CN111812326A - Kit for quantitatively detecting content of PD-L1 and PD-1 conjugate - Google Patents
Kit for quantitatively detecting content of PD-L1 and PD-1 conjugate Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a kit for quantitatively detecting the content of a PD-L1 and PD-1 conjugate, which comprises a magnetic particle reagent, an enzyme-labeled secondary antibody reagent and a chemiluminescent substrate; the magnetic microparticle reagent comprises a streptavidin-magnetic microparticle conjugate with a biotin-labeled PD L1 antibody, labeled M-SA/Bio-Anti-PD L1; the enzyme-labeled secondary antibody reagent comprises an alkaline phosphatase and PD 1 antibody coupling conjugate which is marked as AP-Anti-PD 1; the chemiluminescent substrate comprises: 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt. When the kit provided by the invention is used for detection, the result time for completing all the processes is 20min, and compared with co-immunoprecipitation, the co-localization of immunization shortens the reaction time; and has higher sensitivity, repeatability and stability.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a kit for quantitatively detecting the content of a PD-L1 and PD-1 conjugate.
Background
The two major areas of current tumor immunotherapy are: cell therapy, and immune checkpoint inhibitors represented by PD-1/PD-L1, CTLA-4/CD80, CD47/SIRP alpha. Programmed cell death-1 (PD-1) is mainly expressed in activated T/B lymphocyte, NK cell, monocyte, dendritic cell and mesenchymal stem cell membrane and cytoplasm, and taking T cell as an example, PD-1 can be combined with a ligand PD-L1 (highly expressed in tumor cell) so as to inhibit the activation and proliferation of the T cell and the secretion of cytokine, negatively regulate immune response and enable the tumor cell to have immune escape phenomenon. The immune checkpoint inhibitor can reactivate T cells, enhance immune response reaction, inhibit proliferation of tumor cells and the like by blocking a PD-1/PD-L1 signal pathway.
The combination of immune checkpoint blockers is widely recommended, which also indicates the importance of PD-1/PD-L1 in tumor treatment, at present, scientific experiments all inject PD-1 antibody or PD-L1 antibody into mice, and then determine the curative effect by detecting the content of PD-1 and PD-L1 conjugates, and because the existing methods for detecting the conjugates, such as co-immunoprecipitation, co-localization of immunity, protein mass spectrometry and the like, cannot accurately quantify, a quantitative detection method capable of detecting the content of the mouse PD-1 and PD-L1 conjugates is urgently needed.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention provides a kit for quantitatively detecting the content of a PD-L1 and PD-1 conjugate, which solves the problems and realizes the quantitative detection of the PD-L1 and PD-1 conjugate based on the magnetic particle chemiluminescence detection principle.
The invention is realized by the following technical scheme:
a kit for quantitatively detecting the content of a PD-L1 and PD-1 conjugate comprises a magnetic particle reagent, an enzyme-labeled secondary antibody reagent and a chemiluminescent substrate; the magnetic microparticle reagent comprises a streptavidin-magnetic microparticle conjugate with a biotin-labeled PD L1 antibody, labeled M-SA/Bio-Anti-PD L1; the enzyme-labeled secondary antibody reagent comprises an alkaline phosphatase and PD 1 antibody coupling conjugate which is marked as AP-Anti-PD 1; the chemiluminescent substrate comprises: 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt.
The traditional double-antibody sandwich method only detects one substance in a sample, for example, PD-L1 in the sample is detected, PD-L1 antibody 1 is marked on the magnetic particles in FIG. 1, PD-L1 antibody 2 is marked on the alkaline phosphatase, and PD-L1 antibody 1 and PD-L1 antibody 2 belong to the same kind of antibody but have different structural domains. The kit provided by the invention can simultaneously detect two different substances in a sample and can quantify the contents of the two binders as shown in figure 2. Is greatly different from the traditional double-antibody sandwich method.
More preferably, the magnetic fine particles have a diameter of 500nm to 2500nm and have superparamagnetism.
Further preferably, the concentration of the M-SA/Bio-Anti-PD L1 of the conjugate in the magnetic microparticle reagent is 0.05. mu.g/mL-5. mu.g/mL.
