CN112986575B - Method for verifying DNA coding Miao compound modified antibody in tandem manner - Google Patents
Method for verifying DNA coding Miao compound modified antibody in tandem manner Download PDFInfo
- Publication number
- CN112986575B CN112986575B CN202011598849.5A CN202011598849A CN112986575B CN 112986575 B CN112986575 B CN 112986575B CN 202011598849 A CN202011598849 A CN 202011598849A CN 112986575 B CN112986575 B CN 112986575B
- Authority
- CN
- China
- Prior art keywords
- solution
- antibody
- functional molecule
- amphiphilic functional
- target protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—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
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Abstract
The invention discloses a method for tandem verification of DNA coding screening molecular modification antibody function, which improves the capacity of antibody recognition target spots from biological exogenous and endogenous levels through molecular modification mass spectrum detection experiments, protein combination verification experiments, cell membrane protein combination verification experiments and immunohistochemical verification experiments on obtained amphiphilic functional molecules respectively targeting a target protein and an aldolase antibody. The experimental method can accurately, sensitively and real-timely verify that the antibody enhances the capacity of specifically identifying and recognizing the target protein through the modification of the amphiphilic functional molecule, provides verification data and method support for the modification and optimization of the amphiphilic functional molecule and subsequent in vivo functional experiments, lays a research foundation for the wide application of a DNA coding compound in the research and development of the amphiphilic functional molecule and the clarification of the function, and enables the processes of screening, identifying and verifying the target protein molecule based on the source of the DNA coding compound to be standardized, accurate, digitalized and quantitative.
Description
Technical Field
The invention belongs to the field of drug screening, and particularly relates to a method for verifying modification of a DNA coding head compound and modification of an aldolase antibody by using the molecule, in particular to a method for verifying an amphiphilic functional molecule modified antibody from the DNA coding head compound in a tandem manner.
Background
DNA Encoded Library (DEL) can be obtained in a short time by the synthesis of molecular building blocks of DNA sequence connecting marker compounds according to the combinatorial chemistry mode. The application of Polymerase Chain Reaction (PCR) and Next Generation Sequencing (NGS) can efficiently infer the molecular composition of the compound, and overcomes the dependence on high-sensitivity mass spectrum from the detection level. Thus, DEL is becoming an important tool for the discovery of novel head compounds. The DEL technology is used for identifying the seedling head compound by incubating DEL and target protein, eluting the product, incubating the product with the target protein again, obtaining DEL molecules with high affinity with the target protein through multi-round enrichment, and then obtaining compound molecular information corresponding to a DNA sequence through quantification, amplification, sequencing and translation. And the data analysis link calculates according to the enrichment degree of the compound, and judges the affinity of the binding DEL molecules according to the enrichment value and the number of the molecules identified by sequencing, so as to deduce potential seedling-end compound molecules. After the structure and the biological activity of a parent nucleus of a miaow compound are determined, a lead compound is generally formed through primary structure modification and optimization, so that the selectivity and the biological activity of the lead compound are improved on the basis of the miaow compound, and the physicochemical property and the drug toxicity property of the lead compound are improved.
The antibody is used as a representative of biological macromolecules, and has the function of helping host cells to recognize specific antigens, so that the purpose of eliminating pathogens is achieved through the antibody-antigen immunity. The production of antibodies depends on B lymphocytes, and in the state where helper T cells present antigens, the antibodies specifically and uniquely recognize the antigens through their variable regions, and thus, the antibodies have extremely strong specificity. Engineering of antibodies is usually started from genetic engineering or protein engineering, and antibody expression sequences can be optimized at the DNA level, and specificity and biological activity of antibodies can be enhanced at the protein level by means of cell fusion, chemical modification and the like.
At present, the application of DEL technology and the miao compounds in the field of biochemistry is in the beginning, and few researches report that the miao compounds are used for modifying antibody macromolecules and function researches thereof. The DEL screening technology is combined with antibody engineering modification, potential leptin molecules obtained based on the DEL screening technology are fused with diketone molecules recognized by an aldolase antibody, and the potential leptin molecules can be directly applied to the engineering modification of the aldolase antibody and used for determining the functions of fused amphipathic molecules through a tandem type verification method.
Disclosure of Invention
The invention aims to solve the technical problems of verifying whether the aldolase antibody retains the capability of highly recognizing the target protein after being modified by the amphiphilic functional molecule, avoiding the time-consuming and tedious process of independently constructing the target protein recognition antibody, simultaneously verifying the function of diversified potential DNA coding miaow head compounds, effectively improving the structure-activity relationship and the research on the drug property of the DNA coding miaow head compounds, and reducing the modification and optimization cost of the DNA coding miaow head compounds. The invention utilizes a tandem type verification method to detect the function of the amphiphilic functional molecule modified antibody from the DNA coding miao-cephalin compound, can effectively reduce the interference of negative binding compounds and improve the success rate of the application of the amphiphilic functional molecule modified antibody.
In order to solve the problems in the prior art, the method adopts a tandem type verification method to detect and determine the function of the amphiphilic functional molecule modified antibody from the DNA coding shoot head compound, and can be applied to DEL screened shoot head compounds for antibody modification and function verification research thereof.
The technical scheme adopted by the invention is as follows:
a method for tandem validation of DNA-encoded molecular modification of antibodies to Miao compounds, comprising:
performing affinity screening of a DNA coding compound library aiming at target protein, sequencing the enriched DNA coding compounds from high to low according to the calculated values and the sequencing copy number of the enriched DNA coding compounds, determining the molecular sequence information of potential DNA coding seedling head compounds and designing a synthetic path of the seedling head compounds with DNA labels removed;
a chemical synthesis method is used for redesigning a route to synthesize a potential DNA label-removed seedling-end compound, and an adaptor is used for coupling diketone molecules to form amphiphilic functional molecules with two ends respectively containing the seedling-end compound derived from a DNA coding compound and the diketone molecules. The amphiphilic functional molecule can respectively target a target protein and an aldolase antibody through potential DNA coding of the two ends of the amphiphilic functional molecule;
for said amphiphilic functional molecules obtained targeting the target protein and the aldolase antibody, respectively, the ability of the antibody to recognize the target is enhanced from biologically exogenous and endogenous levels by the following steps, including:
(1) The action mode of the amphiphilic functional molecule and the aldolase antibody is specifically identified through a molecule modification mass spectrum detection experiment;
(2) Detecting the interaction between the target protein and the aldolase antibody through the amphipathic molecule by a protein combination verification experiment;
(3) Verifying the expression of the target protein on the surface of the cell membrane and the capability of coupling the target protein with the aldolase antibody through the amphiphilic functional molecule by a cell membrane protein combination verification experiment;
(4) The expression of the target protein in the tissues is further localized by immunohistochemical validation experiments of the amphiphilic functional molecule assembled antibodies.
