CN107192817A - 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.Provided by the present invention for detecting that PD L1 quick detection 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 immune complex.Provided by the present invention for detect PD L1 quick detection kit have the advantages that detection time is short, consumption is few, high efficient, sensitivity, high specificity, stable repeatability and reliability.
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 is programmed death 1, is a kind of important be immunized
Suppress molecule, be CD28 superfamily members.Immunological regulation using PD-1 as target spot is in antitumor, anti-infective, anti-LADA
There is important meaning in terms of disease and organ transplant survival.Its part PD-L1 also can be 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) is combined, 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 recognizes PD-L1
Afterwards, suppression row signal can be conducted, T cell cannot find tumour cell and send signal to attack to tumour cell.
Chinese patent application CN105579471A provides the antibody of the separation of specific binding human PD-L 1 and such anti-
The antigen-binding fragment of body, and it is used for detection combination human PD-L 1 comprising anti-the 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 had influence on doctor
Treat insurance and society.Because polytype cancer mark is the quick of malignant cell and not modulated propagation, therefore 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 mark 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 PD-L1 tumor patient, for it is personalized block PD-L1 treatment provide instruct and according to
According to.And to there is sensitivity relatively low for the existing kit for being used to detecting PD-L1, and specificity it is not high the shortcomings of.
The content of the invention
In view of the shortcomings 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.Provided by the present invention for detecting that PD-L1 quick detection kit has that detection time is short, consumption
Less, efficiently, sensitivity height, high specificity, stable repeatability and reliability the advantages of.
The technical scheme is that：
A kind of quick detection kit for being used to detect PD-L1, the kit includes luminous substrate solution I, and light 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：The dust technology that concentration is 0.1M is configured to distilled water and nitric acid molten
Liquid, adds hydrogen peroxide, and 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 addition in 1M phosphate buffer
Tween-20, the volume fraction for making Tween-20 is 5%.
The preparation of the PD-L1 standard items comprises the following steps：It is that 1%BSA, volume fraction are with containing volume fraction
The pH of 0.05% thimerosal is 7.4, and concentration is 0.05M phosphate buffer, and PD-L1 is configured into concentration respectively 100pg/
ML, 250pg/mL, 500pg/mL, 1000pg/mL, 2000pg/mL, 3000pg/mL, 5000pg/mL titer, are produced.
The preparation of the PD-L1 quality-control products comprises the following steps：It is that 1%BSA, volume fraction are with containing volume fraction
The pH of 0.05% thimerosal is 7.4, and concentration is 0.05M phosphate buffer, and PD-L1 is configured into concentration is respectively
1500pg/mL, 4000pg/mL solution, are produced.
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, pH adjusts PD- for 9.5 carbonate buffer solution
The concentration of L1 monoclonal antibodies is 1mg/mL, obtains solution A；
(2) acridinium ester is taken, the concentration that addition dimethylformamide makes acridinium ester obtains 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
Mol ratio be 14~22: 1, room temperature reaction, the reaction time be 25~50min, obtain 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
0.05mol/L, pH are 9.5 carbonate buffer solution dialysis 24h, add and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, produce.