Specifically, the M-SA/Bio-Anti-PD L1 of the conjugate in the magnetic particle reagent is prepared by coating PD-L1 antibody magnetic particles and buffer solution, wherein the PD-L1 antibody is a Biotin-labeled PD-L1 antibody. The concentration of M-SA/Bio-Anti-PDL1 was 0.05. mu.g/mL-5. mu.g/mL. It buffersThe liquid comprises: MES buffer, Chinese name morpholine ethanesulfonic acid monohydrate; mainly used for biological buffers; and PBS-phosphate buffer (phosphate buffer saline), which is generally used as a solvent and plays a role of dissolving a protective agent, is the most widely used buffer in biochemical research and mainly contains Na2HPO4、 KH2PO4NaCl and KCl due to Na2HPO4、KH2PO4They have secondary dissociation, the pH value range of the buffer is wide, and NaCl and KCl mainly have the function of increasing the concentration of salt ions; also Tris (hydroxymethyl) aminomethane (Tris) at a concentration of 0.01M to 0.1M; the Tris (hydroxymethyl) aminomethane (Tris) contains 0.1-5 mM of Mg2+0.01M-1mM Zn2+Bovine Serum Albumin (BSA) with the mass fraction of 0.5-5%, Tween 20(Tween-20) with the mass fraction of 0.05-0.5%, trehalose with the mass fraction of 0.5-5% and glycerol with the mass fraction of 0.5-5%.
Further preferably, in the enzyme-labeled secondary antibody reagent, the concentration of the conjugate AP-Anti-PD 1 is 0.05 mu g/mL-1.5 mu g/mL. Specifically, the compound AP-Anti-PD 1 is prepared from an alkaline phosphatase-labeled PD-1 antibody and a buffer solution; wherein the concentration of the AP-Anti-PD 1 is 0.05 mu g/mL-1.5 mu g/mL. The buffer solution comprises: PBS-phosphate buffer (phosphate buffer saline) is generally used as a solvent and plays a role of dissolving a protective agent, and is the most widely used buffer in biochemical research, and the main component of the PBS-phosphate buffer is Na2HPO4、KH2PO4NaCl and KCl due to Na2HPO4、KH2PO4They have secondary dissociation, the pH value range of the buffer is wide, and NaCl and KCl mainly play a role in increasing the salt ion concentration.
Further preferred, a chemiluminescent substrate is also included, said chemiluminescent substrate comprising 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt. The present invention (AP) is alkaline phosphatase; (SA) is streptavidin. The preparation method of the chemiluminescence substrate (AMPPD) comprises the following steps: preparing a substituted phenyl adamantane-2-ketone intermediate by performing Grignard reaction and oxidation on adamantane-2-aldehyde and a Grignard reagent of substituted phenyl, constructing a carbon-carbon double bond by performing oxygen-alkylation on the intermediate, performing deprotection on phenolic hydroxyl to obtain a key intermediate substituted phenyl methoxy methylidene adamantane, and performing conventional photooxidation to obtain the AMPPD.
Further preferably, the kit also comprises a calibrator and a quality control product; the calibrator comprises PD-1 and PD-L1 fusion proteins, and the quality control product comprises PD-1 and PD-L1 fusion proteins.
Specifically, the calibrator, the constructed PD-1 and PD-L1 fusion protein, is used for calibrating a curve. The preparation method of the calibrator comprises the following steps: the constructed PD-1 and PD-L1 fusion proteins are diluted by trihydroxymethyl aminomethane (Tris) buffer solution with the concentration of 0.01M-0.1M and respectively prepared into gradient standard solutions of 400ng/mL, 200ng/mL, 50ng/mL, 20ng/mL, 5ng/mL and 0 ng/mL; the Tris (hydroxymethyl) aminomethane (Tris) contains 0.1-5 mM of Mg2+0.01M-1mM Zn2+Bovine Serum Albumin (BSA) with the mass fraction of 0.5-5%, Tween 20(Tween-20) with the mass fraction of 0.05-0.5%, trehalose with the mass fraction of 0.5-5% and glycerol with the mass fraction of 0.5-5%.
