CN111381024B - Immunocapture composition, preparation method, kit and application - Google Patents

Immunocapture composition, preparation method, kit and application Download PDF

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CN111381024B
CN111381024B CN201811642269.4A CN201811642269A CN111381024B CN 111381024 B CN111381024 B CN 111381024B CN 201811642269 A CN201811642269 A CN 201811642269A CN 111381024 B CN111381024 B CN 111381024B
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blocking
blocking compound
compound
composition
solid phase
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CN111381024A (en
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马志亚
钱婷
汪业红
李会平
刘治志
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Shenzhen Dymind Biotechnology Co Ltd
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Shenzhen Dymind Biotechnology Co Ltd
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    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The application discloses an immunodetection composition, a preparation method, a kit and application, wherein the immunocapture composition comprises: a solid support; at least one first ligand immobilized on the surface of the solid support; and at least a first blocking compound and a second blocking compound modified on the surface of the solid phase carrier, wherein the first blocking compound and the second blocking compound are used for blocking active sites on the surface of the solid phase carrier; wherein the first blocking compound is a polyhydroxy carbohydrate or protein based compound and the second blocking compound is a polyhydroxy carbohydrate or protein based compound, the first blocking compound being different from the second blocking compound. By means of the mode, the sealing effect can be effectively improved, and the accuracy of the detection result is improved.

Description

Immunocapture composition, preparation method, kit and application
Technical Field
The application relates to the technical field of immunodetection, in particular to an immunocapture composition, a preparation method, a kit and application.
Background
In classical immunoassay methods, high affinity, specific recognition between antigen antibodies forms the basis of the analysis. The affinity effect is a general term of a series of non-covalent forces (hydrophilic and hydrophobic effects, electrostatic effects, hydrogen bonds and the like) between a ligand and a receptor under a specific biomolecule structure, and the stable combination of the affinity and the receptor is ensured by the comprehensive effect of multiple weak forces. However, the presence of non-target substances in complex systems may produce adsorption of affinity ligands and receptors to varying degrees, referred to as "non-specific adsorption". In practical experimental operation and related device design, a blocking agent is usually added to bind with active sites of a sensing interface so as to reduce non-specific adsorption and not to interfere normal operation of a target affinity reaction in a system.
The inventor of the present application found in the long-term development process that in the traditional immunoaffinity reaction, cheap and easily available proteins are generally used as a single blocking agent, such as Bovine Serum Albumin (BSA), ovalbumin (OVA), etc. However, for some specific projects, the blocking effect is not good, a high background signal is generated, and the sensitivity and the specificity of the detection result are seriously influenced.
Disclosure of Invention
The technical problem mainly solved by the application is to provide the immunodetection composition, the preparation method, the kit and the application, and the problem that when protein is used as a single sealant, high background signals are generated due to poor sealing effect, so that the sensitivity and the specificity of a detection result are influenced can be solved.
In order to solve the technical problem, the application adopts a technical scheme that: providing an immunocapture composition comprising: a solid support; at least one first ligand immobilized on the surface of the solid support; and at least a first blocking compound and a second blocking compound modified on the surface of the solid phase carrier, wherein the first blocking compound and the second blocking compound are used for blocking active sites on the surface of the solid phase carrier; wherein the first blocking compound is a polyhydroxy carbohydrate or protein based compound and the second blocking compound is a polyhydroxy carbohydrate or protein based compound, the first blocking compound being different from the second blocking compound.
Wherein the immunocapture composition further comprises: a third blocking compound modified on the surface of the solid support; wherein the third blocking compound is a small molecule compound containing a primary amino group.
Wherein the polyhydroxy saccharide compound is at least one of glucose, sucrose, lactose, trehalose, dextran, mannitol or polysucrose; the protein compound is at least one of bovine serum albumin, human serum albumin, casein, gelatin, casein hydrolysate, immunoglobulin, milk powder, and human or animal serum; the micromolecule compound containing primary amino group is at least one of trihydroxymethyl aminomethane, ethanolamine, hydroxylamine, hexylamine or glycine.
Wherein at least one first ligand is an antibody or antigen.
Wherein the solid phase carrier is at least one of a microporous plate, a glass sheet, a microfluidic chip, latex, magnetic or non-magnetic polymer microspheres, metal nanoparticles, metal alloy nanoparticles, inorganic microspheres, inorganic/organic hybrid microspheres, fluorescent nanoparticles, magnetic beads and multifunctional fluorescent magnetic beads; metal nanoparticles include, but are not limited to, gold nanoparticles or silver nanoparticles.
Wherein the solid phase carrier is a polymer microsphere, the particle size of the polymer microsphere is 1-10 μm, the surface of the polymer microsphere is provided with a functional group, and the functional group is used for coupling at least one first ligand; the functional group is at least one of carboxyl, hydroxyl, amino, tosyl, chloromethyl, sulfydryl, aldehyde group, hydrazide, silicon hydroxyl, succinimide ester and epoxy group.
Wherein the at least one first ligand comprises: a first antibody and a second antibody; wherein the first antibody and the second antibody have specific binding effect with the same antigen to be detected.
Wherein the ratio of the sum of the masses of the first blocking compound and the second blocking compound to the mass of the solid phase carrier is (0.01-10): 100.
Wherein the ratio of the sum of the mass of the first blocking compound, the second blocking compound and the third blocking composition to the mass of the solid phase carrier is (0.01-10): 100.
In order to solve the technical problem, the application adopts a technical scheme that: provided is a method for preparing an immunocapture composition, comprising: providing a solid phase carrier; immobilizing at least one first ligand to the surface of the solid support to obtain a solid support coated with at least one first ligand; adding a first blocking solution containing a first blocking compound, and reacting for 1-24 hours to block the active sites on the surface of the solid phase carrier and obtain the solid phase carrier modified with the first blocking compound on the surface; adding a second confining liquid containing a second confining compound, and reacting for 1-24 hours to block active sites on the surface of the solid phase carrier and obtain the solid phase carrier with the surface modified with the second confining compound; wherein the first blocking compound is a polyhydroxy carbohydrate or protein based compound and the second blocking compound is a polyhydroxy carbohydrate or protein based compound, the first blocking compound being different from the second blocking compound.
Wherein, before the steps of adding a first blocking solution containing a first blocking compound, reacting for 1-24 hours to block active sites on the surface of the solid phase carrier and obtain the solid phase carrier modified with the first blocking compound on the surface, the preparation method further comprises the following steps: adding a third sealing solution containing a third sealing compound, reacting for 0.5-2 hours to seal the active sites on the surface of the solid phase carrier and obtain the solid phase carrier modified with the third sealing compound on the surface; the third blocking compound is a small molecule compound containing a primary amine group.
Wherein the first blocking compound is a polyhydroxy carbohydrate; the second blocking compound is a proteinaceous compound.
Wherein the first blocking compound, the second blocking compound and the third blocking compound are modified on the surface of the solid phase carrier through at least one of electrostatic interaction, hydrophobic interaction, hydrogen bond, intermolecular force, specific interaction between biomolecules or covalent bond.
Wherein the step of immobilizing at least one first ligand to the surface of the solid support to obtain a solid support coated with at least one first ligand comprises: immobilizing at least one first ligand directly to a solid support; or indirectly immobilizing at least one first ligand to the solid support by at least one of protein A and/or protein G, a secondary antibody and a primary antibody, or a specific affinity reaction of avidin and biotin.
In order to solve the technical problem, the application adopts a technical scheme that: provides the application of the immunodetection composition in myocardial detection, hypertension detection, thyroid detection, cardiovascular detection, gonadal detection, renal function detection, bone metabolism detection, glycometabolism detection, infectious disease detection, autoimmune disease detection, prenatal screening, drug detection, hepatic fibrosis detection, EB virus detection, inflammation monitoring or tumor detection.
In order to solve the technical problem, the application adopts a technical scheme that: an immunoassay kit is provided, the immunoassay kit comprising: an immunocapture composition as described above; and, an immunodetection composition comprising at least one second ligand and a signal tracer; the signal tracer is at least one of an enzymatic tracer, a chemiluminescent tracer, or a fluorescent tracer.
Wherein the signal tracer is a fluorescent tracer and the fluorescent tracer is different from the fluorescent marker in the immunocapture composition.