As a preferable technical scheme of the invention, the step (1) specifically comprises the following steps: prepare the amphiphilic molecule stock solution, dissolve the amphiphilic molecule with 100% dimethyl sulfoxide, make its final concentration 20mM. According to the mass spectrum detection requirement, the combination of the amphiphilic functional molecules and the aldolase antibody is set to verify an experiment reference group and an experiment group sample, wherein the reference group sample is the aldolase antibody dissolved in the mass spectrum affinity buffer solution, and the protein amount of the sample is 20 mu g; the samples of the experimental group comprise the amphiphilic functional molecules and the aldolase antibodies, wherein the total amount of the aldolase antibodies in each group is 20 mu g, and the final concentration of the amphiphilic functional molecules in the reaction system is 25 mu M.
As a preferred embodiment of the present invention, the components of the mass spectrometry affinity screening buffer are 1 PBS 20 buffer, 1 PBS, pH 7.4,0.05% v/v Tween 20, and each sample is 50. Mu.L.
As a preferred technical solution of the present invention, the step (1) specifically comprises: the samples of the reference group and the experimental group were placed at 37 ℃ and the mixer set to a vertical rotation speed of 50rpm/min for one hour of incubation. Samples were then collected and molecules of compounds that did not react with the aldolase antibody were intercepted using a 7K gel exclusion chromatography column and the eluate collected for mass spectrometric detection.
As a preferable technical scheme of the invention, the following steps are added before the step (2): washing Protein G immunomagnetic beads with a washing buffer solution for three times, and respectively adsorbing the magnetic beads by using a magnetic frame for affinity each time and then discarding the supernatant of the washing buffer solution; after the last washing, subpackaging immunomagnetic beads with 20 mu L of each tube to enable the immunomagnetic beads to correspond to reference group samples and experimental group samples one by one, wherein the reference group samples are respectively aldolase antibody solutions dissolved in a binding buffer solution; a solution of the target protein dissolved in a binding buffer; after mixing, adding no amphiphilic functional molecule solution; and a protein-free group.
As a preferred technical solution of the present invention, the step (2) specifically includes: dissolving 2 mu G of aldolase antibody/group by using a binding buffer solution, and respectively adding a reference group sample and an experimental group sample solution into the washed Protein G immunomagnetic beads respectively; and setting a vertical rotation speed of a blending instrument at 50rpm/min, incubating for half an hour at room temperature to enable the aldolase antibody to coat the magnetic beads, then discarding the supernatant, washing the aldolase antibody coated magnetic beads once by using a binding buffer solution, slightly blowing for resuspension, utilizing a magnetic frame for affinity adsorption of the magnetic beads, then discarding the supernatant, and retaining the aldolase antibody-magnetic bead coated compound.
As a preferred embodiment of the present invention, the washing buffer composition is 1 TBS Tween 20 buffer, 25mM Tris,0.15M NaCl,0.05% Tween 20, pH 7.5, each sample requires 600. Mu.L. The binding buffer was 1 × TBS Tween 20 buffer, 25mM Tris,0.15M NaCl,0.05% v/v Tween 20, pH 7.5, fresh affinity screening buffer was prepared before each experiment, and 200. Mu.L of each sample was used.
As a preferred technical solution of the present invention, the step (2) specifically includes: the parental functional molecule stock was diluted in a gradient of binding buffer to a final concentration of 10. Mu.M. The solutions of the amphipathic molecules dissolved in the binding buffer were added to the samples of the experimental group, and the samples of the reference group were added to solutions of the same concentration of dimethylsulfoxide dissolved in the binding buffer (0.05% v/v), with a total volume of 50. Mu.L for each group. And placing the reference group and the experimental group samples in a 37 ℃ incubator for incubation for one hour, then collecting magnetic beads, discarding supernatant, washing the bifunctional molecule-aldolase antibody coated magnetic beads with a binding buffer solution for three times, gently blowing and resuspending each time, then carrying out affinity adsorption on the magnetic beads by using a magnetic frame, discarding supernatant, and reserving the bifunctional molecule-aldolase antibody-magnetic bead coated compound.
As a preferred technical solution of the present invention, the step (2) specifically includes: dissolving 2 mu g of target protein/group by using a binding buffer solution to enable the final volume to be 50 mu L, respectively adding the target protein solution into a reference group and an experimental group of the amphiphilic functional molecule-aldolase antibody-magnetic bead coating compound, incubating for one hour at room temperature, then collecting magnetic beads, abandoning supernatant, washing the target protein-bifunctional molecule-aldolase antibody coated magnetic beads by using the binding buffer solution for three times, gently blowing and resuspending each time, then utilizing a magnetic frame to perform affinity adsorption on the magnetic beads, abandoning supernatant, and reserving the target protein-bifunctional molecule-aldolase antibody-magnetic bead coating compound. And detecting whether the target protein is subjected to immunoprecipitation with an aldolase antibody by the action of the amphipathic molecule by using a Western Blot method.
As a preferable technical scheme of the invention, the following steps are added before the step (3): constructing a target protein expression plasmid by using a pLV2 vector, cloning a target protein coding sequence into the pLV2 expression vector, sequencing to obtain a plasmid with a correct sequence and the target protein coding sequence, and amplifying for later use. FreeStyle 293 cells were cultured in FreeStyle 293 complete medium, passaged after the cells had grown to a logarithmic phase, and the passaged cells were cultured overnight to overexpress the plasmid. The plasmid constructed with the target protein was mixed with a transfection reagent, added to FreeStyle 293 cells, transfected 6 hours later and replaced with FreeStyle 293 complete medium, and cells were counted 48 hours later. Cells from 1E6 were removed to detect the level of target protein overexpression. Incubating cells and a specific primary antibody of a target protein for one hour at 37 ℃, washing for three times by using a cell affinity buffer solution, re-suspending the collected cells by using the cell affinity buffer solution after each low-speed centrifugation, then carrying out fluorescent staining on the collected cells and a secondary antibody corresponding to the primary antibody of the target protein, incubating for one hour at room temperature, then washing for three times by using the cell affinity buffer solution, re-suspending the cells by using 1mL of the cell affinity buffer solution after collecting the cells, and deducing the expression level of the cell membrane target protein by using the intensity of fluorescent expression in a flow cytometer. The remaining cells were subjected to cell membrane protein binding validation experiments.
