Disclosure of Invention
The invention aims to provide an anti-interference agent aiming at the defects of the prior art, which can well solve the problem of biotin interference in chemiluminescence immunoassay.
In order to achieve the above object, the first aspect of the present invention provides a method for preparing an anti-interference agent.
In some embodiments of the above method, the anti-interference agent comprises a carrier and an active molecule filled in the carrier, the carrier is a porous medium, and the active molecule can specifically bind to a biotin molecule, and the method comprises: step S1, contacting the carrier with the active molecule.
In some embodiments of the above methods, the contacting is performed in a first buffer system.
In some embodiments of the above method, the method further comprises step S0: the carrier is washed with the second buffer system, and step S0 is performed before step S1.
In some embodiments of the above method, the method further comprises step S2: the active molecules not filled in the carrier are removed, and step S2 is performed after step S1.
In some embodiments of the above method, the active molecules not loaded into the carrier are removed by adding a third buffer system to the carrier treated in step S1, and then performing solid-liquid separation.
In some embodiments of the above method, the pH of the first buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, still more preferably from 7.3 to 7.7, further preferably from 7.35 to 7.50, and most preferably 7.40. In still other embodiments of the above method, the pH of the first buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0.
In some embodiments of the above method, the pH of the second buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, still more preferably from 7.3 to 7.7, further preferably from 7.35 to 7.50, and most preferably 7.40. In still other embodiments of the above method, the pH of the second buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0.
In some embodiments of the above methods, the pH of the third buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, even more preferably from 7.3 to 7.7, even more preferably from 7.35 to 7.50, and most preferably 7.40. In still other embodiments of the above method, the pH of the third buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0.
In some embodiments of the above methods, the first buffer system comprises one or more selected from the group consisting of phosphate buffer piperazine-1, 4-diethylsulfonic acid buffer, 3-morpholinopropanesulfonic acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
In some embodiments of the above methods, the second buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a (4-hydroxyethylpiperazine ethanesulfonic acid buffer, a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
In some embodiments of the above methods, the third buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a (4-hydroxyethylpiperazine ethanesulfonic acid buffer, a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer, and tris.
In some embodiments of the above methods, the third buffer system further comprises a surfactant.
In some embodiments of the above methods, the surfactant comprises one or more selected from the group consisting of Tween-20, Tween-80, Triton X-405, Triton X-100, BRIJ 35, and Pluronic L64.
In some embodiments of the above method, the contacting temperature is from 0 to 50 ℃, preferably from 20 to 40 ℃; and/or the contact time is 6 to 24 hours, preferably 8 to 12 hours.
In some embodiments of the above methods, the surfactant comprises one or more selected from the group consisting of Tween-20 (Tween-20), Tween-80, Triton X-405, Triton X-100, BRIJ 35, Pluronic L64.
In some embodiments of the above method, the contacting temperature is from 0 to 50 ℃, preferably from 20 to 40 ℃; and/or the contact time is 1 to 10 hours, preferably 2 to 6 hours.
In some embodiments of the above methods, the anti-interference agent is capable of recognizing a free biotin molecule and a biotin label.
In some embodiments of the above methods, the anti-interference agent is capable of selectively adsorbing free biotin molecules.
In some embodiments of the above method, the free biotin molecule is capable of diffusing into the carrier and specifically binding to the active molecule therein.
In some embodiments of the above method, the biotin label is a conjugate of biotin and a biomacromolecule, wherein the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule, preferably a protein molecule.
In some embodiments of the above method, the proteinaceous molecule is selected from the group consisting of an antigen and/or an antibody; wherein, the antigen refers to a substance with immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.
In some embodiments of the above methods, the anti-interference agent is capable of restricting a biological macromolecule larger in size than the active molecule to its carrier.
In some embodiments of the above methods, the anti-interference agent is capable of being uniformly distributed in the liquid phase reaction system.
In some embodiments of the above method, the carrier has an internal surface area greater than its external surface area; preferably, the internal surface area of the support is more than 5 times, preferably more than 10 times, more preferably more than 20 times the external surface area thereof.
In some embodiments of the above process, the particle size of the support is from 15 to 300nm, preferably from 30 to 250nm, more preferably from 50 to 200 nm.