Wherein at least one second ligand is an antibody or antigen.
Wherein the at least one second ligand comprises: a third antibody and a fourth antibody; the first antibody, the third antibody and the antigen to be detected of the immunocapture composition are specifically combined; the second antibody of the immunocapture composition specifically binds to the fourth antibody and the antigen to be detected.
Wherein the antigen to be detected comprises a protein, a polypeptide, a hormone, a carbohydrate, an enzyme, a drug, a nucleic acid, a small molecule compound, or a metal ion.
Wherein, the immunoassay kit further comprises: at least one reagent tube, wherein a suspension liquid is arranged in the reagent tube; the immunocapture composition and the immunodetection composition are dissolved in the suspension; wherein the suspension comprises a buffer solution, a surfactant, an inorganic salt, a stabilizer and a preservative. In order to solve the technical problem, the application adopts a technical scheme that: there is provided the use of an immunoassay kit as described above in a cardiac assay, a hypertension assay, a thyroid assay, a cardiovascular assay, a gonadal assay, a renal function assay, a bone metabolism assay, a carbohydrate metabolism assay, an infectious disease assay, an autoimmune disease assay, prenatal screening, a drug assay, a liver fibrosis assay, an EB virus assay, an inflammation assay or a tumor assay.
In order to solve the technical problem, the application adopts a technical scheme that: providing an immunoassay method based on the immunoassay kit, wherein the immunoassay method comprises the following steps: providing an immunocapture composition, wherein the immunocapture composition comprises: at least one first ligand, at least one first blocking compound and at least one second blocking compound, wherein the first blocking compound and the second blocking compound are used for blocking active sites on the surface of the solid phase carrier, the first blocking compound is a polyhydroxy carbohydrate compound or a protein compound, the second blocking compound is a polyhydroxy carbohydrate compound or a protein compound, and the first blocking compound is different from the second blocking compound; adding a test sample and an immunocapture composition to a test subject line; adding at least one immunoassay composition to a test system; incubation and magnetic separation; the fluorescent signals of the immunocapture composition and the immunodetection composition are detected separately. Wherein the first ligand is NT-proBNP capture antibody, troponin capture antibody, alpha fetoprotein capture antibody, carcinoembryonic capture antibody, HIV capture antibody, hepatitis B surface capture antibody, thyroglobulin capture antibody, troponin T capture antibody or myoglobin capture antibody.
In order to solve the technical problem, the application adopts a technical scheme that: there is provided an immunofluorescence analysis system comprising: an immunoassay kit as described previously; and an optical detection mechanism for detecting a fluorescent signal of the immunoassay kit; the immunofluorescence analysis system is used for myocardial detection, hypertension detection, thyroid gland detection, cardiovascular detection, gonad detection, renal function detection, bone metabolism detection, glucose metabolism detection, infectious disease detection, autoimmune disease detection, prenatal screening, drug detection, hepatic fibrosis detection, EB virus detection, inflammation monitoring or tumor detection.
The beneficial effect of this application is: in the present application, the surface of the solid phase carrier is modified with a first blocking compound and a second blocking compound, and the first blocking compound and the second blocking compound are used for blocking active sites, wherein the first blocking compound is a polyhydroxy carbohydrate compound, and the second blocking compound is a protein compound, so that most or even all active sites on the surface of the solid phase carrier can be blocked by the two blocking compounds, and thus non-specific adsorption of an interfering substance to the solid phase carrier is effectively inhibited. Particularly, when a plurality of ligands are fixed on the solid phase carrier and classified detection is carried out, the effect of sealing the solid phase carrier by adopting the sealing strategy is obviously superior to that of the traditional sealing strategy in which protein is used as a single sealing agent, the sealing effect is effectively improved, the influence of nonspecific adsorption can be eliminated, the influence on the obtained effective data volume is reduced, and the sensitivity and the specificity of detection are improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:
FIG. 1 is a schematic flow chart of one embodiment of a method of preparing an immunoassay composition of the present disclosure;
FIG. 2 is a schematic flow chart of an embodiment of an immunoassay method of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The present application provides an immunocapture composition comprising: a solid support; at least one first ligand immobilized on the surface of the solid support; and at least a first blocking compound and a second blocking compound modified on the surface of the solid support, the first blocking compound and the second blocking compound being used for blocking active sites on the surface of the solid support; wherein the first blocking compound is a polyhydroxy carbohydrate or protein based compound and the second blocking compound is a polyhydroxy carbohydrate or protein based compound, the first blocking compound being different from the second blocking compound.
Specifically, the number of the solid phase carriers can be multiple, and each first ligand is correspondingly fixed on the surface of each solid phase carrier; alternatively, a plurality of first ligands may correspond to each solid support surface, wherein the plurality of first ligands are the same species, but the performance parameters may be different, including but not limited to affinity constants, etc.
Specifically, the first ligand is usually an antigen or an antibody, and the process of immobilizing the antigen or the antibody is referred to as coating, that is, the process of binding the antigen or the antibody to the surface of the solid support. The antibody or antigen can be bound to the solid support by physical adsorption, covalent binding, affinity interaction (e.g., protein a or/and protein G, secondary antibody and primary antibody, or avidin and biotin), etc., and the physical adsorption is based on the interaction between hydrophobic groups on the coating and hydrophobic groups on the surface of the solid support, or electrostatic interaction. This physical adsorption is non-specific and is affected by the type of coating, molecular weight, isoelectric point, pH, ionic strength, concentration, etc. Covalent bonding is accomplished by covalent bonding of the coating to the active functional groups on the carrier. The affinity interaction is to complete the coating through the affinity interaction between biological molecules.
During the coating process, the coating is immobilized on the surface of the solid phase carrier through the above several actions, but some active sites not occupied by the target coating exist on the surface of the solid phase carrier. The presence of these sites can cause non-specific binding to the solid support, which can result in high background signals that can affect the sensitivity, accuracy, and specificity of the assay. These non-specific binding sites must be blocked for this purpose. Blocking is the process of recoating with a blocking agent, such as the first blocking compound, the second blocking compound or the third blocking compound in this application, at a high concentration subsequent to coating, by allowing a large amount of irrelevant first blocking compound and second blocking compound to occupy these residual sites, thereby inhibiting non-specific adsorption of various interfering substances in the subsequent immunoreaction step, thereby reducing background signals and improving immunoreaction sensitivity, accuracy and specificity.
The closed procedure is similar to the coating process. The most commonly used blocking agent in the prior art is 0.05% -10% of protein, such as Bovine Serum Albumin (BSA), etc., and the inventors of the present application find that in the traditional immunoaffinity reaction, for some specific detection projects, the blocking effect of the protein blocking agent is not good, and a higher background signal is generated, so that the sensitivity, the accuracy and the specificity of the detection result are influenced. The application finds that when the surface of the solid phase carrier is sealed by using polyhydroxy carbohydrate and protein compounds, a good sealing effect can be obtained.
In contrast to the prior art, in the present embodiment, the surface of the solid phase carrier is modified with a first blocking compound and a second blocking compound, and the first blocking compound and the second blocking compound are used for blocking residual active sites, wherein the first blocking compound is a polyhydroxy carbohydrate compound, and the second blocking compound is a protein compound, so that most or even all active sites on the surface of the solid phase carrier can be blocked by the two blocking compounds, and thus non-specific adsorption of an interfering substance to the solid phase carrier is effectively inhibited. The effect of sealing the solid phase carrier by adopting the sealing strategy is obviously superior to that of the traditional sealing strategy in which protein is used as a single sealing agent, so that the sealing effect is effectively improved, and the sensitivity, accuracy and specificity of a detection result are improved.
It is noted that the immunocapture compositions of the present application can be used in at least one of chemiluminescent immunoassay, electrochemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), enzymatic immunoassay, biotin-avidin system assay, radioimmunoassay, immunofluorescent assay. Among them, ELISA is based on immobilization of an antigen or an antibody and enzyme labeling of the antigen or the antibody. It will be appreciated that the primary ligand in the immunocapture compositions include, but are not limited to, a variety of antibodies and/or antigens, which vary in composition depending on the detection methodology. For example, when a double antibody sandwich method is used, the first ligand is a single antibody or a plurality of antibodies; when the competitive method is adopted for detection, the first ligand comprises a single antibody or a plurality of antibodies and a first antigen or a plurality of antigens; when a double antigen sandwich is used, the first ligand comprises a single antibody or multiple antigens. Of course, the specific composition of the first ligand should include, but is not limited to, the above-described means, which should include all detection methodologies in the art, such as indirect methods and the like.