The quality control product is constructed by PD-1 and PD-L1 fusion proteins. Constructing PD-1 and PD-L1 fusion protein, and then using trihydroxymethyl aminomethane (Tris) with the concentration of 0.01M-0.1M; the Tris (hydroxymethyl) aminomethane (Tris) contains 0.1-5 mM of Mg2+0.01M-1mM Zn2+Bovine Serum Albumin (BSA) with the mass fraction of 0.5-5%, Tween 20(Tween-20) with the mass fraction of 0.05-0.5%, trehalose with the mass fraction of 0.5-5% and glycerol with the mass fraction of 0.5-5%.
The invention has the following advantages and beneficial effects:
1. when the kit provided by the invention is used for detection, the result time for completing all the processes is 20min, and compared with co-immunoprecipitation, the co-localization of immunization shortens the reaction time;
2. the invention adopts an Alkaline Phosphatase (AP) -Adamantane (AMPPD) system, and has high sensitivity;
3. the invention is a full-automatic closed operation system, and has high reliability, good stability and good repeatability of detection results;
4. the invention realizes full automation from the steps of dilution, sample adding, incubation, cleaning and detection, and avoids result deviation caused by manual operation.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a diagram illustrating the detection principle of a conventional double antibody sandwich method; the traditional double-antibody sandwich method only detects one substance in a sample, for example, PD-L1 in the sample is detected, PD-L1 antibody 1 is marked on the magnetic particles in FIG. 1, PD-L1 antibody 2 is marked on the alkaline phosphatase, and PD-L1 antibody 1 and PD-L1 antibody 2 belong to the same kind of antibody but have different structural domains.
FIG. 2 shows the detection principle of the kit of the present invention; two different substances in the sample can be detected simultaneously and the amount of the two binders can be quantified. Is greatly different from the traditional double-antibody sandwich method.
FIG. 3 shows the results of conventional immunofluorescence assays; in the figure, PD-1 is a green fluorescence diagram and shows the expression of PD-1; in the figure, PD-L1 is a red fluorescence diagram and shows the expression of PD-L1; DAPI in the figure, blue fluorescence, indicates DAPI-stained nuclei; merge in the figure is a superposition graph of red fluorescence, green fluorescence and blue fluorescence; the red fluorescence and green fluorescence indicate that PD-L1 and PD-1 are expressed in the cell membrane and cytoplasm of mouse mammary cells, and the fluorescence overlap of red and green is seen by combining the orange color of the graph, which also indicates the combination of PD-1 and PD-L1. However, the method cannot detect the content of PD-1 and PD-L1 conjugates, but the kit of the invention can detect.
FIG. 4 is a standard curve based on a calibrator.
FIG. 5 is a detection map; indicating no detection by the addition of dd H2O and detection by the addition of samples containing PD-1 and PD-L1.
FIG. 6 is a graph showing the results of stability tests.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1
The embodiment provides a kit for quantitatively detecting the content of a PD-L1 and PD-1 conjugate, which comprises a magnetic particle reagent, an enzyme-labeled secondary antibody reagent, a chemiluminescent substrate, a cleaning concentrated solution, a calibrator and a quality control product;
the magnetic particle reagent is a conjugate of streptavidin-magnetic particles and biotin-labeled PD L1 antibody, and is labeled as M-SA/Bio-Anti-PD L1. Specifically, before preparation, magnetic particles with the diameter of 500nm are selected, and the magnetic particles have superparamagnetism; coupling Biotin (Biotin) with an antibody PD L1(Anti-PD L1) by using the existing mature coupling technology; the biotin-labeled Anti-PD L1 was conjugated with streptavidin-magnetic microparticles (SA-M) to finally obtain Anti-PDL1 coated magnetic microparticles (M-SA/Bio-Anti-PD L1) with the concentration of conjugate M-SA/Bio-Anti-PD L1 in the magnetic microparticle reagent being 5. mu.g/mL.
The enzyme-labeled secondary antibody reagent is a conjugate of alkaline phosphatase and a PD 1 antibody, and is marked as AP-Anti-PD 1. Specifically, the existing mature coupling technology is utilized to couple Alkaline Phosphatase (AP) with PD 1 antibody (Anti-PD 1); finally obtaining an R2 reagent of enzyme-labeled Anti-PD 1, wherein the concentration of the conjugate AP-Anti-PD 1 in the enzyme-labeled secondary antibody reagent is 1.5 mu g/mL;
the chemiluminescent substrate comprises: 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphoryl-oxy) -phenyl-1, 2-dioxane disodium salt, the main component of which is Adamantane (AMPPD), releases photons under the catalysis of alkaline phosphatase.