In some embodiments of the above method, the support has a specific surface area of 200m2A ratio of,/g or more, preferably400m2More preferably 600 m/g or more2More than g, most preferably 1000m2More than g.
In some embodiments of the above method, the support has a minimum porosity of greater than 40%, preferably greater than 50%, more preferably greater than 60%.
In some embodiments of the above method, the porous medium is selected from one or more of a porous metal material, a porous non-metal material, and a porous polymer material.
In some embodiments of the above method, the support is a mesoporous microsphere, preferably an ordered mesoporous microsphere.
In some embodiments of the above method, the mesoporous microsphere has a pore size of 2 to 50nm, preferably 4 to 30nm, and more preferably 5 to 15 nm.
In some embodiments of the above method, the mesoporous microsphere is a cage-shaped hollow mesoporous microsphere.
In some embodiments of the above method, the mesoporous microspheres are selected from Al2O3Mesoporous material and WO3Mesoporous material and TiO2Mesoporous material, ZrO2At least one of the mesoporous material, the silicon-based mesoporous material and/or the mesoporous carbon material is preferably selected from silicon-based mesoporous materials.
In some embodiments of the above method, the active molecule is selected from avidin and/or streptavidin.
In some embodiments of the above method, the active molecule is loaded into the carrier by physical adsorption, or by direct or indirect chemical crosslinking.
In some embodiments of the above method, the inner surface of the carrier is modified with a chemical group, and the active molecule is filled in the carrier by covalent coupling with the chemical group; wherein the chemical group is selected from one or more of carboxyl, aldehyde group, amino, sulfhydryl and hydroxyl.
In some embodiments of the above method, the carrier has a biotin molecule attached to its inner surface, and the active molecule is filled in the carrier by specific binding to the biotin molecule.
In some embodiments of the above method, further comprising step S3, adding a fourth buffer system. Preferably, the fourth buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulphonic acid buffer, a 3-morpholinopropanesulphonic acid buffer, a 4-hydroxyethylpiperazine ethanesulphonic acid buffer and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulphonic acid buffer.
In some embodiments of the above methods, the total concentration of the carrier and the active molecule filled in the carrier in the anti-interference agent is 5-50ug/mL, preferably 8-30ug/mL, more preferably 10-20 ug/mL.
In a second aspect of the invention, the anti-interference agent prepared by the preparation method is provided for application in chemiluminescence immunoassay.
The invention has the beneficial effects that:
1. according to the preparation method, active molecules such as SA or avidin protein molecules and the like are used as ' guest molecules ' and filled in pores of a porous medium in a proper mode to form a ' mesoporous assembly host-guest ' system, so that the mesoporous assembly host-guest ' system can be used as a novel functional material for resisting biotin interference in immune reaction.
2. The anti-interference agent prepared by the invention can effectively distinguish free biotin molecules from biotin markers, thereby achieving the purpose of interference removal.
3. The anti-interference agent prepared by the invention is used in chemiluminescence immunoassay, can eliminate the interference of free biotin, and avoids false positive and/or false negative results in immunoassay.
4. The anti-interference agent prepared by the invention also has practicability and universality, can be applied to different technical platforms, and has small influence on the performance of the agent.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I. Term(s) for
The term "carrier" as used herein refers to a substance capable of carrying active molecules together to participate in a chemical or physical process. The chemical composition of the carrier in the present invention is not particularly limited, and may be organic or inorganic, such as high molecular polymer, metal, glass, mineral salt, diatom, phospholipid vesicle, silicon particle, microcrystalline dye, etc.
The term "porous medium" as used herein refers to a substance composed of a skeleton composed of a solid substance and a plurality of fine voids densely grouped and partitioned by the skeleton.
The term "active molecule" as used herein refers to a molecule having the ability to specifically bind to a biotin molecule. Exemplary reactive molecules are avidin and streptavidin.
The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, which is the conformational correspondence between the corresponding reactants from a steric standpoint.
Detailed description of the preferred embodiments
The present invention will be described in more detail below.
The invention takes the active molecules capable of being specifically combined with the biotin molecules as the ' guest molecules ' and fills the ' mesoporous assembled host-guest ' system in a proper way to form the ' mesoporous assembled host-guest ' system, so that the mesoporous assembled host-guest ' system can be used for chemiluminescence immunoassay to reduce the interference of free biotin in the detection system and further avoid false positive or false negative results caused by the free biotin.