In one embodiment, the composition further comprises: a third blocking compound modified on the surface of the solid support; wherein the third blocking compound is a compound containing a primary amino group (-NH) 2 ) The small molecule compound of (1).
Specifically, primary amine (-NH) groups are used before modifying the polyhydroxy carbohydrate and protein compounds 2 ) The small molecular weight compound(s) is/are first blocked to inactivate residual reactive groups.
In one embodiment, the polyhydroxylated saccharide compound may be at least one of glucose, sucrose, lactose, trehalose, dextran, mannitol, or polysucrose; the protein compound can be at least one of bovine serum albumin, human serum albumin, casein, gelatin, casein hydrolysate, immunoglobulin, milk powder, and human or animal serum; the primary amino group-containing small molecule compound may be at least one of tris, ethanolamine, hydroxylamine, hexylamine, or glycine.
In one embodiment, the solid phase carrier is at least one of a microporous plate, a glass sheet, a microfluidic chip, latex, polymer microspheres, metal nanoparticles, metal alloy nanoparticles, inorganic microspheres, inorganic/organic hybrid microspheres, fluorescent nanoparticles, magnetic beads, and multifunctional fluorescent magnetic beads; metal nanoparticles include, but are not limited to, gold nanoparticles or silver nanoparticles.
Specifically, a commercial carboxyl modified superparamagnetic microsphere is used as a magnetic bead carrier, and after being washed by a buffer solution, a 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide solution is added according to the volume ratio of 1:1, and the magnetic separation is carried out after the oscillation reaction is carried out for 30-60 minutes at room temperature in a dark place, so as to obtain the activated magnetic bead.
Preferably, the solid phase carrier is a polymer microsphere, the particle size of the polymer microsphere is 1-10 μm, the surface of the polymer microsphere has a functional group, and the functional group is used for covalently coupling the at least one first ligand; the functional group is at least one of carboxyl, hydroxyl, amino, tosyl, chloromethyl, sulfydryl, aldehyde group, hydrazide, silicon hydroxyl, succinimide ester and epoxy group.
In one embodiment, the ratio of the sum of the mass of the first blocking compound and the second blocking compound to the mass of the solid support is (0.01-10) 100, e.g., 0.01.
Specifically, a first blocking compound solution having a concentration of 1 to 100% (e.g., 1%, 5%, 10%, 50%, or 100%) and a second blocking compound solution having a concentration of 1 to 100% (e.g., 1%, 5%, 10%, 50%, or 100%) may be prepared according to the ratio of the sum of the masses of the first blocking compound and the second blocking compound to the mass of the solid phase carrier.
In one embodiment, the ratio of the sum of the mass of the first blocking compound, second blocking compound, and third blocking composition to the mass of the solid support is (0.01-10) 100, e.g., 0.01.
Specifically, a first blocking compound solution having a concentration of 1 to 100% (e.g., 1%, 5%, 10%, 50%, or 100%), a second blocking compound solution having a concentration of 1 to 100% (e.g., 1%, 5%, 10%, 50%, or 100%), and a third blocking compound solution having a concentration of 1 to 100% (e.g., 1%, 5%, 10%, 50%, or 100%) may be prepared according to the ratio of the sum of the masses of the first blocking compound and the second blocking compound to the mass of the solid phase carrier
Preferably, the at least one first ligand is an antibody.
Wherein the at least one first ligand comprises: a first antibody and a second antibody; wherein the first antibody and the second antibody have specific binding effect with the same antigen to be detected.
Specifically, the first antibody and the second antibody may be capture antibodies. In the fluorescence immunoassay multi-pass assay, a first antibody and a second antibody are coated on a polymer microsphere. The first antibody and the second antibody are homogeneous antibodies, and the first antibody and the second antibody have specific binding effect with the homogeneous antigen to be detected.
When the fluorescence immune multi-flux detection is carried out, the added protein compounds, polyhydroxy carbohydrate compounds or primary amino group-containing micromolecule compounds in the antigen to be detected are partially adsorbed on the surface of the polymer microsphere, and the nonspecific adsorption not only can reduce the specific separation effect of the polymer microsphere, but also can increase background signals in the fluorescence immune multi-flux detection, thereby reducing the signal-to-noise ratio. Further, the interior and/or the surface of the polymer microsphere at least contains a fluorescent compound to realize fluorescence immune classification detection, and the polymer microsphere has a relatively complex structure, so that more background signals can be brought. Therefore, the elimination of the influence of nonspecific adsorption is particularly important, the effect of the blocking strategy of the application on the solid phase carrier is obviously superior to that of the traditional blocking strategy of taking protein as a single blocking agent, the blocking effect is effectively improved, the influence of nonspecific adsorption can be eliminated, the influence on the obtained effective data volume is reduced, and the sensitivity and specificity of detection are improved.
Referring to fig. 1, the present application provides a method of preparing an immunoassay composition, the method comprising the steps of:
s101: providing a solid phase carrier.
Specifically, the solid phase carrier is at least one of a microporous plate, a glass sheet, a microfluidic chip, latex, a polymer microsphere, a metal nanoparticle, a metal alloy nanoparticle, an inorganic microsphere, an inorganic/organic hybrid microsphere, a fluorescent nanoparticle, a magnetic bead and a multifunctional fluorescent magnetic bead; metal nanoparticles include, but are not limited to, gold nanoparticles or silver nanoparticles. Preferably, the solid phase carrier is a polymeric microsphere.
The method comprises the steps of selecting commercial carboxyl modified superparamagnetic microspheres as magnetic bead carriers, washing the magnetic bead carriers by using a buffer solution, adding a 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide solution according to the volume ratio of 1:1, carrying out vibration reaction for 30-60 minutes at room temperature in a dark place, and carrying out magnetic separation to obtain activated magnetic beads.
S102: immobilizing at least one first ligand to the surface of the solid support to obtain a solid support coated with at least one first ligand.
Preferably, the ligand is an antibody or antigen.
The antibody can be diluted to 1.0-1000 mug/mL antibody solution by buffer solution, and 100 muL of the antibody solution is added to react for 8-12 hours at 4-6 ℃ to obtain the solid phase carrier coated with the ligand. Wherein the buffer solution can be at least one of sodium carbonate/sodium bicarbonate buffer solution, acetic acid/potassium acetate buffer solution, tris/hydrochloric acid buffer solution, 3- (N-morpholino) propanesulfonic acid sodium salt/hydrochloric acid buffer solution, 2- (N-morpholine) ethanesulfonic acid buffer solution (MES buffer solution), phosphate buffer solution, borate buffer solution, MOPS, HEPES and Good's buffer solution.
S103: adding a first blocking solution containing a first blocking compound, and reacting for 1-24 hours to block the active sites on the surface of the solid phase carrier and obtain the solid phase carrier modified with the first blocking compound on the surface.
In particular, the first blocking compound may be a polyhydroxy carbohydrate compound or a protein compound. The polyhydroxylated saccharide compound may be at least one of glucose, sucrose, lactose, trehalose, dextran, mannitol or polysucrose. The protein compound can be at least one of bovine serum albumin, human serum albumin, casein, gelatin, casein hydrolysate, immunoglobulin, milk powder, and human or animal serum.
The polyhydroxycarbohydrate or proteinaceous compound may be diluted with a buffer solution to a concentration of 0.01% to 10% and added to 1-1000. Mu.L for 1-24 hours (e.g., 1 hour, 4 hours, 12 hours or 24 hours) at 37 ℃ to obtain the first blocking compound-modified solid support. Wherein the buffer solution can be at least one of sodium carbonate/sodium bicarbonate buffer solution, acetic acid/potassium acetate buffer solution, tris/hydrochloric acid buffer solution, 3- (N-morpholino) propanesulfonic acid sodium salt/hydrochloric acid buffer solution, 2- (N-morpholine) ethanesulfonic acid buffer solution (MES buffer solution), phosphate buffer solution, borate buffer solution, MOPS, HEPES and Good's buffer solution.