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 is washed 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 is vibrated, shakes
Speed is swung for 100r/min, is taken out after 12~16min, and the solution obtained with 200 μ L concentrated cleaning solutions according to 1: 100 dilution is washed
4 times, then, it is replaced in above-mentioned constant temperature gas bath shaking table, it is 7.4 containing the pH that volume fraction is 2%BSA to add 1mL, and concentration is
1M phosphate buffer carries out capping, and the capping time is 15~22min, takes out, Magnetic Isolation removes supernatant, uses
The solution that 200 μ L concentrated cleaning solutions are obtained according to 1: 100 dilution is washed 4 times, and it is 7.4 to add pH, and concentration is delayed for 1M phosphate
Fliud flushing is made into 1mg/mL magnetic bead immune complex, is placed in 4 DEG C and is kept in dark place, produces；
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 0.1M phosphate buffer, and sterilizing, sterilising temp is 120 DEG C, sterilization time
For 18min, 1mg Streptavidin is dissolved in the above-mentioned phosphate buffers of 1mL, solution of streptavidin is obtained, 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, and the reaction time is
4h, carries out Magneto separate, is cleaned and is removed after unnecessary glutaraldehyde with water, and it is 7.0, concentration that magnetic microsphere is resuspended in into 5mLpH
In 0.1M phosphate buffer, 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 concussion and cultivate 4h, Magneto separate obtains solids, and it is 7.0 that gained solids is dispersed in into 10mLpH, and concentration is 0.1M phosphate
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 0.1M sodium acetate buffer 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 0.1M sodium acetate buffer and 100mL steps B, is well mixed, obtains mixed liquor；
D adds 100mL concentration into three-necked bottle molten for 6M sodium hydroxide under the enclosed system environment that nitrogen is protected
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, adds rear maintenance reaction 45min, with deionized water cyclic washing products therefrom, until supernatant liquid becomes clarification, uses reagent
AgNO3Sulfate ion is checked untill without precipitation, Magneto separate obtains solid product, and gained solid product is freeze-dried to obtain into 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 interior aqueous phase, weighs 0.6g MPEG-PLAs and is dissolved in 15mL dichloromethane, obtains oily
Phase, above-mentioned interior aqueous phase and oil phase is mixed, ultrasonic emulsification, phaco time is 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 deionized water cyclic washing, Magneto separate obtains solid product, and it is 7.0 that pH is added into gained solids, 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 pH adjusts PD-L1 for 9.5 carbonate buffer solution
The concentration of monoclonal antibody is 1mg/mL, obtains solution A；
II is dissolved in biotin, dicyclohexylcarbodiimide and n-hydroxysuccinimide in dimethylformamide, biological
The mol 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, the reaction time is 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 is 1h, precipitation is collected by filtration, obtains the plain ester crude product of active bio, above-mentioned activity is raw
Thing element ester crude product is dissolved with hot isopropanol, is placed in ice bath, standing time is 3h, collected by filtration, uses dimethylformamide
Ether is added after dissolving, is placed in ice bath, standing time is 1h, precipitation is collected by filtration, is dried in vacuo, the plain ester of active bio is obtained；
III adds the plain ester of active bio obtained by step II into step I resulting solution A, and active bio element ester and PD-L1 are mono-
The mol 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
0.05mol/L, pH are 9.5 carbonate buffer solution dialysis 24h, add and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, produce.
In addition, present invention also offers the detection method for being used to detect PD-L1 quick detection kit, 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 patted 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
The μ L of pipe 50, mix rearmounted 37 DEG C and are incubated 15 minutes；
S5 repeat steps S3 operation；
S6 adds luminous substrate solution I into flat based tubes, and the addition of luminous substrate solution I is per the μ of flat based tubes 50
Luminous substrate solution II is added after L, 1S, the addition of luminous substrate solution II is per the μ L of flat based tubes 50；
S7 detects the luminous value of often pipe with Chemiluminescence Apparatus, and standard curve is drawn according to each standard liquid luminous value, you can
Draw the concentration of PD-L1 in sample to be tested.
Provided by the present invention for detecting that the PD-L1 principle of quick detection kit is：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, prepare magnetic bead immune complex.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 is excessive, therefore compound relative to determined antigen in reaction system
Forming amount is directly proportional to the content of determined antigen.The luminous value that the acridinium ester in compound is acted on after the substrate of addition is determined,
It can determine that determined antigen content.
Compared with prior art, provided by the present invention for detecting that PD-L1 quick detection kit has the advantage that：
(1) provided by the present invention for detecting that PD-L1 quick detection kit has stable repeatability and reliability.
(2) provided by the present invention for detecting that PD-L1 quick detection kit has efficient, sensitivity high, specific
Strong the characteristics of, not with other soluble structure analogue cross reactions.
(3) provided by the present invention for detect PD-L1 quick detection kit be applicable serum, blood plasma, tissue homogenate,
The a variety of specimen types of cell culture supernatant, urine etc..
(4) provided by the present invention for detect PD-L1 quick detection kit detection time is short, consumption 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 in the presence 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, reaches the purpose of separation 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 are according to the basic thought of the present invention, and 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 is purchased from Shanghai Lian Shuo bio tech ltd, and article No. 448877, Streptavidin is purchased from Shanghai Rong
River bend up to Industrial Co., Ltd., article No. 21122, MPEG-PLA is purchased from the auspicious auspiciousness limited public affairs of biotechnology in Xi'an
Department, article No. 32423, polyvinyl alcohol is 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 PD-L1 quick detection kit
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 is 2%.