In a preferred embodiment of the present invention, the cell affinity buffer is 1 × hbss buffer, pH 6.7-7.8, and fresh cell affinity screening buffer is prepared before each experiment, wherein 600 μ L of each sample is required.
As a preferred technical solution of the present invention, the step (3) specifically includes: the number of reaction tubes required for the reference and experimental groups was calculated, and 20. Mu.g of aldolase antibody per group was dissolved in cell affinity buffer to give a final volume of 25. Mu.L, while the amphipathic functional molecule stock was diluted with a gradient of cell affinity buffer to give a final concentration of 50. Mu.M and a final volume of 25. Mu.L. Mixing the aldolase antibody solution and the amphiphilic functional molecule diluent in equal volume to ensure that the final volume of the mixed solution is 50 mu L and the final concentration of the amphiphilic functional molecule solution is 25 mu M, placing the samples of the reference group and the experimental group at 37 ℃, setting the vertical rotation speed of a mixing instrument to be 50rpm/min, and incubating for one hour. Samples were then collected and the incubation solution was filtered using a 7K gel exclusion chromatography column, the molecules of the compound that did not react with the aldolase antibody were intercepted, and the eluate (i.e. the solution of the amphiphilic molecule and aldolase antibody complex) was collected for cell incubation.
As a preferred technical solution of the present invention, the step (3) specifically includes: collecting FreeStyle 293 cells expressing substrate protein and completing counting, centrifuging 1E6 cells/group and then discarding supernatant, mixing the eluted amphiphilic functional molecule-aldolase antibody compound solution with the cells, wherein the final volume of the mixed solution is 50 mu L, shaking and incubating for one hour in a mixer at 37 ℃, collecting the cells, centrifuging at low speed, then discarding supernatant, washing the cells once with cell affinity buffer solution, centrifuging and then discarding supernatant, labeling aldolase antibody with fluorescent secondary goat anti-mouse IgG, and shaking and incubating for one hour in a mixer at room temperature. And then collecting the cell, centrifuging at a low speed, discarding the supernatant, washing the cell once by using a cell affinity buffer solution, resuspending the cell by using a cell affinity buffer solution after centrifuging at a low speed to prepare a cell suspension, detecting the intensity of fluorescence expression by using a flow cytometer, and deducing the interaction between the target protein expressed on the surface of the cell membrane and the aldolase antibody through the connection of the amphiphilic functional molecule and the difference between the obtained fluorescence expression intensity in a reference group and an experimental group.
As a preferred technical solution of the present invention, the step (4) specifically comprises: the number of reaction tubes required for the reference and experimental groups was calculated and 20. Mu.g of aldolase antibody/group was dissolved in mass-spectrometric affinity buffer to give a final volume of 25. Mu.L, while the amphiphilic functional molecule stock was diluted with a mass-spectrometric affinity buffer gradient to give a final concentration of 50. Mu.M and a final volume of 25. Mu.L. Mixing the aldolase antibody solution and the amphiphilic functional molecule diluent in equal volume to ensure that the final volume of the mixed solution is 50 mu L and the final concentration of the amphiphilic functional molecule solution is 25 mu M, placing the samples of the reference group and the experimental group at 37 ℃, setting the vertical rotation speed of a mixing instrument to be 50rpm/min, and incubating for one hour. Samples were then collected and incubated solutions were filtered using 7K gel exclusion chromatography plates to intercept molecules of the compound that did not react with aldolase antibody and the eluate (i.e. solution of amphiphilic molecule and aldolase antibody complex) was collected for immunohistochemistry.
As a preferred technical solution of the present invention, the step (4) specifically includes: the immunohistochemical material is prepared by selecting human placenta tissue, slicing the purchased human placenta tissue paraffin block, baking for one hour in a constant temperature oven at 60 ℃, then performing immersion dewaxing operation in xylene and gradient alcohol, and washing and hydrating the slices with distilled water. Repairing the antigen by using EDTA antigen repairing solution in a high-temperature water bath at 93 ℃ and 98 ℃ for 20 minutes respectively, cooling to room temperature, and then cleaning with distilled water. And then 3% hydrogen peroxide is used for incubation for 10 minutes at room temperature, and the inactivation of the endogenous peroxidase of the tissue section is completed after the tissue section is washed by distilled water.
As a preferred technical solution of the present invention, the step (4) specifically includes: and (3) dropwise adding the eluted amphiphilic functional molecule-aldolase antibody compound solution to the center of the section and completely covering the section tissue, incubating for one hour at room temperature, and washing with PBS for three times. After the PBS solution is completely absorbed, CRF anti-multiple species (rabbit, rat/mouse, guinea pig) HRP polymer labeled secondary antibody is added to completely cover the tissue, the tissue is incubated at room temperature for half an hour to one hour, then PBS is washed for three times, and distilled water is washed for one time.
As a preferred technical solution of the present invention, the step (4) specifically includes: staining the finished section with hematoxylin after DAB (diaminobenzidine) color development, incubating for 2 minutes at room temperature, washing the section with PBS or tap water, soaking gradient alcohol for dehydration after the section turns blue, soaking dimethylbenzene to make the section transparent, sealing the section with neutral gum, and taking a picture by microscopic examination to facilitate subsequent data collection.
The adaptor (Linker) in the present invention refers to a portion between a conjugated diketone molecule and a headpiece compound that binds to a target protein in an amphiphilic functional molecule. Specifically, it may be a DNA strand, a polyethylene glycol strand, an alkyl chain or the like.
The Diketone molecule (Diketone) in the present invention refers to an organic compound containing two carbonyl groups specifically recognized by an aldolase antibody in the present invention. The simplest diketone is butanedione, i.e., 2,3-butanedione.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for effectively identifying an amphiphilic functional molecule modified antibody and verifying the function of the antibody in tandem. By utilizing the mass spectrum identification technology, the immunoprecipitation technology, the flow cytometry detection technology and the immunohistochemical technology, the specific recognition capability of the amphiphilic functional molecules on the target protein and the aldolase antibody can be directly applied to the universal antibody combined with the target protein, the operation of simplification and repetition of directly synthesizing the specific engineered antibody is avoided, the physicochemical property and the biological activity of the potential DNA coding vaccine head compound combined with the target protein with high reliability are provided, the interaction mode of the amphiphilic functional molecules, the target protein and the universal antibody is further favorably applied in vivo, the application of the DNA coding compound library screening to the modification and optimization of the lead compound is enlarged, the application range of the screening is improved, and the cost of chemically synthesized molecules is reduced. Compared with the traditional method for expressing and purifying the engineered antibody, the method is connected with the screening method of the DNA coding compound library in series, so that the flux of drug screening and the universal range of the universal antibody can be greatly promoted, the application range of the screened compound is improved, and the time is reduced to 3-6 months from 8-10 months; meanwhile, the screening cost including labor cost and material cost can be reduced. At present, the development of the technology is not reported in the prior art, so that the method has larger potential market space and application value.