S104: adding a second blocking solution containing a second blocking compound, and reacting for 1-24 hours to block the active sites on the surface of the solid phase carrier and obtain the solid phase carrier modified with the second blocking compound on the surface.
In particular, the second blocking compound may be a polyhydroxy carbohydrate compound or a proteinaceous compound, the first blocking compound being different from the second blocking compound. When the first blocking compound is a polyhydroxycarbohydrate compound, the second blocking compound is a proteinaceous compound. When the first blocking compound is a proteinaceous compound, the second blocking compound is a polyhydroxycarbohydrate compound.
The polyhydroxycarbohydrate or proteinaceous compound may be diluted with a buffer solution to a concentration of 0.01-10% and added to 1-1000. Mu.L for 1-24 hours (e.g., 1 hour, 4 hours, 12 hours or 24 hours) at 37 ℃ to provide a solid support modified with a second blocking compound. Wherein the buffer solution can be at least one of sodium carbonate/sodium bicarbonate buffer solution, acetic acid/potassium acetate buffer solution, tris/hydrochloric acid buffer solution, 3- (N-morpholino) propanesulfonic acid sodium salt/hydrochloric acid buffer solution, 2- (N-morpholine) ethanesulfonic acid buffer solution (MES buffer solution), phosphate buffer solution, borate buffer solution, MOPS, HEPES and Good's buffer solution.
In contrast to the prior art, in the present embodiment, the surface of the solid phase carrier is modified with a first blocking compound and a second blocking compound, and the first blocking compound and the second blocking compound are used for blocking active sites, wherein the first blocking compound is a polyhydroxy carbohydrate compound, and the second blocking compound is a protein compound, so that most or even all active sites on the surface of the solid phase carrier can be blocked by the two blocking compounds, and thus non-specific adsorption of an interfering substance to the solid phase carrier is effectively inhibited. Particularly, when a plurality of ligands are immobilized on the solid phase carrier and classification detection is carried out, the effect of blocking the solid phase carrier by adopting the blocking strategy of the embodiment is obviously superior to that of the traditional blocking strategy of taking protein as a single blocking agent, the blocking effect is effectively improved, the influence of nonspecific adsorption can be eliminated, the influence on the obtained effective data volume is reduced, and the sensitivity and specificity of detection are improved.
In an embodiment, before step S103, the preparation method further includes the steps of: adding a third sealing solution containing a third sealing compound, and reacting for 0.5-2 hours to seal the active sites on the surface of the solid phase carrier and obtain the solid phase carrier modified with the third sealing compound on the surface. Wherein the third blocking compound is a small molecule compound containing a primary amino group.
Specifically, the primary amine group-containing small molecule compound may be at least one of tris, ethanolamine, hydroxylamine, hexylamine, or glycine. It may be diluted to a concentration of 0.01% to 10% with a buffer solution, added to 1 to 1000. Mu.L, and reacted at 37 ℃ for 0.5 to 2 hours (e.g., 0.5 hour, 1 hour, or 2 hours) to obtain a solid support modified with a third blocking compound. Wherein the buffer solution can be at least one of sodium carbonate/sodium bicarbonate buffer solution, acetic acid/potassium acetate buffer solution, tris/hydrochloric acid buffer solution, 3- (N-morpholino) propanesulfonic acid sodium salt/hydrochloric acid buffer solution, 2- (N-morpholine) ethanesulfonic acid buffer solution (MES buffer solution), phosphate buffer solution, borate buffer solution, MOPS, HEPES and Good's buffer solution.
By the mode, the blocking effect can be further improved by pre-blocking with the primary amine group-containing small molecular weight compound before surface modification of the polyhydroxy carbohydrate compound and the protein compound.
In one embodiment, the first blocking compound, the second blocking compound, and the third blocking compound are modified on the surface of the solid support by at least one of electrostatic interactions, hydrophobic interactions, hydrogen bonding, intermolecular forces, specific interactions between biomolecules, or covalent bonds.
In one embodiment, the step of immobilizing the ligand to the surface of the solid support comprises: the ligand is directly immobilized on a solid support. Or indirectly fixing the ligand on the solid phase carrier through specific affinity reaction of protein A and/or protein G, secondary antibody and primary antibody, avidin and biotin.
Specifically, the coating method includes a direct coating method in which a ligand is directly immobilized on a solid support, and an indirect coating method. The indirect coating method is to fix the ligand on the solid phase carrier indirectly through the specific affinity reaction of protein A and/or protein G, secondary antibody and primary antibody, avidin and biotin.
The present application provides for the use of an immunoassay composition as described above in a cardiac assay, a hypertension assay, a thyroid assay, a cardiovascular assay, a gonadal assay, a renal function assay, a bone metabolism assay, a carbohydrate metabolism assay, an infectious disease assay, an autoimmune disease assay, prenatal screening, a drug assay, a liver fibrosis assay, an epstein barr virus assay, an inflammation assay, or a tumor assay.
The present application provides an immunoassay kit comprising, e.g., an immunocapture composition; and, an immunodetection composition comprising at least one second ligand and a signal tracer; the signal tracer is at least one of an enzymatic tracer, a chemiluminescent tracer, or a fluorescent tracer. In particular, the at least one second ligand is an antibody or an antigen.
Preferably, the second ligand may be a detection antibody. When the immunoassay kit is used, firstly, the capture antibody is combined with an antigen to be detected, the formed antigen-antibody complex is combined with a detection antibody, and a solid phase carrier-capture antibody-antigen to be detected-detection antibody complex is formed in the reaction process.
For a detailed description of the immunocapture composition, reference is made to the immunocapture composition in the above embodiments, which are not described herein again. The effect of sealing by adopting the immunoassay kit is obviously superior to the sealing strategy of taking the traditional protein as a single sealing agent, the sealing effect is effectively improved, the influence of non-specific adsorption can be eliminated, the influence on the obtained effective data volume is reduced, and the sensitivity and specificity of detection are improved.
Preferably, the signal tracer is a fluorescent tracer and the fluorescent tracer is different from the fluorescent marker in the immunocapture composition. The fluorescent tracer and the fluorescent label may have different fluorescent intensities, for example, the fluorescent tracer and the fluorescent label may be different kinds of fluorescent compounds, or the fluorescent tracer and the fluorescent label may be the same kind of fluorescent compound having different particle sizes.
Further, the immunoassay composition may comprise a fluorescent label and a fluorescence-enhancing particle, each linked to a detection antibody, wherein the fluorescence-enhancing particle comprises a nano-metal particle. According to the embodiment, the detection antibody is connected with the nano metal particles and the fluorescent marker, and the plasma resonance generated by the metal nano particles enables the fluorescent signal emitted by the fluorescent marker to be enhanced, so that the detection sensitivity can be effectively improved.
Wherein the at least one second ligand comprises: a third antibody and a fourth antibody; the first antibody, the third antibody and the antigen to be detected of the immunocapture composition are specifically combined; the second antibody of the immunocapture composition specifically binds to the fourth antibody, the antigen to be detected.
Specifically, the third antibody and the fourth antibody may be detection antibodies. When the immunoassay kit is used, firstly, the first antibody is combined with an antigen to be detected, the formed antigen-antibody complex is combined with the third antibody, and a solid phase carrier-first antibody-antigen to be detected-third antibody complex is formed in the reaction process. The second antibody is combined with the antigen to be detected, the formed antigen-antibody complex is combined with the fourth antibody, and a solid phase carrier-second antibody-antigen to be detected-fourth antibody complex is formed in the reaction process.
In one embodiment, the immunoassay kit further comprises: at least one reagent tube, wherein a suspension liquid is arranged in the reagent tube; the immunocapture composition and the immunodetection composition are dissolved in the suspension; wherein the suspension comprises a buffer solution, a surfactant, an inorganic salt, a stabilizer and a preservative. It will be appreciated that the suspension may comprise a plurality of capture immune compositions and a plurality of detection immune compositions, each of which may in turn comprise a plurality of single first ligands and a plurality of single second ligands, respectively.