In the preparation method of the anti-interference agent provided by the invention, the anti-interference agent comprises a carrier and an active molecule filled in the carrier, the carrier is a porous medium, and the active molecule can be specifically combined with a biotin molecule, and the method comprises the following steps: step S1, contacting the carrier with the active molecule. According to the present invention, the phrase "the active molecule is filled in the carrier" means that the active molecule is located in the void in the carrier, and may or may not be in contact with the skeleton.
According to some embodiments, the contacting is performed in a first buffer system. In some embodiments of the above method, the pH of the first buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, still more preferably from 7.3 to 7.7, further preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of the pH of the first buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like.
In still other embodiments of the above method, the pH of the first buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH of the first buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, and the like.
According to some embodiments, the method further comprises step S0: the carrier is washed with the second buffer system, and step S0 is performed before step S1. In some embodiments of the above method, the pH of the second buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, still more preferably from 7.3 to 7.7, further preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of the pH of the first buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like. In still other embodiments of the above method, the pH of the second buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH of the first buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, and the like.
According to some embodiments, the method further comprises step S2: the active molecules not filled in the carrier are removed, and step S2 is performed after step S1. Preferably, the active molecules not filled in the carrier are removed by adding a third buffer solution system to the carrier treated in step S1 and then performing solid-liquid separation.
In some embodiments of the above methods, the pH of the third buffer system is from 7.0 to 9, preferably from 7.1 to 8.0, more preferably from 7.2 to 7.8, even more preferably from 7.3 to 7.7, even more preferably from 7.35 to 7.50, and most preferably 7.40. In this embodiment, examples of the pH of the third buffer system include 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9.0, and the like. In still other embodiments of the above method, the pH of the third buffer system is from 3.0 to 7.0, preferably from 3.5 to 6.8, more preferably from 4.0 to 6.5, even more preferably from 5.0 to 6.4, even more preferably from 5.5 to 6.3, and most preferably 6.0. In this embodiment, examples of the pH of the third buffer system include 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, and the like.
In some embodiments of the above methods, the first buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer. In some embodiments of the above methods, the second buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer. In some embodiments of the above methods, the third buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulfonic acid buffer, a 3-morpholinopropanesulfonic acid buffer, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulfonic acid buffer.
In some embodiments of the above methods, the third buffer system further comprises a surfactant. According to some embodiments, the surfactant comprises one or more selected from the group consisting of Tween-20, Tween-80, Triton X-405, Triton X-100, BRIJ 35 and Pluronic L64. According to some embodiments, the surfactant comprises tween-20.
In some embodiments of the above method, the contacting temperature is 0-50 ℃, preferably 20-40 ℃, such as 25-30 ℃ (i.e., room temperature) in step S1; and/or the contact time is 6-24 hours, preferably 8-12 hours, such as 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc. In some other embodiments of the above method, the contacting temperature is from 0 to 50 ℃, preferably from 20 to 40 ℃, e.g., from 25 to 30 ℃ (i.e., room temperature); and/or the contact time is 1 to 10 hours, preferably 2 to 6 hours, such as 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc.
In some embodiments of the above method, further comprising step S3, adding a fourth buffer system. Preferably, the fourth buffer system comprises one or more selected from the group consisting of a phosphate buffer, a piperazine-1, 4-diethylsulphonic acid buffer, a 3-morpholinopropanesulphonic acid buffer, a 4-hydroxyethylpiperazine ethanesulphonic acid buffer and a 3- (hydroxyethylpiperazine) -2-hydroxypropanesulphonic acid buffer.
In some embodiments of the invention, the anti-interference agent is capable of recognizing a free biotin molecule and a biotin label. In the present invention, "recognition" may mean that the active molecule in the anti-interference agent and the free biotin molecule and/or biotin label are combined with each other through the synergistic effect of intermolecular forces.
In some embodiments of the invention, the anti-interference agent is capable of selectively adsorbing free biotin molecules. In the present invention, "selective adsorption" may mean that free biotin molecules are accumulated in the pores (which may or may not be in contact with the scaffold) in the carrier.
