 5mLpH, concentration 0.1M Phosphate buffer in, obtain magnetic microsphere liquid；2.3 are added to step 2.2 gained magnetic microsphere liquid in EP pipes of the step 2.1 equipped with 400 μ L solution of streptavidin, Room temperature shake culture 4h, Magneto separate, obtains solids, and it is 7.0 that obtained solid thing is dispersed in 10mLpH, and concentration is the phosphoric acid of 0.1M In salt buffer, the concentration for making solids is 10mg/mL, obtains Streptavidin MagneSphere；The preparation of the chitosan magnetic microsphere of modification described in step 2.2 comprises the following steps：3.1 weigh 4.02g anhydrous sodium acetates, add 2.9mL glacial acetic acid, and stirring adds deionized water and be diluted to being completely dissolved 500mL, obtains the sodium acetate buffer that concentration is 0.1M；3.2 weigh 2.5g chitosans, add 1.5mL glacial acetic acid, stirring to being completely dissolved, add 250mL deionized waters and 250mL step 3.1 gained concentration is the sodium acetate buffer of 0.1M, shakes up, obtains chitosan acetate buffer；3.3 weigh 4.7g iron ammonium sulfates and 9.67g ammonium ferric sulfates, are ground, and add 250mL step 3.1 gained concentration and are The sodium acetate buffer and 100mL step 3.2 gained chitosan acetate buffers of 0.1M, is uniformly mixed, obtains mixed liquor；3.4 under the enclosed system environment of nitrogen protection, and the sodium hydroxide solution that 100mL concentration is 6M is added into three-necked bottle, Stirring, is heated to 60 DEG C, average to add step 3.3 gained mixed liquor in three times, and the time interval added every time is 30 points Clock, maintains reaction 45min, products therefrom is washed repeatedly with deionized water, until supernatant liquid becomes clarification, use reagent after adding AgNO3Sulfate ion is checked untill no precipitation, Magneto separate, obtains solid product, and obtained solid product is freeze-dried to obtain shell Glycan magnetic nano-particle；3.5 take 0.5g step 3.4 gained chitosan magnetic nano particles, add the polyvinyl alcohol that 10mL mass concentrations are 0.1% Solution, ultrasonic disperse, obtains inner aqueous phase, weighs 0.6g methoxy polyethylene glycol-polylactic acid and is dissolved in 15mL dichloromethane, obtains oily Phase, above-mentioned inner aqueous phase and oil phase is mixed, ultrasonic emulsification, phaco time 16min, obtains emulsion；3.6 are added to step 3.5 gained emulsion in the poly-vinyl alcohol solution that 20mL mass concentrations are 0.1%, emulsifying, Matter emulsifying rate is 6000r/min, and the emulsifying time is 18min, dichloromethane is volatilized complete, obtains product, gained is produced Thing is washed repeatedly with deionized water, and Magneto separate, obtains solid product, and it is 7.0 that pH is added into obtained solid thing, and concentration is 0.1M's Phosphate buffer, the concentration for making solid product is 20mg/mL, obtains the chitosan magnetic microsphere of modification；The preparation of the biotinylation PD-L1 monoclonal antibodies comprises the following steps：4.1 take PD-L1 monoclonal antibodies, are 0.05mol/L with concentration, it is mono- that the carbonate buffer solution that pH is 9.5 adjusts PD-L1 The concentration of clonal antibody is 1mg/mL, obtains solution A；4.2 are dissolved in biotin, dicyclohexylcarbodiimide and n-hydroxysuccinimide in dimethylformamide, biotin, The molar ratio of dicyclohexylcarbodiimide and n-hydroxysuccinimide is 1: 1: 1.2, and magnetic agitation is reacted in 50 DEG C of water-baths, Reaction time is 12h, filters off the white precipitate dicyclohexylurea (DCU) generated in reaction solution, collects filtrate, adds ether and extremely precipitates not It is further added by, is placed in ice bath, standing time 1h, is collected by filtration precipitation, active bio element ester crude product is obtained, by above-mentioned active bio Plain ester crude product is dissolved with hot isopropanol, is placed in ice bath, standing time 3h, collected by filtration, molten with dimethylformamide Ether is added after solution, is placed in ice bath, standing time 1h, is collected by filtration precipitation, and vacuum drying, obtains active bio element ester；4.3 add step 4.2 gained active bio element ester into step 4.1 resulting solution A, and active bio element ester and PD-L1 are mono- The molar ratio of clonal antibody is 11~14: 1, and room temperature reaction, the reaction time is 35~50min, obtains reaction solution；4.4 move to step 4.3 gained reaction solution in the bag filter that molecular cut off is 7500~95000, are with concentration The carbonate buffer solution dialysis 24h that 0.05mol/L, pH are 9.5, addition and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C Preserve, to obtain the final product.
- 2. the quick detection kit as claimed in claim 1 for being used to detect PD-L1, it is characterised in that the luminous substrate The preparation method of liquid I is：The dilute nitric acid solution that concentration is 0.1M is configured to distilled water and nitric acid, hydrogen peroxide is added, made The concentration of hydrogen oxide is 0.08M；The preparation method of the luminous substrate liquid II is：Concentration is configured to distilled water and sodium hydroxide For the sodium hydroxide solution of 0.2M, TritonX-100 is added, the mass concentration for making TritonX-100 is 2%；Concentrated cleaning solution Preparation method be：It is 7.4 to pH, concentration is to add Tween-20 in the phosphate buffer of 1M, makes the volume of Tween-20 Fraction is 5%.
- 3. the quick detection kit as claimed in claim 1 for being used to detect PD-L1, it is characterised in that the PD-L1 standards The preparation of product comprises the following steps：Be 1%BSA containing volume fraction, the pH that volume fraction is 0.05% thimerosal be 7.4, Concentration be 0.05M phosphate buffer, by PD-L1 be configured to concentration be respectively 100pg/mL, 250pg/mL, 500pg/mL, The titer of 1000pg/mL, 2000pg/mL, 3000pg/mL, 5000pg/mL, to obtain the final product.
- 4. the quick detection kit as claimed in claim 1 for being used to detect PD-L1, it is characterised in that the PD-L1 Quality Controls The preparation of product comprises the following steps：Be 1%BSA containing volume fraction, the pH that volume fraction is 0.05% thimerosal be 7.4, Concentration is the phosphate buffer of 0.05M, PD-L1 is configured to the solution that concentration is respectively 1500pg/mL, 4000pg/mL, i.e., .
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| CN108152484A (en) * | 2017-12-07 | 2018-06-12 | 珠海霍普金斯医药研究院股份有限公司 | A kind of chemiluminescence magnetic bead enzyme-linked immunization rapid quantitative detection reagent box of PD-L1 |
| CN108997475A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Improve the dialysis purification technique of acridinium ester label protein recovery |
| CN111812326A (en) * | 2020-07-23 | 2020-10-23 | 四川携光生物技术有限公司 | Kit for quantitatively detecting content of PD-L1 and PD-1 conjugate |
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Effective date of registration: 20200820 Address after: Jiangsu province Suzhou City Road 215000 Hong Kong port of Taicang economic and Technological Development Zone No. 52 Patentee after: Nevonos (Suzhou) Bioengineering Co.,Ltd. Address before: 510530, 84, two, science Road, hi tech Industrial Development Zone, Guangdong, Guangzhou Province, 101 Patentee before: GUANGZHOU HUAHONG BIOTECHNOLOGY Co.,Ltd. |