The calibrator, the constructed PD-1 and PD-L1 fusion protein is used for calibrating a curve. The preparation method of the calibrator comprises the following steps: the constructed PD-1 and PD-L1 fusion proteins are diluted by trihydroxymethyl aminomethane (Tris) buffer solution with the concentration of 0.01M-0.1M and respectively prepared into gradient standard solutions of 400ng/mL, 200ng/mL, 50ng/mL, 20ng/mL, 5ng/mL and 0 ng/mL; the Tris (hydroxymethyl) aminomethane (Tris) contains 2mM of Mg2+0.1mM Zn2+Bovine Serum Albumin (BSA) with the mass fraction of 3%, Tween 20(Tween-20) with the mass fraction of 0.09%, trehalose with the mass fraction of 1% and glycerol with the mass fraction of 2%.
The quality control product is constructed by PD-1 and PD-L1 fusion proteins. Constructing PD-1 and PD-L1 fusion protein, and then using trihydroxymethyl aminomethane (Tris) with the concentration of 0.01M-0.1M; the Tris (hydroxymethyl) aminomethane (Tris) contains 2mM of Mg2+0.1mM Zn2+Bovine Serum Albumin (BSA) with the mass fraction of 3%, Tween 20(Tween-20) with the mass fraction of 0.09%, trehalose with the mass fraction of 1% and glycerol with the mass fraction of 2%.
The method comprises the steps of combining a PD L1 antibody (Anti-PD L1) marked by Biotin (Biotin) with streptavidin-magnetic particles (SA-M), finally obtaining magnetic particles M-SA/Bio-Anti-PD L1, then adding a sample to be detected, and finally adding an enzyme-labeled secondary antibody for reaction.
The method comprises the steps of combining a PD L1 antibody (Anti-PD L1) marked by Biotin (Biotin) with streptavidin-magnetic particles (SA-M), finally obtaining magnetic particles M-SA/Bio-Anti-PD L1, then adding a sample to be detected, and finally adding an enzyme-labeled secondary antibody for reaction.
Example 2
In this embodiment, taking a tumor tissue sample of mouse breast cancer (4T1) as an example, a tumor tissue of a mouse is taken out, and then a whole protein is extracted, and then the detection method of the present invention is used to perform subsequent detection, specifically including the following steps:
1. preparing immunomagnetic beads: the magnetic material is prepared by a chemical coprecipitation method, brown round magnetic particles with the particle size of 500nm are selected and washed by 0.05mol/L MES buffer solution, then 1.25% glutaraldehyde and 0.05mol/L PBS with the PH of 6.0 are added for oscillation reaction for 1 hour, and then 0.05mol/L MES buffer solution is used for washing to obtain activated magnetic beads; then 4mg of PD-L1 antibody was pipetted into a 1.5ml centrifuge tube, 10mM NHS 53.2ul was added, mixed well, and left to react at room temperature for 30 min. Adding 1M Tris buffer solution to the final concentration of 10mM, terminating the reaction, standing at room temperature for 10min, taking out the labeled biotin antibody, measuring the volume, and adding the same volume of glycerol for preservation at-20 ℃. Taking out the activated magnetic beads and the PD-L1 antibody, incubating for 1h at 25 ℃ according to the proportion of 1mg of magnetic beads and 250 mu g of antibody, magnetically separating, adding 2% BSA to block free radicals, and washing with MES again to obtain immunomagnetic beads;
2. preparation of enzyme-labeled antibody: taking out 1mg of HRP, dissolving in 0.5mL of distilled water, fully dissolving, taking 0.125mL for marking, adding 0.125mL of freshly prepared 12mmol/L sodium periodate distilled water solution, mixing uniformly, standing at 2-8 ℃ for 30min, adding into 0.125mL of 32mmol/L ethylene glycol distilled water solution, mixing uniformly, standing at room temperature in a dark place for 30min to stop the action of sodium periodate, adding 0.25mg of PD-1 antibody into the solution, mixing uniformly, filling into a dialysis bag, and dialyzing for 16-24h at 2-8 ℃ by using 0.05mol/L sodium carbonate buffer solution (PH is 9.5). Finally, taking out the solution, adding a sodium borohydride solution, standing for 2 hours, dialyzing by using a PBS solution with the concentration of 0.02mol/LPH7.4, finally taking out, centrifuging, measuring the volume, and adding glycerol with the same volume for storage;
3. detection of PD-1 and PD-L1 conjugates in mouse breast cancer tumor tissue: tumor tissues are taken out, whole protein is extracted, protein quantification is carried out by using the Kamasie brilliant blue, the same amount of protein is taken out from each group, then magnetic particles coated with PD-L1 antibody with the concentration of 0.05 mu g/mL are added, and enzyme labeled PD-1 with the concentration of 0.05 mu g/mL is added.