In some embodiments of the invention, the free biotin molecule is capable of diffusing into the carrier and specifically binding to the active molecule therein. In the present invention, the "diffusion" may mean that free biotin molecules are dispersed into a carrier due to random movement of the molecules.
In some embodiments of the invention, the biotin label is a conjugate of biotin and a biomacromolecule, wherein the biomacromolecule is selected from the group consisting of a protein molecule, a nucleic acid molecule, a polysaccharide molecule, and a lipid molecule, preferably a protein molecule.
In some embodiments of the invention, the protein molecule is selected from an antigen and/or an antibody; wherein, the antigen refers to a substance with immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.
In some embodiments of the invention, the anti-interference agent is capable of restricting the entry of a biological macromolecule larger in size than the active molecule into its carrier.
In some embodiments of the invention, the anti-interference agent is capable of being uniformly distributed in a liquid phase reaction system.
In some embodiments of the invention, the vector satisfies at least one of the following conditions: a) the inner pores of the carrier have a sufficiently large surface area (far exceeding the surface area of the carrier), and the voids can only allow the entry of active molecules, but limit the larger proteins of the active molecules, such as antibodies or large antigens, etc.; b) active molecules such as SA or avidin can be filled in the carrier, for example, inside the voids, by a chemical or physical adsorption method; c) the carrier can be stably and uniformly distributed in a solution (e.g., an aqueous solution) without precipitation.
In some embodiments of the invention, the support has an internal surface area that is greater than its external surface area. Preferably, the internal surface area of the support is more than 5 times, preferably more than 10 times, more preferably more than 20 times the external surface area thereof. In some preferred embodiments of the invention, the internal surface area of the support is a multiple of its external surface area including, but not limited to: 5 times, 6 times, 8 times, 10 times, 12 times, 16 times, 18 times, 20 times, 22 times, 24 times, 26 times, 28 times, or 30 times. .
In some embodiments of the invention, the support has a specific surface area of 200m2Per g or more, e.g. 200m2/g、400m2/g、600m2/g、800m2/g、1000m2/g、1200m2/g、1500m2G, etc., preferably 400m2More preferably 600 m/g or more2More than g, most preferably 1000m2More than g.
In some embodiments of the invention, the particle size of the support is 15-300nm, such as 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 100nm, 250nm, 300nm, etc., preferably 30-250nm, more preferably 50-200 nm. Too large a carrier particle size can cause the carrier to settle too quickly, which is not conducive to forming a stable, uniform solution.
In some embodiments of the invention, the support has a minimum porosity of greater than 40%, preferably greater than 50%, more preferably greater than 60%.
In some embodiments of the invention, the porous medium is selected from one or more of a porous metal material, a porous non-metal material, and a porous polymer material.
In some embodiments of the present invention, the support is a mesoporous microsphere, preferably an ordered mesoporous microsphere.
In some embodiments of the present invention, the pore size of the mesoporous microsphere is 2 to 50nm, such as 2nm, 5nm, 10, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 50nm, etc., preferably 4 to 30nm, and more preferably 5 to 15 nm.
In some embodiments of the present invention, the mesoporous microsphere is a cage-shaped hollow mesoporous microsphere.
In some embodiments of the present invention, the mesoporous microspheres are selected from Al2O3Mesoporous material and WO3Mesoporous material and TiO2Mesoporous material, ZrO2At least one of the mesoporous material, the silicon-based mesoporous material and/or the mesoporous carbon material is preferably selected from silicon-based mesoporous materials.
The silicon-based mesoporous material is made of SiO2(CH2)2Periodic mesoporous material composed of tetrahedral structural units. The mesoporous silica materials can be microscopically classified into two types: one is disordered mesoporous solids represented by silica xerogels and aerogels. The disordered mesoporous silica can be powder, block, sheet or film in macroscopic view. The other is an ordered mesoporous silica represented by MCM 41. The ordered mesoporous silica has the structural characteristics that the pore size is uniform, the ordered mesoporous silica is arranged in a hexagonal order, and the pore size of the mesoporous silica can be adjusted between 2nm and 10 nm. Because the hole wall is thin, the silicon-based unit has low alternating current degree and poor hydrothermal stability. The specific surface area can reach 1000m2(ii) in terms of/g. Also SBA series, HMM series, TUD series, FSM series, KIT series, CMK series, FDU series, starbon series, etc. Among them, SBA-15 has more research, and the hydrothermal stability of the material is better than that of MCM series. The aperture is adjustable between 5nm and 30 nm. HMM is a spherical mesoporous material, the aperture of which is 4-15nm, and the outer diameter of which is 20-80nm and is adjustable.