Drawings
FIG. 1 is a schematic flow diagram of the process of the present invention.
FIG. 2 is a schematic diagram showing the result of mass spectrometric identification of the antibody against the amphiphilic functional molecule-modified aldolase of example 1 in accordance with the present invention.
FIG. 3 is a graph showing the results of in vitro interaction of the amphiphilic functional molecule of example 1 of the present invention with an aldolase antibody.
FIG. 4 is a schematic diagram showing the interaction between the amphiphilic functional molecule of example 1 of the present invention and the antibody against aldolase on the surface of cell membrane.
FIG. 5 is a graphical representation of the immunohistochemical results of the interaction of the amphiphilic functional molecule of example 1 of the present invention with the aldolase antibody.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1: tandem verification of function of amphiphilic functional molecule modified antibody derived from DNA coding Miao-headed compound on PD-L1 target protein to confirm feasibility of method
1. Background: PD-L1 plays an important regulatory role in tumor immunity as an immune checkpoint inhibitor, and in this case, purchased His-tagged PD-L1 was used as a target protein to validate the method. Aldolase antibodies murine 38C2 protein was selected as the engineered antibody target.
2. The implementation method comprises the following steps:
as shown in fig. 1, the method specifically includes the following steps:
1. buffer preparation
10 XPBS (Thermo fisher, USA), 100% Tween 20 (Sigma-Aldrich, USA), 100% dimethyl sulfoxide (Sigma-Aldrich, USA); 1 hbss (Invitrogen, usa); lipofectamine 3000 (Thermo fisher, USA); freeStyle 293 complete medium (Thermo fisher, usa); bovine serum albumin (Sigma-Aldrich, usa); PD-L1 monoclonal antibody (Abcam, USA); goat anti-mouse fluorescent secondary antibody (Abcam, usa); goat anti-rabbit fluorescent secondary antibody (Abcam, USA).
Preparing a mass spectrum affinity buffer: 1 × PBS,0.05% tween 20;
preparing a binding buffer solution: 25mM Tris,0.15M NaCl,0.05% v/v Tween 20, pH 7.5;
preparing a cell affinity buffer solution: HBSS buffer, pH 6.7-7.8
2. Aldolase antibody solution preparation
Aldolase antibody 38C2 was dissolved in mass-spectrometric affinity buffer in a total amount of (number of groups + 1) × 20 μ g and distributed to the reference group and the experimental group.
3. Bifunctional molecular solution preparation
Bifunctional molecules (10150-18-34-40-diketone and 10150-185-34-40-diketone) were weighed and the required volume of solution for the compound molecule at a final concentration of 20mM was calculated from the weighed mass = molarity volume molecular weight. The weighed compound was dissolved in diluted 100% dimethylsulfoxide and the stock was diluted to 50 μ M with a mass-spectrometric affinity buffer gradient.
4. Affinity incubation
The compounds were mixed with the aldolase antibody at equal volumes such that the final volume of each group was 50. Mu.L and the final concentration of the compound was 25. Mu.M. Incubate at 37 ℃ for one hour with tumbling.
5. Post incubation complex collection
The 7K gel exclusion chromatography column was rinsed with distilled water 200. Mu.L/lot, 1. Mu.L PBS 200. Mu.L/lot, and 1. Mu.L PBS 200. Mu.L/lot in that order, during which time 1200rpm, and the rinse was removed by centrifugation for 2 minutes. After incubation, the samples were loaded on gel exclusion chromatography columns, and the eluates were collected by centrifugation at 1200rpm for 2 minutes.
6. Mass spectrometric detection
The eluted product was analyzed for the degree of modification of the aldolase antibody 38C2 by the amphipathic molecule using a BioAccord LC-MS System (Waters, USA) liquid mass spectrometer and the number of the modified molecules was counted. The results of the detection are shown in FIG. 2. As can be seen from FIG. 2, the results of protein mass spectrometry of the aldolase antibody group only showed that the aldolase antibody contained the molecular weights of 150730/150933/151093/151256/151409 Da; after the amphiphilic functional molecules 101050-18-34-40-diketone are added, except for the original molecular weight, the molecular weight of combining one amphiphilic functional molecule (DAR =1, +1330 Da) 152263/152422/152586Da and the like and the molecular weight of combining two amphiphilic functional molecules (DAR =2, +2660 Da) 153593/153752/153916Da and the like appear; after addition of the amphiphilic molecules 101050-185-34-40-diketone, in addition to the original molecular weight, there appear molecular weights (DAR =1, +1402 Da) 152335/152495/152662Da, etc. binding to one amphiphilic molecule, and molecular weights (DAR =2, +2804 Da) 153738/153900/154064Da, etc. binding to two amphiphilic molecules. The above results indicate that the aldolase antibody can form a stable complex with the amphiphilic functional molecule.
7. Magnetic bead preparation
Protein G immunomagnetic beads (Thermo fisher, usa) were placed in a vortex shaker (Scientific Industries, usa) and shaken for 30 seconds, 10 μ L to 1.5ml low adsorption centrifuge tubes (Eppendorf, germany) were pipetted (number of sets + 1) and the centrifuge tubes were placed in a magnetic rack (Thermo fisher, usa) to adsorb the beads and the supernatant was pipetted off. The beads were washed three times with 50 μ L (number of sets + 1) of binding buffer, each time the centrifuge tube was placed on a magnetic rack to adsorb the beads and the supernatant was discarded, and the bead storage buffer was completely removed (packing buffer).
8. Aldolase antibody 38C2 immobilization
Mu.g/group of aldolase antibody 38C2 was dispensed according to the reference group 1 only PD-L1 target Protein group, the reference group 2 only aldolase antibody 38C2 group, the reference group 3 no Protein group, the reference group 4 no amphipathic functional molecule group, the experimental group 1 no amphipathic functional molecule group, the experimental group 2 positive control group (BMS 202-diketone), the experimental group 3 amphipathic functional molecule group (10150-18-34-40-diketone), the experimental group 4 amphipathic functional molecule group (10150-185-34-40-diketone), and added to the corresponding washed Protein G immunomagnetic bead group with a final volume of 50. Mu.L. Incubate for 30 minutes with inversion at room temperature. Placing the centrifugal tube on a magnetic frame, and sucking supernatant liquid by using a liquid-transfering gun; the beads were washed once with 200. Mu.L/set of binding buffer, the centrifuge tube was placed on a magnetic rack to adsorb the beads and the supernatant was discarded.