Specifically, the suspension comprises a buffer, a surfactant, an inorganic salt, a stabilizer and a preservative. Wherein the pH range of the buffer solution is between 7.0 and 9.0, and the concentration range of the buffer solution is between 10 and 100mmol/L. The buffer is at least one of 3- (N-morpholino) propanesulfonic acid-sodium hydroxide buffer (MOPS-NaOH), 3- [ N, N-bis (hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid-sodium hydroxide buffer (DIPSO-NaOH), 4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid-sodium hydroxide buffer (HEPPS-NaOH), tris (hydroxymethyl) aminomethane-HCl buffer (Tris-HCl), N- (2-hydroxyethyl) piperazine-N' -2-ethanesulfonic acid-NaOH buffer (HEPES-NaOH), phosphate buffer, borate buffer, imidazole buffer, citric acid buffer, glycine-NaOH buffer, or barbiturate buffer. The stabilizer is gelatin, bovine serum albumin or casein. The inorganic salt is at least one of sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium chloride, sodium sulfate or potassium sulfate. The surfactant is Tween20 or Triton X-100. The stabilizer is at least one of sucrose, trehalose, glycerol, mannitol, polyethylene glycol, polyvinylpyrrolidone or disodium ethylene diamine tetraacetate with the concentration of 1-5%. The antiseptic is at least one of sodium azide, thimerosal or Proclin-300.
In order to further improve the convenience of the application of the immunoassay kit in an automated immunoassay device, in a preferred embodiment of the present application, the kit further comprises a calibrator and a quality control material. The calibrator and the quality control material are commonly used in the existing detection immunoassay kit.
The present application provides the use of an immunoassay composition as described above in an immunoassay.
Referring to fig. 2, the present application provides an immunoassay method based on the aforementioned immunoassay kit, the immunoassay method comprising the following steps:
s201: an immunocapture composition is provided.
Specifically, the immunocapture compositions comprise: the solid phase carrier comprises at least one first ligand, at least one first blocking compound and at least one second blocking compound, wherein the first blocking compound and the second blocking compound are fixed on the surface of the solid phase carrier and modified on the surface of the solid phase carrier and are used for blocking active sites on the surface of the solid phase carrier, the first blocking compound is polyhydroxy carbohydrate or protein compound, the second blocking compound is polyhydroxy carbohydrate or protein compound, and the first blocking compound is different from the second blocking compound. For a detailed description of the preparation of the immunocapture composition, reference is made to the preparation method of the immunocapture composition in the above embodiments, which are not described herein again.
S202: adding the test sample and the immunocapture composition to the test line.
Specifically, the immunocapture composition prepared in step S102 and the sample to be tested are added to the test system, and after 10-30 minutes (e.g., 10 minutes, 20 minutes, or 30 minutes) of reaction, the following reaction results: antigen to be detected-primary antibody-solid phase carrier-secondary antibody-antigen to be detected.
S203: adding at least one immunoassay composition to the test system.
Specifically, the second ligand may be a biotinylated detection antibody, for example, the detection antibody is reacted with biotin to prepare a biotin-labeled detection antibody, that is, the biotinylated detection antibody. The preparation of the biotinylated antibody is a conventional technical means in the art, and is not described herein. Adding a biotinylation detection antibody, and incubating at room temperature to form a solid phase carrier-first antibody-antigen to be detected-detection antibody-biotin compound, a solid phase carrier-second antibody-antigen to be detected-detection antibody-biotin compound, or biotin-detection antibody-antigen to be detected-first antibody-solid phase carrier-second antibody-antigen to be detected-detection antibody-biotin compound.
Specifically, the detection antibody is reacted with biotin to prepare a biotin-labeled detection antibody, namely a biotinylated detection antibody. The preparation of the biotinylated detection antibody is a conventional technical means in the art, and is not described herein.
The signal tracer is at least one of an enzymatic tracer, a chemiluminescent tracer, or a fluorescent tracer. Preferably, the signal tracer may be streptavidin-phycoerythrin, which is added to the detection system to allow the detection antibody to bind to the streptavidin-phycoerythrin.
Streptavidin-phycoerythrin (SAPE) is added, and incubation is carried out at room temperature, so that a solid phase carrier-first antibody-antigen to be detected-detection antibody-biotin-SAPE compound, a solid phase carrier-second antibody-antigen to be detected-detection antibody-biotin-SAPE compound or SAPE-biotin-detection antibody-antigen to be detected-first antibody-solid phase carrier-second antibody-antigen to be detected-detection antibody-biotin-SAPE compound can be formed.
The signal tracer can be at least one of fluorescein isothiocyanate, phycoerythrin-texas red or phycoerythrin-anthocyanin.
S204: and (5) incubating and magnetically separating.
S205: the fluorescent signals of the immunocapture composition and the immunodetection composition are detected separately.
Specifically, the signal intensity generated by the photosensitive substance SA-PE can be detected by using a flow cytometer as a detection tool and adopting a double-antibody sandwich method as a methodology mode.
Wherein the first ligand is NT-proBNP capture antibody, troponin capture antibody, alpha fetoprotein capture antibody, carcinoembryonic capture antibody, HIV capture antibody, hepatitis B surface capture antibody, thyroglobulin capture antibody, troponin T capture antibody or myoglobin capture antibody.
The present application is further described below with reference to examples:
example 1
The immunoassay composition 1 comprises the following components:
(1) Solid phase carrier: surface carboxylated magnetic beads with the diameter of 1-10 mu m;
(2) Ligand: a nitrogen-terminal brain natriuretic peptide capture antibody;
(3) First blocking compound: trehalose;
(4) A second blocking compound: bovine serum albumin.
The preparation of the immunoassay composition 1 comprises the following steps:
step (1): after magnetic separation, 1mg of surface-carboxylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with MES buffer (50mM, pH 6), and after magnetic separation, the magnetic beads were resuspended in MES buffer, and 100. Mu.g each of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added, wherein the NHS and EDC were dissolved in 50mM, pH6 MES buffer, respectively, and activated at room temperature for 30 minutes. Magnetic separation was performed by washing twice with PBS buffer (10mM, 0.1% Tween 20). 40. Mu.g of the N-terminal brain natriuretic peptide capture antibody (dissolved in PBS buffer) was added thereto, and the reaction was carried out at room temperature for 2 hours. After magnetic separation, the mixture was washed twice with PBS buffer (10 mM,0.1% Tween 20) to obtain magnetic beads coated with the nitrogen-terminal BNP capture antibody.
Step (2): 0.5mL of the first blocking compound solution (5% trehalose dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 2 hours.
And (3): 0.5mL of a second blocking compound solution (1% bovine serum albumin, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 1 hour.
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10 mM,0.05% Tween20, 0.1% BSA, pH 7.4) buffer was added to the cells, and the cells were stored at a magnetic bead concentration of 0.2mg/mL and protected from light at 2 to 8 ℃.
Example 2
The immunoassay composition 2 comprises the following components:
(1) Solid phase carrier: surface carboxylated magnetic beads with the diameter of 6 mu m;
(2) Ligand: a nitrogen-terminal brain natriuretic peptide capture antibody;
(3) First blocking compound: bovine serum albumin;
(4) A second blocking compound: trehalose.
The preparation of the immunoassay composition 2 comprises the following steps:
step (1): after magnetic separation, 1mg of surface-carboxylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with MES buffer (50mM, pH 6), and after magnetic separation, the magnetic beads were resuspended in MES buffer, and 100. Mu.g each of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added, wherein the NHS and EDC were dissolved in 50mM, pH6 MES buffer, respectively, and activated at room temperature for 30 minutes. Magnetic separation was performed by washing twice with PBS buffer (10mM, 0.1% Tween 20). 40. Mu.g of the N-terminal brain natriuretic peptide capture antibody (dissolved in PBS buffer) was added thereto, and the reaction was carried out at room temperature for 2 hours. After magnetic separation, washing was performed twice with PBS buffer (10 mM,0.1% Tween 20) to obtain magnetic beads coated with the nitrogen-terminal brain natriuretic peptide capture antibody.
Step (2): 0.5mL of a first blocking compound solution (1% bovine serum albumin, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 1 hour.
And (3): 0.5mL of a second blocking compound solution (5% trehalose dissolved in 10mM PBS buffer, pH 7.4) was added and blocked for 2 hours at room temperature.
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10 mM,0.05% Tween20, 0.1% BSA, pH 7.4) buffer was added to the cells, and the cells were stored at a magnetic bead concentration of 0.2mg/mL and protected from light at 2 to 8 ℃.