In some embodiments of the invention, the active molecule is selected from avidin and/or streptavidin. Avidin is a glycoprotein extracted from egg white, has a molecular weight of about 60kD, is composed of 4 subunits per molecule, and can be closely bound to 4 biotin molecules. Such avidin includes, but is not limited to: avidin, streptavidin, vitellin, and avidin-like. Streptavidin (SA), a protein with biological properties similar to those of avidin (A), is a protein product secreted by Streptomyces avidins during culture, and SA can also be produced by genetic engineering means. The molecular weight of SA is 65000, and consists of 4 peptide chains with the same sequence, and each SA peptide chain can combine with 1 biotin molecule. Thus, like avidin, each SA molecule also has 4 binding sites for biotin moleculesWith a binding constant of 10 as avidin15mol/L。
In some embodiments of the invention, the active molecule is packed in the carrier by physical adsorption. Physical adsorption, also known as van der waals adsorption, is caused by intermolecular forces between the adsorbate and the adsorbent, also known as van der waals forces.
In other embodiments of the invention, the active molecule is loaded into the carrier by contacting the carrier in a system comprising a buffer.
In some embodiments of the invention, the active molecule is loaded into the carrier by direct or indirect chemical crosslinking.
In some embodiments of the present invention, the inner surface of the carrier is modified with a chemical group, and the active molecule is filled in the carrier by covalent coupling with the chemical group; wherein the chemical group is selected from carboxyl (-COOH), aldehyde (-CHO), amino (-NH)2) One or more of mercapto (-SH) and hydroxyl (-OH).
In some embodiments of the present invention, the carrier has a biotin molecule attached to its inner surface, and the active molecule is filled in the carrier by specific binding to the biotin molecule.
In some embodiments of the invention, the total concentration of the carrier and the active molecule filled in the carrier in the anti-interference agent is 5-50ug/mL, such as 5ug/mL, 10ug/mL, 15ug/mL, 20ug/mL, 25ug/mL, 30ug/mL, 35ug/mL, 40ug/mL, 45ug/mL, 50ug/mL, etc., preferably 8-30ug/mL, more preferably 10-20 ug/mL.
In a second aspect, the invention provides the use of an anti-interference agent prepared by the method as described above in a chemiluminescent immunoassay.
Example III
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
Reagents and instrumentation:
SA (Sigma Aldrich Co.), carboxyl-functionalized silica-based microspheres (particle size 15-200nm, pore size 2-15nm, Sigma Aldrich Co.), phosphate buffer (0.02M PBS, pH 7.4), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDAC (thermo fisher), Tween-20, 0.1M MES buffer (pH 6.0), biotin (D-biotin), serotonin serum, triiodothyronine (T3) kit, (general liquid GG-NA (photosphere solution) for light-activated chemical laser analysis systems.
LiCA HT (Shanghai Boyang Biotechnology Co., Ltd.), Hitachi high speed refrigerated centrifuge.
The anti-interference agent is prepared by a physical adsorption mode
Example 8 (covalent coupling mode)
In the first step, 10mg of carboxyl functionalized silica-based microspheres were taken in a 2mL centrifuge tube and washed once with 0.1M MES (pH 6.0) buffer at 4 ℃ at 10000rpm for 15 min.
In the second step, 200uL of 0.1M MES (pH 6.0) buffer was added and dispersed by sonication, followed by 150uL of 10mg/mL SA in water, followed by 100uL of 10mg/mLEDAC (0.1M MES) solution and stirring at room temperature for 4 h.
Thirdly, the SA microspheres are centrifugally washed three times by using 0.02M PBS (pH 7.4) buffer solution containing 0.5% Tween-20 to remove unadsorbed SA, and finally, the volume is adjusted to 10mg/mL by using PBS buffer solution.
Evaluation of Effect of the anti-interference agent of the invention
The experimental steps are as follows:
1. a concentrated solution of T3 was added to the serum of degerming serum to prepare a solution of T3 at concentrations of 1nmol/L and 2 nmol/L.