9. Amphiphilic functional molecule incubation
Adding the bifunctional molecules (BMS 202-diketone/10150-18-34-40-diketone/10150-185-34-40-diketone) into the corresponding tubes according to a reference group 1 only PD-L1 target protein group, a reference group 2 only aldolase antibody 38C2 group, a reference group 3 no protein group, a reference group 4 no bifunctional molecule group, an experimental group 1 no amphiphilic molecule group, an experimental group 2 positive control group (BMS 202-diketone), an experimental group 3 bifunctional molecule group (10150-18-34-40-diketone), and an experimental group 4 bifunctional molecule group (10150-185-34-40-diketone), wherein the final volume is 50 muL. Incubate at 37 ℃ for one hour with tumbling. Placing the centrifugal tube on a magnetic frame, and sucking supernatant liquid by using a liquid-transfering gun; the beads were washed three times with 200. Mu.L/set of binding buffer, each time placing the centrifuge tube on a magnetic rack to adsorb the beads and discarding the supernatant.
PD-L1 target protein incubation
Mu.g/group of PD-L1 target protein was dispensed according to the reference group 1 only PD-L1 target protein group, the reference group 2 only aldolase antibody 38C2 group, the reference group 3 no protein group, the reference group 4 no amphipathic molecule group, the test group 1 no amphipathic molecule group, the test group 2 positive control group (BMS 202-diketone), the test group 3 bifunctional molecule group (10150-18-34-40-diketone), the test group 4 amphipathic molecule group (10150-185-34-40-diketone), and the final volume was 50. Mu.L. At room temperature, after one hour incubation with tumbling, 2.5. Mu.L of each group was collected as Input samples. Placing the centrifugal tube on a magnetic frame, and sucking supernatant liquid by using a liquid-transfering gun; the beads were washed three times with 200. Mu.L/set of binding buffer each time, the centrifuge tubes were placed on a magnetic rack to adsorb the beads and the supernatant was discarded.
Western Blot assay
mu.L of loading buffer was added to each group, the samples were boiled at 95 ℃ for 10 minutes, subjected to 120V electrophoresis for 40 minutes, and then subjected to PVDF membrane transfer operation, 1.3A-25V for 7 minutes, after which the PVDF membrane was blocked with 5% Bovine Serum Albumin (BSA) solution for one hour, and anti-PD-L1 primary antibody was added thereto, and incubated at 4 ℃ overnight. After three washes with PBST, secondary antibody was added and incubated for 1 hour at room temperature. After three washes with PBST, the reaction mixture was developed with ECL. The results of the detection are shown in FIG. 3. As can be seen from FIG. 3, PD-L1 target proteins are added to the PD-L1 target protein group, the amphipathic functional molecule group and the amphipathic functional molecule group (BMS 202-diketone,10150-18-34-40-diketone, 10150-185-34-40-diketone) in the left Input sample; in the right-hand Pull-down sample, only the band of the PD-L1 target protein was detected in the group of amphipathic functional molecules (BMS 202-diketone,10150-18-34-40-diketone, 10150-185-34-40-diketone). The above results indicate that the amphiphilic functional molecule can mediate the interaction between the PD-L1 target protein and the aldolase antibody 38C2.
PD-L1 plasmid transfected cells
And connecting the expression vector pLV2 subjected to enzyme digestion with the PCR and the PD-L1 coding fragment subjected to the same enzyme digestion, converting, sequencing to obtain a PD-L1 plasmid with a correct sequence, and performing large-scale extraction and quantification for later use. After passaging, freeStyle 293 cells were cultured in FreeStyle 293 complete medium and transfected with the PD-L1 expression plasmid after overnight culture. The PD-L1 plasmid and transfection reagent Lipo3000 are mixed and added into FreeStyle 293 cells, and the complete culture medium of FreeStyle 293 is replaced after 6 hours of transfection, and the cells are kept for later use after 48 hours of culture.
PD-L1 cell expression assay
Taking PD-L1 overexpression cells of 1E6 to detect the expression level of PD-L1. FreeStyle 293-PD-L1 overexpression cells with 1 HBSS solution after washing, and PD-L1 specific primary antibody at 37 ℃ were incubated for one hour, through 1 HBSS washing three times, each 1000rpm,3 minutes low speed centrifugation to collect cell body and then 1 HBSS heavy suspension. And (3) staining the washed cells for three times with a fluorescent goat-anti-rabbit secondary antibody corresponding to the PD-L1 primary antibody, incubating for one hour at room temperature, washing for three times with 1 x HBSS, collecting cell bodies, then re-suspending the cells with 1mL of cell affinity buffer solution, determining the analyzed cell groups by using forward reflected light (FSC) and lateral reflected light (SSC) in a flow cytometer, detecting the expression of cell surface fluorescence after circling a door, and deducing the expression level of cell membrane target protein according to the intensity of the fluorescence expression.
14. Cell-interactive in vitro affinity incubation
Aldolase antibody 38C2 was dissolved in the cell affinity buffer in a total amount of (number of groups + 1) × 20. Mu.g, and dispensed to the reference and experimental groups, and the parental functional molecule stocks (10150-18-34-40-diketone and 10150-185-34-40-diketone) were diluted with a gradient of cell affinity buffer to 50. Mu.M. The compounds were mixed with the aldolase antibody at equal volumes and the amphipathic molecules (10150-18-34-40-diketone and 10150-185-34-40-diketone) were added to the experimental groups such that the final volume of each group was 50. Mu.L and the final concentration of the compound was 25. Mu.M. Incubate at 37 ℃ for one hour with tumbling.
15. Post incubation complex collection
The 7K gel exclusion chromatography column was rinsed with distilled water 200. Mu.L/lot, 1. Mu.L PBS 200. Mu.L/lot, and 1. Mu.L PBS 200. Mu.L/lot in that order, during which time 1200rpm, and the rinse was removed by centrifugation for 2 minutes. After incubation, the samples were loaded on 7K gel exclusion chromatography columns, centrifuged at 1200rpm for 2 minutes and the eluates were collected.
16. Cell incubation:
mu.L (about 1500 ng) of 38C2 antibody-small molecule complex was diluted to 50. Mu.L with PBS, added to PD-L1-overexpressed FreeStyle 293 cells (suspension), and incubated at 37 ℃ for 1 hour. Cells were washed once with 200 μ L HBSS and cells were collected by low speed centrifugation and discarding supernatant. After that, a fluorescently-labeled secondary antibody against murine 38C2 was added and incubated for 1 hour at normal temperature. Cells were washed twice with 200 μ L HBSS and cells were collected by low speed centrifugation and discarding supernatant.