Example 3
The immunoassay composition 3 comprises the following components:
(1) Solid phase carrier: surface carboxylated magnetic beads with the diameter of 6 μm;
(2) Ligand: a nitrogen-terminal brain natriuretic peptide capture antibody;
(3) First blocking compound: trehalose;
(4) Third blocking compound: ethanolamine.
The preparation of the immunoassay composition 3 comprises the following steps:
step (1): after magnetic separation, 1mg of surface-carboxylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with MES buffer (50mM, pH 6), and after magnetic separation, the magnetic beads were resuspended in MES buffer, and 100. Mu.g each of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added, wherein the NHS and EDC were dissolved in 50mM, pH6 MES buffer, respectively, and activated at room temperature for 30 minutes. Washed twice with PBS buffer (10 mM,0.1% Tween 20) to perform magnetic separation. 40. Mu.g of the N-terminal brain natriuretic peptide capture antibody (dissolved in PBS buffer) was added thereto, and the reaction was carried out at room temperature for 2 hours. After magnetic separation, the mixture was washed twice with PBS buffer (10 mM,0.1% Tween 20) to obtain magnetic beads coated with the nitrogen-terminal BNP capture antibody.
Step (2): 0.5mL of a third blocking compound solution (5% ethanolamine, 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 0.5 hour.
And (3): 0.5mL of the first blocking compound solution (5% trehalose dissolved in 10mM PBS buffer, pH 7.4) was added and blocked for 3 hours at room temperature.
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10 mM,0.05% Tween20, 0.1% BSA, pH 7.4) buffer was added to the cells, and the cells were stored at a magnetic bead concentration of 0.2mg/mL and protected from light at 2 to 8 ℃.
Example 4
The immunoassay composition 4 comprises the following components:
(1) Solid phase carrier: surface carboxylated magnetic beads with the diameter of 6 mu m;
(2) Ligand: a nitrogen-terminal brain natriuretic peptide capture antibody;
(3) First blocking compound: bovine serum albumin;
(4) Third blocking compound: ethanolamine.
The preparation of the immunoassay composition 4 comprises the following steps:
step (1): after magnetic separation, 1mg of surface-carboxylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with MES buffer (50mM, pH 6), and after magnetic separation, the magnetic beads were resuspended in MES buffer, and 100. Mu.g each of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added, wherein the NHS and EDC were dissolved in 50mM, pH6 MES buffer, respectively, and activated at room temperature for 30 minutes. Magnetic separation was performed by washing twice with PBS buffer (10mM, 0.1% Tween 20). 40. Mu.g of the N-terminal brain natriuretic peptide capture antibody (dissolved in PBS buffer) was added thereto, and the reaction was carried out at room temperature for 2 hours. After magnetic separation, washing was performed twice with PBS buffer (10 mM,0.1% Tween 20) to obtain magnetic beads coated with the nitrogen-terminal brain natriuretic peptide capture antibody.
Step (2): 0.5mL of a third blocking compound solution (5% ethanolamine, 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 0.5 hour.
And (3): 0.5mL of the first blocking compound solution (1% bovine serum albumin, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked for 3 hours at room temperature.
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10mM, 0.05% Tween20, 0.1% BSA, pH7.4) buffer was added, the concentration of magnetic beads was 0.2mg/mL, and the mixture was stored at 2-8 ℃ in the dark.
Example 5
The immunoassay composition 5 comprises the following components:
(1) Solid phase carrier: surface carboxylated magnetic beads with the diameter of 6 mu m;
(2) Ligand: a nitrogen-terminal brain natriuretic peptide capture antibody;
(3) First blocking compound: bovine serum albumin;
(4) A second blocking compound: trehalose;
(5) Third blocking compound: ethanolamine.
The preparation of the immunoassay composition 5 comprises the following steps:
step (1): after magnetic separation, 1mg of surface-carboxylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with MES buffer (50mM, pH 6), and the magnetic beads were resuspended in MES buffer, 100. Mu.g each of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added, wherein the NHS and EDC were dissolved in 50mM, pH6 MES buffer, respectively, and activated at room temperature for 30 minutes. Magnetic separation was performed by washing twice with PBS buffer (10mM, 0.1% Tween 20). 40. Mu.g of the N-terminal brain natriuretic peptide capture antibody (dissolved in PBS buffer) was added thereto, and the reaction was carried out at room temperature for 2 hours. After magnetic separation, the mixture was washed twice with PBS buffer (10 mM,0.1% Tween 20) to obtain magnetic beads coated with the nitrogen-terminal BNP capture antibody.
Step (2): 0.5mL of a third blocking compound solution (5% ethanolamine, 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 0.5 hour.
And (3): 0.5mL of the first blocking compound solution (1% bovine serum albumin, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked for 1 hour at room temperature.
And (4): 0.5mL of a second blocking compound solution (5% trehalose dissolved in 10mM PBS buffer, pH 7.4) was added and blocked for 2 hours at room temperature.
And (5): after the sealing is completed, the magnetic separation is washed twice.
And (6): PBS-TBN (10 mM,0.05% Tween20, 0.1% BSA, pH 7.4) buffer was added to the cells, and the cells were stored at a magnetic bead concentration of 0.2mg/mL and protected from light at 2 to 8 ℃.
Example 6
The immunoassay composition 6 comprises the following components:
(1) Solid phase carrier: surface carboxylated magnetic beads with the diameter of 6 mu m;
(2) Ligand: a nitrogen-terminal brain natriuretic peptide capture antibody;
(3) First blocking compound: trehalose;
(4) A second blocking compound: bovine serum albumin;
(5) Third blocking compound: ethanolamine.
The preparation of the immunoassay composition 6 comprises the following steps:
step (1): after magnetic separation, 1mg of surface-carboxylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with MES buffer (50mM, pH 6), and after magnetic separation, the magnetic beads were resuspended in MES buffer, and 100. Mu.g each of N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added, wherein the NHS and EDC were dissolved in 50mM, pH6 MES buffer, respectively, and activated at room temperature for 30 minutes. Washed twice with PBS buffer (10 mM,0.1% Tween 20) to perform magnetic separation. 40. Mu.g of the N-terminal brain natriuretic peptide capture antibody (dissolved in PBS buffer) was added thereto, and the reaction was carried out at room temperature for 2 hours. After magnetic separation, the mixture was washed twice with PBS buffer (10 mM,0.1% Tween 20) to obtain magnetic beads coated with the nitrogen-terminal BNP capture antibody.
Step (2): 0.5mL of a third blocking compound solution (5% ethanolamine, 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 0.5 hour.
And (3): 0.5mL of the first blocking compound solution (5% trehalose dissolved in 10mM PBS buffer, pH 7.4) was added and blocked for 2 hours at room temperature.
And (4): 0.5mL of a second blocking compound solution (1% bovine serum albumin, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked for 1 hour at room temperature.
And (5): after the sealing is completed, the magnetic separation is washed twice.
And (6): PBS-TBN (10 mM,0.05% Tween20, 0.1% BSA, pH 7.4) buffer was added to the cells, and the cells were stored at a magnetic bead concentration of 0.2mg/mL and protected from light at 2 to 8 ℃.
Example 7
The immunodetection compositions prepared in examples 1 to 6 were used in immunodetection methods for nitrogen-terminal brain natriuretic peptides.
The immunodetection method of the N-terminal brain natriuretic peptide comprises the following steps:
step (1): preparation of biotinylated N-terminal brain natriuretic peptide detection antibody
Taking 0.1mg nitrogen terminal brain natriuretic peptide detection antibody, using NaHCO 3 (100mM, pH8.3) the solution was diluted to a concentration of 2mg/mL; mu.L of a 10mM DMSO solution of (+) Biotin-N-succinimidyl ester (Biotin-NHS) was added and the reaction was carried out at 37 ℃ for 2 hours. Purifying the biotinylated nitrogen terminal brain natriuretic peptide detection antibody through a Sephadex G-25 chromatographic column; 1mL of PBS-TBN buffer solution was added, and the mixture was stored at 2-8 ℃ in pH7.4.