(3) preparation of concentrated cleaning solution：
Preparation method：It is 7.4 to pH, concentration makes Tween-20's to add Tween-20 in 1M phosphate buffer
Volume fraction is 5%.
(4) preparation of PD-L1 standard items：
Preparation method：It is 7.4, concentration with containing the pH that volume fraction is 1%BSA, volume fraction is 0.05% thimerosal
For 0.05M phosphate buffer, by PD-L1 be configured to concentration be respectively 100pg/mL, 250pg/mL, 500pg/mL,
1000pg/mL, 2000pg/mL, 3000pg/mL, 5000pg/mL titer, are produced.
(5) preparation of PD-L1 quality-control products：
Preparation method：It is 7.4, concentration with containing the pH that volume fraction is 1%BSA, volume fraction is 0.05% thimerosal
For 0.05M phosphate buffer, PD-L1 is configured to the solution that concentration is respectively 1500pg/mL, 4000pg/mL, produced.
(6) preparation of antibody acridinium ester label：
Preparation method：
(1) PD-L1 monoclonal antibodies are taken, are 0.05mol/L with concentration, pH adjusts PD- for 9.5 carbonate buffer solution
The concentration of L1 monoclonal antibodies is 1mg/mL, obtains solution A；
(2) acridinium ester is taken, the concentration that addition dimethylformamide makes acridinium ester obtains 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
Mol ratio be 14~22: 1, room temperature reaction, the reaction time be 25~50min, obtain 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
0.05mol/L, pH are 9.5 carbonate buffer solution dialysis 24h, add and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, produce.
(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 0.1M sodium acetate buffer 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 0.1M sodium acetate buffer and 100mL steps B, is well mixed, obtains mixed liquor；
D adds 100mL concentration into three-necked bottle molten for 6M sodium hydroxide under the enclosed system environment that nitrogen is protected
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, adds rear maintenance reaction 45min, with deionized water cyclic washing products therefrom, until supernatant liquid becomes clarification, uses reagent
AgNO3Sulfate ion is checked untill without precipitation, Magneto separate obtains solid product, and gained solid product is freeze-dried to obtain into 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 interior aqueous phase, weighs 0.6g MPEG-PLAs and is dissolved in 15mL dichloromethane, obtains oily
Phase, above-mentioned interior aqueous phase and oil phase is mixed, ultrasonic emulsification, phaco time is 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 deionized water cyclic washing, Magneto separate obtains solid product, and it is 7.0 that pH is added into gained solids, 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 0.1M phosphate buffer, and sterilizing, sterilising temp is 120 DEG C, sterilization time
For 18min, 1mg Streptavidin is dissolved in the above-mentioned phosphate buffers of 1mL, solution of streptavidin is obtained, 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, and the reaction time is
4h, carries out Magneto separate, is cleaned and is removed after unnecessary glutaraldehyde with water, and it is 7.0, concentration that magnetic microsphere is resuspended in into 5mLpH
In 0.1M phosphate buffer, 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 concussion and cultivate 4h, Magneto separate obtains solids, and it is 7.0 that gained solids is dispersed in into 10mLpH, and concentration is 0.1M phosphate
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 pH adjusts PD-L1 for 9.5 carbonate buffer solution
The concentration of monoclonal antibody is 1mg/mL, obtains solution A；
II is dissolved in biotin, dicyclohexylcarbodiimide and n-hydroxysuccinimide in dimethylformamide, biological
The mol 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, the reaction time is 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 is 1h, precipitation is collected by filtration, obtains the plain ester crude product of active bio, above-mentioned activity is raw
Thing element ester crude product is dissolved with hot isopropanol, is placed in ice bath, standing time is 3h, collected by filtration, uses dimethylformamide
Ether is added after dissolving, is placed in ice bath, standing time is 1h, precipitation is collected by filtration, is dried in vacuo, the plain ester of active bio is obtained；
III adds the plain ester of active bio obtained by step II into step I resulting solution A, and active bio element ester and PD-L1 are mono-
The mol 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
0.05mol/L, pH are 9.5 carbonate buffer solution dialysis 24h, add and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, produce.