And (4) analyzing results: after the magnetic particles are activated, centrifuged and coupled with the antibody, the magnetic particles can be uniformly dispersed in the solution without agglomeration. The binding of PD-1 and PD-L1 was detected by immunofluorescence in-aid before the content of the conjugate was determined, orange fluorescence of the conjugate as shown in FIG. 2 illustrates the immunological co-localization, i.e., there was binding between PD-1 and PD-L1, and the binding content of PD-1 and PD-L1 was detected by the kit of the present invention as shown in FIG. 5, dd H was added2O was not detected, while samples with the addition of PD-1 and PD-L1 detected an average concentration of 15.64 ng/ml.
Example 3
This example performed an effect test on the detection result of example 2.
1. And (3) testing precision: respectively measuring the quality control substances of 400ng/mL, 50ng/mL and 5ng/mL twice a day, repeating the measurement for 4 times each time, measuring for 10 days totally, measuring each concentration for 80 times totally, calculating the variation coefficient, and measuring the precision of the detection reagent in the samples of 400ng/mL, 50ng/mL and 5 ng/mL. The results show that the coefficient of variation is within 8 percent, and the repeatability is good.
TABLE 1 results of precision measurement
2. Stability: the detection kit (M, R2) of the invention is respectively placed at 37 ℃ for 7 days, and the signal retention rates of the three concentrations of 400ng/mL, 50ng/mL and 5ng/mL are measured. As shown in the results of FIG. 6, the results are all more than 95%, which indicates that the kit is stable well.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A kit for quantitatively detecting the content of a PD-L1 and PD-1 conjugate is characterized by comprising a magnetic particle reagent, an enzyme-labeled secondary antibody reagent and a chemiluminescent substrate;
the magnetic microparticle reagent comprises a streptavidin-magnetic microparticle conjugate with a biotin-labeled PD L1 antibody, labeled M-SA/Bio-Anti-PD L1;
the enzyme-labeled secondary antibody reagent comprises an alkaline phosphatase and PD 1 antibody coupling conjugate which is marked as AP-Anti-PD 1;
the chemiluminescent substrate comprises: 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt.
2. The kit for quantitatively detecting the content of the PD-L1 and PD-1 conjugate according to claim 1, wherein the magnetic particles have a diameter of 500nm to 2500nm and are superparamagnetic.
3. The kit for quantitatively detecting the content of the PD-L1 and PD-1 conjugate according to claim 1, characterized in that the concentration of the M-SA/Bio-Anti-PD L1 of the conjugate in the magnetic particle reagent is 0.05 μ g/mL-5 μ g/mL.
4. The kit for quantitatively detecting the content of the PD-L1 and PD-1 conjugate according to claim 1, characterized in that the concentration of the conjugate AP-Anti-PD 1 in the enzyme-labeled secondary antibody reagent is 0.05 μ g/mL-1.5 μ g/mL.
5. The kit for quantitatively detecting the content of the PD-L1 and PD-1 conjugate according to claim 1, characterized by further comprising a chemiluminescent substrate comprising 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt.
6. The kit for quantitatively detecting the content of the PD-L1 and PD-1 conjugate according to claim 1, characterized by further comprising a calibrator and a quality control; the calibrator comprises PD-1 and PD-L1 fusion proteins, and the quality control product comprises PD-1 and PD-L1 fusion proteins.
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