Step (2): immunoassay
mu.L of the sample to be tested was added to the reaction cuvette, 50. Mu.L of the immunodetection compositions of examples 1 to 6 and the control were added, and after 20 minutes of reaction, 100. Mu.L of the biotinylated N-terminal brain natriuretic peptide detection antibody was added, and after 10 minutes of reaction, 100. Mu.L of streptavidin-labeled phycoerythrin (SA-PE) solution was finally added, and the reaction was carried out for 5 minutes. After magnetic separation, 100. Mu.L of PBS-TBN buffer solution was added, and the mixture was put into a flow cytometer to detect fluorescence intensity (MFI). The results of the experiment are shown in table 1.
The immunoassay composition of the control group was prepared by blocking only bovine serum albumin blocking solution (1% bovine serum albumin, dissolved in 10mM PBS buffer, pH 7.4) at room temperature for 3 hours.
TABLE 1 fluorescence intensity measurement results of immunodetection method for N-terminal brain natriuretic peptide using the immunodetection compositions prepared in examples 1 to 6
Figure BDA0001931366140000171
Example 8
The immunoassay composition 7 comprises the following components:
(1) Solid phase carrier: magnetic beads with tosylated surface, diameter 1 μm;
(2) Ligand: an alpha-fetoprotein capture antibody;
(3) First blocking compound: mannitol;
(4) A second blocking compound: casein.
The preparation of the immunoassay composition 7 comprises the following steps:
step (1): 50mg of surface tosylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with 0.1M borate buffer, after magnetic separation, the magnetic beads were resuspended in 0.8mL of borate buffer, and 2mg of AFP antibody was added. Finally, 0.4mL of 3M ammonium sulfate solution was added and the reaction was carried out at 37 ℃ for 24 hours. Magnetic separation was performed, and washing was performed twice with PBS buffer (10 mM,0.05% Tween 20) to obtain magnetic beads coated with the alpha-fetoprotein capture antibody.
Step (2): 0.5mL of the first blocking compound solution (10% mannitol dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (3): 0.5mL of a second blocking compound solution (1% casein, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10mM, 0.05% Tween20, 0.1% bovine serum albumin, pH7.4) buffer was added, the concentration of the magnetic beads was 0.5mg/mL, and the mixture was stored at 2 to 8 ℃ in the dark.
Example 9
The immunoassay composition 8 comprises the following components:
(1) Solid phase carrier: magnetic beads with tosylated surface, diameter 1 μm;
(2) Ligand: an alpha-fetoprotein capture antibody;
(3) First blocking compound: casein;
(4) A second blocking compound: mannitol.
The preparation of the immunoassay composition 8 comprises the steps of:
step (1): 50mg of surface tosylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with 0.1M borate buffer, after magnetic separation, the magnetic beads were resuspended in 0.8mL of borate buffer, and 2mg of AFP antibody was added. Finally, 0.4mL of 3M ammonium sulfate solution was added and the reaction was carried out at 37 ℃ for 24 hours. Magnetic separation was performed, and washing was performed twice with PBS buffer (10 mM,0.05% Tween 20) to obtain magnetic beads coated with the alpha-fetoprotein capture antibody.
Step (2): 0.5mL of the first blocking compound solution (1% casein, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (3): 0.5mL of a second blocking compound solution (10% mannitol dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10mM, 0.05% Tween20, 0.1% bovine serum albumin, pH7.4) buffer was added, the concentration of the magnetic beads was 0.5mg/mL, and the mixture was stored at 2 to 8 ℃ in the dark.
Example 10
The immunoassay composition 9 comprises the following components:
(1) Solid phase carrier: magnetic beads with tosylated surface, diameter 1 μm;
(2) Ligand: an alpha-fetoprotein capture antibody;
(3) First blocking compound: mannitol;
(4) Third blocking compound: glycine.
The preparation of the immunoassay composition 9 comprises the following steps:
step (1): 50mg of surface tosylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with 0.1M borate buffer, after magnetic separation, the magnetic beads were resuspended in 0.8mL of borate buffer, and 2mg of AFP antibody was added. Finally, 0.4mL of 3M ammonium sulfate solution was added and the reaction was carried out at 37 ℃ for 24 hours. Magnetic separation was performed, and washing was performed twice with PBS buffer (10 mM,0.05% Tween 20) to obtain magnetic beads coated with the alpha-fetoprotein capture antibody.
Step (2): 0.5mL of a third blocking compound solution, (10 mM glycine, pH 8.3) was added and blocked for 0.5 hours at room temperature.
And (3): 0.5mL of the first blocking compound solution (10% mannitol dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 8 hours.
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10mM, 0.05% Tween20, 0.1% bovine serum albumin, pH7.4) buffer was added, the concentration of the magnetic beads was 0.5mg/mL, and the mixture was stored at 2 to 8 ℃ in the dark.
Example 11
The immunoassay composition 10 includes the following components:
(1) Solid phase carrier: magnetic beads with tosylated surface, diameter 1 μm;
(2) Ligand: an alpha-fetoprotein capture antibody;
(3) First blocking compound: casein protein;
(4) Third blocking compound: glycine.
The preparation of the immunoassay composition 10 comprises the steps of:
step (1): 50mg of surface tosylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with 0.1M borate buffer, after magnetic separation, the magnetic beads were resuspended in 0.8mL of borate buffer, and 2mg of AFP antibody was added. Finally, 0.4mL of 3M ammonium sulfate solution was added and the reaction was carried out at 37 ℃ for 24 hours. Magnetic separation was performed, and washing was performed twice with PBS buffer (10 mM,0.05% Tween 20) to obtain magnetic beads coated with the alpha-fetoprotein capture antibody.
Step (2): 0.5mL of a third blocking compound solution (10 mM glycine, pH 8.3) was added and blocked at room temperature for 0.5 hours. .
And (3): 0.5mL of a first blocking compound solution (1% casein, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 8 hours
And (4): after the sealing is completed, the magnetic separation is washed twice.
And (5): PBS-TBN (10mM, 0.05% Tween20, 0.1% bovine serum albumin, pH7.4) buffer was added, the concentration of the magnetic beads was 0.5mg/mL, and the mixture was stored at 2 to 8 ℃ in the dark.
Example 12
The immunoassay composition 11 comprises the following components:
(1) Solid phase carrier: magnetic beads with tosylated surface, diameter 1 μm;
(2) Ligand: an alpha-fetoprotein capture antibody;
(3) First blocking compound: mannitol;
(4) A second blocking compound: casein;
(5) Third blocking compound: glycine.
The preparation of the immunoassay composition 11 comprises the following steps:
step (1): 50mg of surface tosylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with 0.1M borate buffer, after magnetic separation, the magnetic beads were resuspended in 0.8mL of borate buffer, and 2mg of AFP antibody was added. Finally, 0.4mL of 3M ammonium sulfate solution was added and the reaction was carried out at 37 ℃ for 24 hours. Magnetic separation was performed, and washing was performed twice with PBS buffer (10 mM,0.05% Tween 20) to obtain magnetic beads coated with the alpha-fetoprotein capture antibody.
Step (2): 0.5mL of a third blocking compound solution (10 mM glycine, pH 8.3) was added and blocked at room temperature for 0.5 hours.
And (3): 0.5mL of a second blocking compound solution (1% casein, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (4): 0.5mL of a first blocking compound solution (10% mannitol dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (5): after the sealing is completed, the magnetic separation is washed twice.
And (6): PBS-TBN (10mM, 0.05% Tween20, 0.1% bovine serum albumin, pH7.4) buffer was added, the concentration of the magnetic beads was 0.5mg/mL, and the mixture was stored at 2 to 8 ℃ in the dark.
Example 13
The immunoassay composition 12 comprises the following components:
(1) Solid phase carrier: magnetic beads with tosylated surface, diameter 1 μm;
(2) Ligand: an alpha-fetoprotein capture antibody;
(3) First blocking compound: casein protein;
(4) A second blocking compound: mannitol;
(5) Third blocking compound: glycine.
The preparation of the immunoassay composition 12 comprises the steps of:
step (1): 50mg of surface tosylated magnetic beads (hereinafter referred to as magnetic beads) were washed twice with 0.1M borate buffer, after magnetic separation, the magnetic beads were resuspended in 0.8mL of borate buffer, and 2mg of AFP antibody was added. Finally, 0.4mL of 3M ammonium sulfate solution was added and the reaction was carried out at 37 ℃ for 24 hours. Magnetic separation was performed, and washing was performed twice with PBS buffer (10 mM,0.05% Tween 20) to obtain magnetic beads coated with the alpha-fetoprotein capture antibody.