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 is washed 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 is vibrated, shakes
Speed is swung for 100r/min, is taken out after 12~16min, and the solution obtained with 200 μ L concentrated cleaning solutions according to 1: 100 dilution is washed
4 times, then, it is replaced in above-mentioned constant temperature gas bath shaking table, it is 7.4 containing the pH that volume fraction is 2%BSA to add 1mL, and concentration is
1M phosphate buffer carries out capping, and the capping time is 15~22min, takes out, Magnetic Isolation removes supernatant, uses
The solution that 200 μ L concentrated cleaning solutions are obtained according to 1: 100 dilution is washed 4 times, and it is 7.4 to add pH, and concentration is delayed for 1M phosphate
Fliud flushing is made into 1mg/mL magnetic bead immune complex, is placed in 4 DEG C and is kept in dark place, produces；
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 patted 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
The μ L of pipe 50, mix rearmounted 37 DEG C and are incubated 15 minutes；
S5 repeat steps S3 operation；
S6 adds luminous substrate solution I into flat based tubes, and the addition of luminous substrate solution I is per the μ of flat based tubes 50
Luminous substrate solution II is added after L, 1S, the addition of luminous substrate solution II is per the μ L of flat based tubes 50；
S7 detects the luminous value of often pipe with Chemiluminescence Apparatus, and standard curve is drawn according to each standard liquid 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 the composition of the kit of comparative example 1 and preparation and the kit of embodiment 1 and prepare essentially identical, difference exists
In, the magnetic bead immune complex is prepared from 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 carries out performance test, the performance indications of test kit.Accelerate the failure steady
Qualitative results are as shown in table 1.
Table 1：Kit accelerates the failure stability result
Calculate and accelerate the failure 10 days at the empirical method of the kit term of validity, 37 DEG C according to the experiment that accelerates the failure, 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 transportation, kit each component multigelation is to stabilization of kit under low temperature environment
Influence, being used for of preparing of embodiment 1 is detected into the PD-L1 quick detection kit freeze overnight in a low temperature of -20 DEG C, 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.As a result it is as shown in table 2.
Table 2：Kit freeze-thaw stability result
As can be seen from Table 2, the present invention is used for the quick detection kit for detecting PD-L1 after multigelation 5 times, reagent
Sensitivity, accuracy and accuracy of box etc. are satisfied by requiring.This explanation, kit of the present invention has preferable freeze-thaw stability
Property.
3rd, transportation stability is simulated
Be simulation kit in transportation, vehicle jolts and influence of the hot environment to stabilization of kit,
Quick detection kit for detecting PD-L1 prepared by embodiment 1 is fixed on micro oscillator, is positioned over 37 DEG C of constant temperature
Case vibrates 7 days.As a result it is as shown in table 3.
Table 3：Kit simulates transportation stability result
As can be seen from Table 3, the present invention is used to detect that PD-L1 quick detection kit to be positioned over 37 DEG C of insulating box vibrations
After 7 days, sensitivity, accuracy and accuracy of kit etc. are satisfied by requiring.
Prepared by test example two, the kit of comparative example 1 and the embodiment of the present invention 1 is used to detect that PD-L1 quick detection to be tried
The results contrast that agent box is detected to PD-L1
Quick detection kit for detecting 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 quick detection reagent for being used to detect PD-L1 prepared by the kit and embodiment 1 of the preparation of comparative example 1 is respectively adopted
Box, while being detected to Serum of Patients with Lung Cancer sample 100 parts (PD-L1 positive rates are 100%), as a result as shown in table 4.
Table 4：Testing result compares
As can be seen from Table 4, the sensitivity decrease that the kit prepared using comparative example 1 is detected to PD-L1 in serum, batch
It is interior and batch between accuracy reduce.This explanation, prepared by the present invention is used to detect that PD-L1 quick detection kit is remarkably improved
Detection sensitivity and detection accuracy.
Claims (10)
1. a kind of 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.
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 0.2M sodium hydroxide solution, TritonX-100 is added, the mass concentration for making TritonX-100 is 2%；The concentration is washed
The preparation method for washing liquid is：It is 7.4 to pH, concentration makes Tween-20's to add Tween-20 in 1M phosphate buffer
Volume 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：With being 7.4 containing the pH that volume fraction is 1%BSA, volume fraction is 0.05% thimerosal,
Concentration be 0.05M phosphate buffer, by PD-L1 be configured to concentration be respectively 100pg/mL, 250pg/mL, 500pg/mL,
1000pg/mL, 2000pg/mL, 3000pg/mL, 5000pg/mL titer, are produced.