17. Cell detection
Resuspend cells at 1 × HBSS, 1000 μ L/group, pass cells through Becton Dickinson FACSAria TM III flow cytometer (BD Biosciences) flow cytometry. Forward reflected light (FSC) and sideDetermining the cell population analyzed from the reflected light (SSC), and detecting the expression of cell surface fluorescence after gating; the difference of the fluorescence expression of the reference group and the experiment group is measured by taking the average fluorescence expression intensity as a contrast value, so that the fact that the cell membrane target protein acts on the aldolase antibody 38C2 through the affinity of the amphiphilic functional molecule is inferred, and the expression and the affinity action specificity of the cell membrane target protein are indicated by taking the cell membrane target protein as an indication. The results are shown in FIG. 4. As can be seen from FIG. 4, the mean fluorescence intensity of PD-L1 cells added with the amphipathic molecule (10150-18-34-40-diketone and 10150-185-34-40-diketone) -aldolase antibody 38C2 complex is significantly higher than that of PD-L1 cells added with only aldolase antibody 38C2, and also significantly higher than that of the cells of the control group of the same group not expressing PD-L1. The above results indicate that the cell membrane target protein PD-L1 can act on the aldolase antibody 38C2 through the affinity of the amphipathic molecule.
18. Immunohistochemical in vitro affinity incubation
Aldolase antibody 38C2 was dissolved in the cell affinity buffer in a total amount of (number of groups + 1) × 20. Mu.g, and dispensed to the reference and experimental groups, and the parental functional molecule stocks (10150-18-34-40-diketone and 10150-185-34-40-diketone) were diluted with a gradient of cell affinity buffer to 50. Mu.M. The compounds were mixed with the aldolase antibody at equal volumes and the amphipathic molecules (10150-18-34-40-diketone and 10150-185-34-40-diketone) were added to the experimental groups such that the final volume of each group was 50. Mu.L and the final concentration of the compound was 25. Mu.M. Incubate at 37 ℃ for one hour with tumbling.
15. Post incubation complex collection
The 7K gel exclusion chromatography column was rinsed with distilled water 200. Mu.L/lot, 1. Mu.L PBS 200. Mu.L/lot, and 1. Mu.L PBS 200. Mu.L/lot in that order, during which time 1200rpm, and the rinse was removed by centrifugation for 2 minutes. After incubation, the samples were loaded on 7K gel exclusion chromatography columns, centrifuged at 1200rpm for 2 minutes and the eluates were collected.
20. Immunohistochemical tissue preparation
Human placental tissue wax blocks were purchased from the southern changsha family. Slicing the paraffin block, baking for one hour in a constant temperature box at 60 ℃, then performing soaking dewaxing operation, and performing gradient dewaxing by soaking for 10 minutes in xylene twice and soaking for 5 minutes in absolute ethyl alcohol, 95% ethyl alcohol, 85% ethyl alcohol and 70% ethyl alcohol respectively; the sections were then rinsed with distilled water for 2 washes for 3 minutes each. Followed by antigen retrieval. Using EDTA antigen repairing solution to repair in high temperature water bath at 93 ℃ and 98 ℃ for 20 minutes, cooling to room temperature, then washing with distilled water for 2 times, and 3 minutes each time. And then 3% hydrogen peroxide is used for incubation for 10 minutes at room temperature, and the tissue section is washed and rinsed for 2 times with distilled water for 3 minutes each time, so that the endogenous peroxidase of the tissue section is inactivated.
21. Immunohistochemical affinity incubation
The eluted reference and experimental complex solutions were each pipetted at 3.75 μ L (about 1500 ng) diluted to 50 μ L with 1 × PBS, and then added dropwise to the center of the paraffin section and completely cover the section tissue, and incubated at room temperature for one hour and then washed three times with PBS for 5 minutes each. After the PBS solution was aspirated, CRF anti-multiple species (rabbit, rat/mouse, guinea pig) HRP multimeric labeled secondary antibody was added to completely cover the tissue, incubated at room temperature for half an hour, washed with PBS three times for 3 minutes each, and washed with distilled water once for 3 minutes.
22. Immunohistochemical staining and detection
Adding prepared DAB solution, developing for 2.5 min according to color change, washing with distilled water for 3 times, each time for 3 min. And lining-staining the slices with hematoxylin, incubating at room temperature for 2 minutes, washing the slices with PBS or tap water for 10-15 minutes, soaking in 70% ethanol, 85% ethanol, 95% ethanol and 100% absolute ethanol after the slices turn blue, and dehydrating the slices 3 minutes each time. Then, xylene soaking is carried out for 10 minutes twice for transparency, and the transparent film is sealed by neutral gum and then photographed by microscopic examination. The results of the detection are shown in FIG. 5. As can be seen from FIG. 5, the trophoblast cells of human placental tissue to which the amphiphilic molecules (10150-18-34-40-diketone and 10150-185-34-40-diketone) -aldolase antibody 38C2 complex was added were more intensely colored, similar to the group using PD-L1 antibodies, while the trophoblast cells of human placental tissue to which only the aldolase antibody was added were less colored. And it is known that trophoblast cells of human placental tissue highly express the PD-L1 target protein. The above results indicate that the aldolase antibody 38C2 can specifically recognize the PD-L1 target protein in tissue sections by the amphipathic molecule.
The experimental method can accurately, sensitively and real-timely verify that the antibody enhances the capacity of specifically identifying and recognizing the target protein through the modification of the amphiphilic functional molecule, provides verification data and method support for the modification and optimization of the amphiphilic functional molecule and subsequent in vivo functional experiments, lays a research foundation for the wide application of a DNA coding compound in the research and development of the amphiphilic functional molecule and the clarification of the function, and enables the screening, identifying and verifying processes of the target protein molecule based on the source of the DNA coding compound to be standardized, accurate, digitalized and quantitative.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (16)
1. A method for tandem verifying the function of a DNA coding vaccine head compound molecule modified antibody for non-disease diagnosis and treatment purposes is characterized in that,
according to the result of screening of the DNA coding compound library based on the PD-L1 target protein, performing descending order sorting according to the enrichment calculation value through data analysis to obtain a potential DNA coding seedling-end compound with high reliability;
synthesizing a potential DNA coding head compound, and connecting the potential DNA coding head compound and diketone molecules through an adaptor to form an amphiphilic functional molecule;
for the obtained amphiphilic functional molecules respectively targeting a PD-L1 target protein and an aldolase antibody, the capacity of the antibody for identifying a target point is improved from the biological exogenous and endogenous levels through a molecular modification mass spectrum detection experiment, a protein combination verification experiment, a cell membrane protein combination verification experiment and an immunohistochemical verification experiment, and the capacity comprises the following steps:
(1) The action mode of the amphiphilic functional molecules and the aldolase antibody is specifically identified through the molecular modification mass spectrometry detection experiment;
(2) Detecting the interaction between the PD-L1 target protein and the aldolase antibody through the amphiphilic functional molecule by the protein binding verification experiment;
(3) Verifying the expression of the PD-L1 target protein on the surface of the cell membrane and the capability of the PD-L1 target protein to be coupled with the aldolase antibody through an amphiphilic functional molecule by the cell membrane protein binding verification experiment;
(4) The expression of the PD-L1 target protein is positioned through an immunohistochemical verification experiment of the amphipathic functional molecule assembled antibody.