Step (2): 0.5mL of a third blocking compound solution (10 mM glycine, pH 8.3) was added and blocked at room temperature for 0.5 hours.
And (3): 0.5mL of a second blocking compound solution (10% mannitol dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (4): 0.5mL of a first blocking compound solution (1% casein, dissolved in 10mM PBS buffer, pH 7.4) was added and blocked at room temperature for 4 hours.
And (5): after the sealing is completed, the magnetic separation is washed twice.
And (6): PBS-TBN (10mM, 0.05% Tween20, 0.1% bovine serum albumin, pH7.4) buffer was added, the concentration of the magnetic beads was 0.5mg/mL, and the mixture was stored at 2 to 8 ℃ in the dark.
Example 14
Use of the immunoassay compositions prepared in examples 7-13 in a method for immunoassay of alpha-fetoprotein.
The immunodetection method of alpha-fetoprotein comprises the following steps:
step (1): preparation of tracer antibody
1mg of alpha-fetoprotein detection antibody is prepared into 5mg/mL by 0.05MPBS buffer solution with the pH value of 8.0, 5 mu L of 2-iminothiolane hydrochloride is added, and the reaction is carried out for 2 hours at the temperature of 37 ℃. The amino group on the alpha-fetoprotein detection antibody is converted to a thiol group. Purifying the antibody by a SephadexG-25 chromatographic column; 1mg of alkaline phosphatase was activated with 10. Mu.L of succinimidyl-4- (N-maleimide) cyclohexane-1-carboxylate (SMCC coupling agent) (10 mg/mL) at room temperature for 0.5h. The thiol-modified alpha-fetoprotein detection antibody was added to the activated alkaline phosphatase, and the reaction was carried out at 4 ℃ for 12 hours.
Step (2): immunoassay
After 20. Mu.L of the sample to be tested and 50. Mu.L of each of the immunodetection compositions prepared in examples 7 to 13 and the control were added to the reaction cup, and reacted for 20 minutes, magnetic separation was performed and washing was performed 2 times. Then 50. Mu.L of tracer antibody is added, after reacting for 20 minutes, 50. Mu.L of substrate solution AMPPD (3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) benzene-1,2-dioxetane) is finally added, reacting for 15 minutes, and the mixture is placed into a multifunctional microplate reader for detecting a luminescence value (RLU). The results of the experiment are shown in table 2.
The immunoassay composition of the control group was prepared by blocking only bovine serum albumin blocking solution (1% bovine serum albumin, dissolved in 10mM PBS buffer, pH 7.4) at room temperature for 3 hours.
TABLE 2 results of luminescence value measurement of the immunoassay method for alpha-fetoprotein using the immunoassay compositions prepared in examples 8 to 13
Figure BDA0001931366140000211
As can be seen from tables 1-2 above:
(1) In examples 1-6 and 8-13, the signal-to-noise ratios of examples 1-6 were all higher than the signal-to-noise ratio of the control group, i.e., the background luminescence signals of examples 1-6 were all lower than the background luminescence signal of the control group, which indicates that compared with the case where a single protein compound was used as a blocking agent, the background signal was significantly reduced, and the detection sensitivity was effectively improved.
(2) In examples 1-2 and 8-9, when blocking was performed using a polyhydroxy carbohydrate and then using a protein compound, the signal-to-noise ratio was higher, which indicates that when the preferred blocking order was polyhydroxy carbohydrate and protein compound, the background signal was significantly reduced, and the sensitivity of detection could be effectively improved.
(3) In examples 3-4 and 10-11, the signal-to-noise ratio of the blocking using the primary amino group-containing small molecule compound and the polyhydroxycarbohydrate compound was higher than that of the blocking using the primary amino group-containing small molecule compound and the protein compound, indicating that when the blocking order is preferably the primary amino group-containing small molecule compound and the polyhydroxycarbohydrate compound, the background signal is significantly reduced, and the detection sensitivity can be effectively improved.
(4) In examples 5 to 6 and 12 to 13, when the primary amine group-containing small molecular compound, the polyhydroxy carbohydrate compound, and the protein compound were used in this order as the blocking agent, nonspecific adsorption was significantly reduced, and compared with the other examples, the background signal was significantly reduced, and the detection sensitivity was improved.
The above description is only an embodiment of the present application, and is not intended to limit the scope of the present application, and all modifications, equivalents, flow charts, and other related technical fields that are made by the present application are included in the scope of the present application.

Claims (7)

1. A method of preparing an immunocapture composition, comprising:
step (1): providing a solid phase carrier, and directly fixing a first ligand on the surface of the solid phase carrier to obtain the solid phase carrier coated with the first ligand; the solid phase carrier is a surface carboxylated magnetic bead with the diameter of 6 mu m; the first ligand is a nitrogen-terminal brain natriuretic peptide capture antibody;
step (2): adding 0.5mL of a third blocking compound solution, wherein the third blocking compound solution is 5% ethanolamine, 10mM PBS buffer solution and pH7.4, and blocking for 0.5 hour at room temperature;
and (3): adding 0.5mL of a first blocking compound solution, wherein the first blocking compound solution is formed by dissolving 5% trehalose in a 10mMPBS buffer solution with a pH value of 7.4, and blocking for 2 hours at room temperature;
and (4): adding 0.5mL of a second blocking compound solution, wherein the second blocking compound solution is formed by dissolving 1% bovine serum albumin in 10mMPBS buffer solution at pH7.4, and blocking for 1 hour at room temperature;
and (5): after the sealing is finished, performing magnetic separation and cleaning twice;
and (6): adding PBS-TBN buffer solution which is 10mM PBS, 0.05% Tween20, 0.1% BSA, pH7.4, the concentration of magnetic beads is 0.2mg/mL, and storing at 2-8 ℃ in a dark place.
2. A method of preparing an immunocapture composition, comprising:
step (1): providing a solid phase carrier, and directly fixing a first ligand on the surface of the solid phase carrier to obtain the solid phase carrier coated with the first ligand; the solid phase carrier is a magnetic bead with the surface being tosylated, and the diameter is 1 mu m; the first ligand is an alpha fetoprotein capture antibody;
step (2): adding 0.5mL of a third blocking compound solution, wherein the third blocking compound solution is 10mM glycine, pH8.3, and blocking for 0.5 hour at room temperature;
and (3): adding 0.5mL of a second blocking compound solution, dissolving 10% mannitol in a 10mMPBS buffer solution, adjusting the pH value to 7.4, and blocking at room temperature for 4 hours;
and (4): adding 0.5mL of a first blocking compound solution, wherein the first blocking compound solution is formed by dissolving 1% casein in 10mMPBS buffer solution, and blocking at room temperature and pH7.4 for 4 hours;
and (5): after the sealing is finished, performing magnetic separation and cleaning twice;
and (6): adding PBS-TBN buffer solution which is 10mM PBS, 0.05% Tween20, 0.1% bovine serum albumin, pH7.4, and the concentration of magnetic beads is 0.5mg/mL, and storing at 2-8 ℃ in a dark place.
3. An immunocapture composition obtained by the production method according to claim 1 or 2.
4. An immunoassay kit, comprising:
the immunocapture composition of any one of claims 1-3;
and an immunodetection composition comprising at least one second ligand and a signal tracer;
the signal tracer is at least one of an enzymatic tracer, a chemiluminescent tracer, or a fluorescent tracer.
5. The immunoassay kit of claim 4, wherein the immunoassay kit further comprises:
at least one reagent tube, wherein a suspension liquid is arranged in the reagent tube;
the immunocapture composition and the immunodetection composition are dissolved in the suspension;
wherein the suspension comprises a buffer, a surfactant, an inorganic salt, a stabilizer and a preservative.
6. An immunoassay method based on the immunoassay kit according to claim 4 or 5, characterized in that the immunoassay method comprises:
providing the immunocapture composition of claim 4 or 5;
adding a test sample and the immunocapture composition to a test subject line;
adding at least one immunoassay composition to the test system;
incubation and magnetic separation;
detecting a fluorescent signal of the immunoassay composition.
7. An immunofluorescence analysis system, comprising:
the immunoassay kit of claim 4 or 5;
and an optical detection mechanism for detecting the fluorescent signal of the immunoassay kit.
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