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：With being 7.4 containing the pH that volume fraction is 1%BSA, volume fraction is 0.05% thimerosal,
Concentration is 0.05M phosphate buffer, PD-L1 is configured into the solution that concentration is respectively 1500pg/mL, 4000pg/mL, i.e.,
.
5. the quick detection kit as claimed in claim 1 for being used to detect PD-L1, it is characterised in that the antibody acridine
The preparation of ester label comprises the following steps：
(1) PD-L1 monoclonal antibodies are taken, are 0.05mol/L with concentration, pH is mono- for 9.5 carbonate buffer solution adjustment PD-L1
The concentration of clonal antibody is 1mg/mL, obtains solution A；
(2) acridinium ester is taken, the concentration that addition dimethylformamide makes acridinium ester obtains solution B to 2mg/mL；
(3) step (2) resulting solution B is added in step (1) resulting solution A, acridinium ester rubs with PD-L1 monoclonal antibodies
You are than being 14~22: 1, and 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
0.05mol/L, pH are 9.5 carbonate buffer solution dialysis 24h, add and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, produce.
6. the quick detection kit as claimed in claim 1 for being used to detect PD-L1, it is characterised in that the magnetic bead is immunized
The preparation of compound 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 is washed 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 is vibrated, vibration speed
Spend for 100r/min, taken out after 12~16min, the solution obtained with 200 μ L concentrated cleaning solutions according to 1: 100 dilution is washed 4 times,
Then, it is replaced in above-mentioned constant temperature gas bath shaking table, it is 7.4 containing the pH that volume fraction is 2%BSA to add 1mL, concentration is 1M's
Phosphate buffer carries out capping, and the capping time is 15~22min, is taken out, Magnetic Isolation removes supernatant, with 200 μ L
The solution that concentrated cleaning solution is obtained according to 1: 100 dilution is washed 4 times, and it is 7.4 to add pH, and concentration is matched somebody with somebody for 1M phosphate buffer
Into 1mg/mL magnetic bead immune complex, it is placed in 4 DEG C and is kept in dark place, produce；
Wherein, the preparation method of the concentrated cleaning solution is：It is 7.4 to pH, concentration is addition in 1M phosphate buffer
Tween-20, the volume fraction for making Tween-20 is 5%.
7. the quick detection kit as claimed in claim 6 for being used to detect PD-L1, it is characterised in that the strepto- is affine
The preparation of biscuit porcelain pearl comprises the following steps：
A. it is 7.0 to prepare pH, and concentration is 0.1M phosphate buffer, and sterilizing, sterilising temp is 120 DEG C, and sterilization time is
18min, 1mg Streptavidin is dissolved in the above-mentioned phosphate buffers of 1mL, solution of streptavidin is obtained, and takes 400 μ L strepto-s
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, is entered
Row Magneto separate, is cleaned with water and removed after unnecessary glutaraldehyde, and it is 7.0 that magnetic microsphere is resuspended in into 5mLpH, and concentration is 0.1M
Phosphate buffer in, 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 temperature shake
Culture 4h is swung, Magneto separate obtains solids, it is 7.0 that gained solids is dispersed in into 10mLpH, and concentration is 0.1M phosphate-buffered
In liquid, the concentration for making solids is 10mg/mL, obtains Streptavidin MagneSphere.