2. The method according to claim 1, characterized in that in step (1) comprises in particular: dissolving the amphiphilic functional molecules in 100% dimethyl sulfoxide solution to prepare the amphiphilic functional molecule stock solution, so that the final concentration of the amphiphilic functional molecule stock solution is 20mM, and diluting the amphiphilic functional molecule stock solution in a mass spectrum affinity buffer solution in a gradient manner, so that the final concentration of the amphiphilic functional molecule stock solution is 50 mu M.
3. The method according to claim 2, characterized in that in step (1) comprises in particular: dissolving 20 mu g of aldolase antibody by using a mass spectrum affinity buffer solution, mixing the solution with the amphiphilic functional molecule solution in equal volume, and incubating for one hour at 37 ℃; and filtering the incubation solution by using a gel exclusion chromatographic column, and collecting an elution solution for mass spectrum detection.
4. The method of claim 2, wherein the mass spectrometry affinity buffer composition is 1 × PBS Tween 20 buffer, 1 × PBS, pH 7.4,0.05% Tween 20, and 50 μ L is required for each set of samples.
5. The method according to claim 1, characterized in that in step (2) comprises in particular: washing Protein G immunomagnetic beads for three times by using a washing buffer solution, and discarding the washing buffer solution; after 2. Mu.g of aldolase antibody per set was dissolved in binding buffer, both were incubated at room temperature for half an hour to coat the aldolase antibody on the magnetic beads, the supernatant was discarded and washed once with binding buffer, and the aldolase antibody-magnetic bead-coated complex was retained by discarding the supernatant.
6. The method of claim 5, wherein the wash buffer composition is 1 × TBS Tween 20 buffer, 25mM Tris,0.15M NaCl,0.05% Tween 20, pH 7.5, requiring 600 μ L per set of samples; the components of the binding buffer solution are 1 TBS Tween 20 buffer solution, 25mM Tris,0.15M NaCl,0.05% Tween 20 and pH 7.5, fresh affinity screening buffer solution is prepared before each experiment, and each group of samples needs 200 muL.
7. The method according to claim 5, characterized in that in step (2) comprises in particular: diluting the amphiphilic functional molecule stock solution in a gradient manner by using a binding buffer solution to ensure that the final concentration of the amphiphilic functional molecule stock solution is 10 mu M; and mixing the diluted amphiphilic functional molecules with the aldolase antibody-magnetic bead coating compound, incubating for one hour at 37 ℃, then discarding the supernatant, washing the incubated magnetic beads with a binding buffer solution for three times, and discarding the supernatant to retain the amphiphilic functional molecule-aldolase antibody-magnetic bead coating compound.
8. The method according to claim 7, characterized in that in step (2) comprises in particular: dissolving 2 mu g of PD-L1 target protein/group by using a binding buffer solution to enable the final volume of the PD-L1 target protein/group to be 50 mu L, mixing the amphiphilic functional molecule-aldolase antibody-magnetic bead coating compound with the PD-L1 target protein solution, incubating for one hour at room temperature, then abandoning the supernatant, washing the incubated magnetic beads by using the binding buffer solution for three times, abandoning the supernatant, and reserving the PD-L1 target protein-amphiphilic functional molecule-aldolase antibody-magnetic bead coating compound; and detecting whether the PD-L1 target protein interacts with an aldolase antibody through bridging of an amphiphilic functional molecule by using a Western Blot method.
9. The method according to claim 1, characterized in that in step (3) comprises in particular: carrying out overexpression operation in FreeStyle 293 cells by using a plasmid constructed with a PD-L1 PD-L1 target protein coding sequence; mixing the plasmid with a transfection reagent, adding the mixture into FreeStyle 293 cells, culturing for 48 hours, counting the cells, and taking out the number of the 1E6 cells to detect the over-expression level of the PD-L1 target protein; after carrying out fluorescent staining on the specific primary antibody of the PD-L1 target protein and a corresponding secondary antibody, deducing the expression level of the cell membrane target through the intensity of fluorescent expression in a flow cytometer; the remaining cells were subjected to cell membrane protein binding validation experiments.
10. The method according to claim 9, characterized in that in step (3) comprises in particular: dissolving 20 mu g of aldolase antibody by using a cell affinity buffer solution, and diluting an amphiphilic functional molecule storage solution by using a cell affinity buffer solution in a gradient manner to ensure that the final concentration of the amphiphilic functional molecule storage solution is 50 mu M; mixing the aldolase antibody solution and the amphiphilic functional molecule diluent in equal volume to ensure that the final volume of the mixed solution is 50 mu L and the final concentration of the amphiphilic functional molecule solution is 25 mu M, and incubating for one hour at 37 ℃; and filtering the incubation solution by using a gel exclusion chromatographic column, and collecting an elution solution, namely a solution of the amphiphilic functional molecule and aldolase antibody complex.
11. The method of claim 10, wherein the cell affinity buffer composition is 1 × HBSS buffer, pH 6.7-7.8, and fresh cell affinity screening buffer is prepared before each experiment, requiring 600 μ L per group of samples.
12. The method according to claim 10, characterized in that in step (3) comprises in particular: centrifuging 1E6 cells/group, then discarding supernatant, mixing the eluted amphiphilic functional molecules and aldolase antibody compound solution with the cells, incubating for one hour at 37 ℃, centrifuging, then discarding supernatant, washing the cells once with cell affinity buffer solution, centrifuging, then discarding supernatant, labeling the aldolase antibody with fluorescent secondary antibody, and incubating for one hour at room temperature; and after centrifugation, discarding the supernatant, washing the cells once by using a cell affinity buffer solution, suspending the cells by using the cell affinity buffer solution after centrifugation to prepare a cell suspension, and detecting the intensity of fluorescence expression by using a flow cytometer so as to infer the interaction between the PD-L1 target protein expressed on the surface of the cell membrane and the aldolase antibody through the connection of the amphiphilic functional molecule.