2. A40 ug/mL solution of beads was prepared and solutions of different concentrations (diluted in PBS) were prepared using the anti-interference agents prepared in examples 1-7, as shown in Table 1.
3. Biotin was added to the above T3 solution to prepare a sample solution with a Biotin concentration of 0,128 ng/ml.
4. Adding 25uL of sample solution, sequentially adding 25uL of reagent I and reagent II in the T3 kit, manually adding 25uL of sample and 25uL of reagent according to a reaction mode, and adding 25uL of solution prepared in step 2 according to the following table 1, wherein no anti-interference agent is added under the conditions 1 and 2.
5. Put into LICA HT, a first stage incubation is performed: incubate at 37 ℃ for 17 min.
6. Add 175ul of photosensing bead solution by hand.
7. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
8. And (6) reading. See tables 2 and 3.
TABLE 1
And (3) data analysis:
when the concentration of T3 is 1nM/L, Biotin is 128ng/mL, the signal drops by 89%, and biotin interference is more severe. When the microspheres with the pore diameter of 2nm (condition 3) are added, signals are almost not obviously changed, and when the microspheres with the particle diameter of 50nm, the pore diameter of 5nm and the particle diameter of 10nm (condition 4 and condition 5) are adopted, the signals are improved to a certain extent, and the falling amplitude is 50-70%.
When the particle size and the aperture are not changed, the concentration of the anti-interference agent is increased to 10ug/mL (compared with the condition 5 and the condition 6), the signal is further increased, and the falling amplitude is about 25%. When the aperture is 10nm and the concentration is 10ug/ml, and the particle size of the microsphere is increased to 100nm (condition 6 and condition 7), the drop amplitude deviation is within 10%, and the biotin interference phenomenon disappears. When the concentration increased to 20ug/ml (condition 7 and condition 8), the signal dropped within 10% of the deviation. When the concentration is not changed by 20ug/mL and the particle size and the aperture are increased, the signal falls to a certain extent, and the falling amplitude is 20-40%.
And (4) experimental conclusion:
when the particle size of the microsphere filled with SA is 100nm, the pore diameter is 10nm, and the concentration is 10-20ug/mL, the anti-biotin interference capability is strongest. When the pore diameter is smaller, 2nm, the anti-biotin interference capability is not available. When the particle size is larger than 100nm and the pore size is 10nm, the anti-biotin interference ability is decreased as the particle size and pore size are further increased.
Effect evaluation of the anti-interference agent of the invention II
The experimental steps are as follows:
1. a concentrated solution of T3 was added to the serum of degerming serum to prepare a solution of T3 at concentrations of 1nmol/L and 2 nmol/L.
2. A40 ug/mL solution of beads was prepared, and different concentrations of the anti-interference agent prepared in examples 5 and 8 (diluted in PBS) were prepared, as shown in Table 4 (diluted in PBS) at 10ug/mL and 20 ug/mL.
3. Biotin was added to the above T3 solution to prepare a sample solution with a Biotin concentration of 0,128ng/ml
4. Adding 25uL of sample solution, sequentially adding 25uL of reagent I and reagent II in the T3 kit, manually adding 25uL of sample and 25uL of reagent according to a reaction mode, adding 25uL of the anti-interference agent prepared in the step 2 according to the following table, and adding no anti-interference agent under the conditions 1 and 2.
5. Put into LICA HT, a first stage incubation is performed: incubate at 37 ℃ for 17 min.
6. Add 175ul of photosensing bead solution by hand.
7. Performing a second stage incubation: incubate at 37 ℃ for 15 min.
8. And (6) reading. See tables 5 and 6.
TABLE 4
And (3) data analysis:
compared with the condition 3 and the condition 5, the anti-biotin interference capability of the anti-interference agent prepared by the physical adsorption mode is strong, the signal drops by about 10%, and the signal drops by about 50% of the anti-interference agent prepared by the covalent coupling mode. Compared with the condition 6, the anti-biotin interference capability of the anti-interference agent prepared by the physical adsorption mode is better than that of the anti-interference agent prepared by the covalent coupling mode in the condition 4.
And (4) experimental conclusion:
the anti-interference agent prepared by the physical adsorption mode with the same particle size, aperture, concentration, specific surface area and anti-biotin interference capability is better than that prepared by the covalent coupling mode.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.