8. the quick detection kit as claimed in claim 7 for being used to detect PD-L1, it is characterised in that the step b modifications
The preparation of chitosan magnetic microsphere comprise the following steps：
A weighs 4.02g anhydrous sodium acetates, adds 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；
B weighs 2.5g chitosans, adds 1.5mL glacial acetic acid, and stirring adds 250mL deionized waters and 250mL to being completely dissolved
Concentration is 0.1M sodium acetate buffer obtained by step A, shakes up, obtains chitosan acetate buffer；
C weighs 4.7g iron ammonium sulfates and 9.67g ammonium ferric sulfates, is ground, and it is 0.1M to add concentration obtained by 250mL steps A
Sodium acetate buffer and 100mL steps B obtained by chitosan acetate buffer, be well mixed, obtain mixed liquor；
D adds the sodium hydroxide solution that 100mL concentration is 6M into three-necked bottle, stirred under the enclosed system environment that nitrogen is protected
Mix, be heated to 60 DEG C, average to add mixed liquor obtained by step C in three times, the time interval added every time is 30 minutes, plus
Complete rear maintenance reaction 45min, with deionized water cyclic washing products therefrom, until supernatant liquid becomes clarification, uses reagent A gNO3Inspection
Sulfate ion is looked into untill without precipitation, Magneto separate obtains solid product, gained solid product is freeze-dried to obtain chitosan magnetic
Nano-particle；
E takes chitosan magnetic nano particle obtained by 0.5g steps D, adds the poly-vinyl alcohol solution that 10mL mass concentrations are 0.1%,
Ultrasonic disperse, obtains interior aqueous phase, weighs 0.6g MPEG-PLAs and is dissolved in 15mL dichloromethane, obtains oil phase, will be upper
Interior aqueous phase and oil phase mixing are stated, ultrasonic emulsification, phaco time is 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, homogenized milk
Change speed is 6000r/min, and the emulsifying time is 18min, dichloromethane is volatilized complete, obtains product, products therefrom is used
Deionized water cyclic washing, Magneto separate obtains solid product, and it is 7.0 that pH is added into gained solids, and concentration is 0.1M phosphoric acid
Salt buffer, the concentration for making solid product is 20mg/mL, obtains the chitosan magnetic microsphere of modification.
9. the quick detection kit as claimed in claim 6 for being used to detect PD-L1, it is characterised in that the biotinylation
The preparation of PD-L1 monoclonal antibodies comprises the following steps：
I takes PD-L1 monoclonal antibodies, is 0.05mol/L with concentration, and pH adjusts PD-L1 Dan Ke for 9.5 carbonate buffer solution
The concentration of grand antibody is 1mg/mL, obtains solution A；
II is dissolved in biotin, dicyclohexylcarbodiimide and n-hydroxysuccinimide in dimethylformamide, biotin, two
The mol ratio of carbodicyclo hexylimide and n-hydroxysuccinimide is 1: 1: 1.2, and magnetic agitation is reacted in 50 DEG C of water-baths, instead
It is 12h between seasonable, filters off the white precipitate dicyclohexylurea (DCU) generated in reaction solution, collects filtrate, addition ether to precipitation is no longer
Increase, is placed in ice bath, standing time is 1h, precipitation is collected by filtration, and obtains the plain ester crude product of active bio, by above-mentioned active bio element
Ester crude product is dissolved with hot isopropanol, is placed in ice bath, and standing time is 3h, collected by filtration, is dissolved with dimethylformamide
After add ether, be placed in ice bath, standing time is 1h, precipitation be collected by filtration, be dried in vacuo, obtain the plain ester of active bio；
III adds the plain ester of active bio obtained by step II, active bio element ester and PD-L1 monoclonals into step I resulting solution A
The mol ratio of 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
0.05mol/L, pH are 9.5 carbonate buffer solution dialysis 24h, add and the isometric glycerine of above-mentioned reaction solution, put -25 DEG C
Preserve, produce.
10. a kind of being used for as described in claim any one of 1-9 detects the detection method of PD-L1 quick detection kit,
It is characterised in that it includes following steps：
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 in each flat based tubes, and 37 DEG C incubate 10 points
Clock, adds 25 μ L magnetic bead immune complexs, after mixing, and 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 Magneto separate
Device is together upside down on blotting paper and patted 3 times, the liquid in hole is removed completely, and 200 μ L concentrations are added in each flat based tubes
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, which is together upside down on blotting paper, 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 based tubes 50
μ L, mix rearmounted 37 DEG C and are incubated 15 minutes；
S5 repeat steps S3 operation；
S6 adds luminous substrate solution I into flat based tubes, and the addition of luminous substrate solution I is per flat based tubes 50 μ L, 1S
Luminous substrate solution II is added afterwards, and the addition of luminous substrate solution II is per the μ L of flat based tubes 50；
S7 detects the luminous value of often pipe with Chemiluminescence Apparatus, draws standard curve according to each standard liquid luminous value, you can draw
PD-L1 concentration in sample to be tested.
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| CN107192817B (en) | 2018-04-24 |
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