13. The method according to claim 1, characterized in that in step (4) comprises in particular: dissolving 20 mu g of aldolase antibody by using a mass spectrum affinity buffer solution, and diluting an amphiphilic functional molecule storage solution by using the mass spectrum affinity buffer solution in a gradient manner to enable the final concentration to be 50 mu M; mixing the aldolase antibody solution and the amphiphilic functional molecule diluent in equal volume to ensure that the final volume of the mixed solution is 50 mu L and the final concentration of the amphiphilic functional molecule solution is 25 mu M, and incubating for one hour at 37 ℃; and filtering the incubation solution by using a gel exclusion chromatographic column, and collecting an elution solution, namely a solution of the amphiphilic functional molecule and aldolase antibody complex.
14. The method according to claim 13, characterized in that in step (4) comprises in particular: taking a human placenta tissue paraffin block for slicing, then respectively dewaxing and hydrating, performing antigen retrieval and endogenous peroxidase inactivation, incubating the solution of the compound of the amphiphilic functional molecule and the aldolase antibody and the tissue slice at room temperature for one hour, and then flushing the slice by using a PBS solution for three times.
15. The method according to claim 14, characterized in that in step (4) comprises in particular: tissue sections were incubated with HRP polymer-labeled secondary antibody for one hour at room temperature, after which the sections were rinsed three times with PBS solution and once with distilled water.
16. The method according to claim 15, characterized in that in step (4) comprises in particular: DAB color development, hematoxylin lining dyeing and dehydrated transparent sealing are respectively utilized to facilitate subsequent data collection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011598849.5A CN112986575B (en) | 2020-12-29 | 2020-12-29 | Method for verifying DNA coding Miao compound modified antibody in tandem manner |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011598849.5A CN112986575B (en) | 2020-12-29 | 2020-12-29 | Method for verifying DNA coding Miao compound modified antibody in tandem manner |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112986575A CN112986575A (en) | 2021-06-18 |
| CN112986575B true CN112986575B (en) | 2023-03-17 |
Family
ID=76345150
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011598849.5A Active CN112986575B (en) | 2020-12-29 | 2020-12-29 | Method for verifying DNA coding Miao compound modified antibody in tandem manner |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112986575B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021115319A1 (en) * | 2019-12-10 | 2021-06-17 | 成都先导药物开发股份有限公司 | Screening method for dna encoded compound library simultaneously binding to multiple biological targets |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8293714B2 (en) * | 2008-05-05 | 2012-10-23 | Covx Technology Ireland, Ltd. | Anti-angiogenic compounds |
| KR101640697B1 (en) * | 2011-12-22 | 2016-07-18 | 화이자 인코포레이티드 | Anti-diabetic compounds |
| WO2013137737A1 (en) * | 2012-03-16 | 2013-09-19 | Flexgen B.V. | Methods of creating and screening of dna encoded combinatorial libraries of chemical antibodies |
| JP6236540B2 (en) * | 2014-01-27 | 2017-11-22 | ファイザー・インク | Bifunctional cytotoxic agent |
| US11028427B2 (en) * | 2015-08-18 | 2021-06-08 | Purdue Research Foundation | Systems and methods for proteomic activity analysis using DNA-encoded probes |
| CN109023535B (en) * | 2018-07-19 | 2020-11-17 | 上海药明康德新药开发有限公司 | Method for screening DNA coding compound by using antibody to non-recognition marker protein or cell lysate |
| CN111733459A (en) * | 2020-07-08 | 2020-10-02 | 佰奥思特生物医药科技(上海)有限公司 | DNA coding compound library and screening method thereof |
| CN111965295A (en) * | 2020-07-28 | 2020-11-20 | 上海药明康德新药开发有限公司 | DNA coding shoot-head compound identification method based on LC-MS (liquid chromatography-mass spectrometry) technology verification |
-
2020
- 2020-12-29 CN CN202011598849.5A patent/CN112986575B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112986575A (en) | 2021-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3361252A1 (en) | Pivka-ii assay method and method for manufacturing reagent or kit for pivka-ii immunoassay | |
| CA2978998A1 (en) | Method and kit for measuring component to be assayed in specimen | |
| CN114807054B (en) | Mouse anti-human IgG monoclonal antibody hybridoma cell strain, antibody composition and kit | |
| CN111004324A (en) | Calprotectin monoclonal antibody and application thereof | |
| CN112986575B (en) | Method for verifying DNA coding Miao compound modified antibody in tandem manner | |
| CN114594262A (en) | Mycotoxin magnetic chemiluminescence immunoassay kit based on bifunctional fusion protein and application thereof | |
| US9632086B2 (en) | Method and kit for determining-antibody sensitivity and clone cell strain | |
| CN113166235A (en) | Antibody conjugates | |
| AU2016223431A1 (en) | Antibody detection method and system | |
| CN112430569B (en) | Application of protein SFTPC as lung cancer diagnosis marker and kit | |
| KR102320436B1 (en) | Biomarker composition for diagnosis of glioblastoma comprising glioblastoma stem cell exosome-derived peptide | |
| JP7153493B2 (en) | Method for measuring bioparticles, method for detecting non-specific signals, method for measuring bioparticles, and reagent kit for detecting bioparticles | |
| CN116041520B (en) | Rabbit monoclonal antibody aiming at Human CD63, and preparation method and application thereof | |
| CN114686443B (en) | Hybridoma cell, antithrombotic regulatory protein monoclonal antibody, preparation method and application thereof | |
| JP7153494B2 (en) | Method for measuring bioparticles, method for detecting non-specific signals, method for measuring bioparticles, and reagent kit for detecting bioparticles | |
| CN113563479B (en) | Echinococcosis diagnostic kit | |
| CN117187069B (en) | Escherichia coli HCP detection method for alkaline hydrolysis process | |
| CN113960313B (en) | Exosome ALK fusion protein magnetic immunochemiluminescence detection kit | |
| US20020019004A1 (en) | Method for isolating molecules, cells and other particles which are specifically bound to a large particle | |
| EP3599466B1 (en) | Bioparticle measuring method | |
| CN116482375A (en) | Human membranous nephropathy related index detection kit and preparation method and application method thereof | |
| CN117867070A (en) | Detection method and kit | |
| CN114686444A (en) | Hybridoma cell, anti-thrombomodulin monoclonal antibody, and preparation method and application thereof | |
| CN115894680A (en) | C-reactive protein monoclonal antibody and preparation method and application thereof | |
| CN117417450A (en) | Myeloperoxidase binding proteins, methods of preparation and uses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |