CN116735871A - Acceptor reagent and application thereof - Google Patents
Acceptor reagent and application thereof Download PDFInfo
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- CN116735871A CN116735871A CN202310842967.3A CN202310842967A CN116735871A CN 116735871 A CN116735871 A CN 116735871A CN 202310842967 A CN202310842967 A CN 202310842967A CN 116735871 A CN116735871 A CN 116735871A
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- acceptor
- receptor
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
Abstract
The application discloses a receptor reagent and application thereof, wherein the receptor reagent comprises a first buffer solution and receptor particles which are suspended in the buffer solution and can react with active oxygen to generate a chemiluminescent signal, the surface of the receptor particles is combined with biomolecules, and the variation coefficient C.V value of the particle size distribution of the receptor particles in the receptor reagent is not lower than 5% and not higher than 20%; the sugar content in the acceptor particle is not higher than 40 mug per milligram by mass. The beneficial effects of the application are as follows: the receptor reagent provided by the application has the advantages that the receptor particle size distribution variation coefficient C.V value is not lower than 5% and not higher than 20%; the acceptor reagent can meet the commercial requirement of mass production, has ultrahigh sensitivity and has a wide detection range. Meanwhile, the application controls the sugar content in the receptor particles, reduces the influence of sugar on detection signals, improves the detection precision and reduces the production cost.
Description
Technical Field
The technical scheme relates to the field of chemiluminescence detection, in particular to a receptor reagent and application thereof.
Background
Immunoassays have been developed for over half a century. The separation of the test substance from the reaction system in the measurement process may be classified into heterogeneous (heterogenic) immunoassays and Homogeneous (homogenic) immunoassays. Heterogeneous immunoassay refers to the main method in the prior immunoassay, wherein various related reagents are required to be separated after mixing reaction in the operation process of marking a probe, and the detection is performed after separating an object to be detected from a reaction system. Such as enzyme-linked immunosorbent assay (ELISA) and magnetic particle chemiluminescence, which are widely known. Homogeneous immunoassay refers to a method in which an analyte is mixed with a reagent in a reaction system and then directly measured in a measurement process without redundant separation or washing steps. Up to now, various sensitive detection methods are applied to homogeneous immunoassays, such as optical detection methods, electrochemical detection methods, and the like.
For example, photo-activated chemiluminescence detection (Light Initiated Chemiluminescent Assay, liCA) is a typical homogeneous immunoassay. It is based on antigens or antibodies coated on the surface of two microspheres, and two microspheres are pulled together by forming immune complexes in the liquid phase. Under the excitation of laser, the transfer of singlet oxygen between the microspheres occurs, and then high-energy red light is generated, and the photon number is converted into the target molecule concentration through a single photon counter and mathematical fitting. When the sample does not contain target molecules, immune complexes cannot be formed between the two microspheres, the distance between the two microspheres exceeds the propagation range of singlet oxygen, the singlet oxygen is rapidly quenched in a liquid phase, and no high-energy level red light signal is generated during detection. It has the characteristics of quick, homogeneous phase (no flushing), high sensitivity and simple operation. Photo-activated chemiluminescence has been applied to a number of detection projects.
According to the conventional optical detection theory knowledge, the more uniform the particle size of the microsphere used in the homogeneous chemiluminescence detection, the better the chemiluminescence detection performance by using the microsphere. Thus, those skilled in the art tend to strive to obtain monodisperse microspheres of a more uniform particle size. However, with the progress of the detection industry, the requirement for a hypersensitive reagent is more and more, the sensitivity requirement is extremely high, the detection range is extremely wide, and the existing homogeneous chemiluminescence detection method is difficult to meet the detection conditions.
Therefore, there is a need to develop a receptor reagent that can be mass-produced, has low cost, qualified quality, and stable performance, and can meet the sensitivity requirement and the linearity range requirement.
Disclosure of Invention
The technical problem to be solved by the application is to provide a receptor reagent for homogeneous chemiluminescence aiming at the defects of the prior art. When a kit comprising the acceptor reagent is applied to homogeneous chemiluminescent assay detection, the inventors of the present application have unexpectedly found that the kit has both ultra-high sensitivity and a very wide detection range.
Based on this, the present application provides in one aspect a receptor reagent comprising a first buffer solution and, suspended therein, receptor particles capable of generating a chemiluminescent signal upon action of active oxygen, the surface of the receptor particles having biomolecules bound thereto, the receptor particles having a particle size distribution coefficient of variation C.V value in the receptor reagent of not less than 5% and not more than 20%; the sugar content in the acceptor particle is not higher than 40 mug per milligram by mass.
In a preferred embodiment of the application, the acceptor particle comprises a carrier, the interior of which is filled with a luminescent composition, the surface of which is bound with biomolecules.
In a preferred embodiment of the application, the carrier surface is coated with polysaccharide molecules, which are indirectly bound to the surface of the receptor particles by chemical bonding to the polysaccharide molecules.
In a preferred embodiment of the application, the sugar content per milligram of mass of the acceptor particle is not higher than 20. Mu.g, preferably not higher than 10. Mu.g.
In a preferred embodiment of the application, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose, most preferably dextran or dextran derivatives.
In a preferred embodiment of the application, the acceptor particles have a particle size distribution coefficient of variation C.V value in the acceptor agent of not more than 15%.
In a preferred embodiment of the application, the acceptor particles have a particle size distribution coefficient of variation C.V value in the acceptor agent of not less than 8%.
In a preferred embodiment of the present application, the sugar content in the first buffer solution per liter of volume is not less than 0.010g and not more than 0.30g.
In a preferred embodiment of the present application, the sugar content in the first buffer solution per liter of volume is not less than 0.015g and not more than 0.20g.
In a preferred embodiment of the application, the surface of the support is not coated with sugar molecules but is directly bonded to biological molecules.
In a preferred embodiment of the present application, the surface of the carrier has a bonding functional group for directly chemically bonding a biomolecule to the surface of the carrier, the bonding functional group being at least one selected from the group consisting of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, an epoxy group, a maleimide group and a thiol group; preferably selected from aldehyde groups, carboxyl groups, epoxy groups and maleimide groups.
In a preferred embodiment of the application, the aforementioned sugar content is detected using the anthrone method.
The second aspect of the application also provides a homogeneous chemiluminescent assay kit comprising the acceptor reagent as described above.
The kit comprises a plurality of reagent strips, wherein each reagent strip is provided with a plurality of reagent hole slots for containing reagents, and at least one reagent hole slot is used for containing the acceptor reagents.
In a third aspect, the application provides the use of the above receptor reagent or the above kit in a chemiluminescent analyzer. In a fourth aspect, the present application provides an application of the above-mentioned kit for POCT detection.
The beneficial effects of the application are as follows: the receptor reagent provided by the application has the advantages that the receptor particle size distribution variation coefficient C.V value is not lower than 5% and not higher than 20%; the acceptor reagent can meet the commercial requirement of mass production, has ultrahigh sensitivity and has a wide detection range. Meanwhile, the application controls the sugar content in the receptor particles, reduces the influence of sugar on detection signals, improves the detection precision and reduces the production cost.
Drawings
FIG. 1 is a Gaussian distribution diagram of acceptor particle a prepared in example 1 in acceptor reagent.
FIG. 2 is a Gaussian distribution diagram of receptor particles after coupling anti-HBsAg antibodies prepared in example 1.
FIG. 3 is a graph of a standard for sugar content measurement.
Detailed Description
In order that the application may be readily understood, the application will be described in detail. Before the present application is described in detail, it is to be understood that this application 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. The practice of the application is not limited to the following examples, but is intended to be within the scope of the application in any form and/or modification thereof.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the application.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present application, the preferred methods and materials are now described.
I terminology
The term "active oxygen" as used herein refers to a substance which is composed of oxygen in the body or in the natural environment, contains oxygen and is active in nature, and is mainly an excited oxygen molecule, including an electron reduction product of oxygen, superoxide anion (O 2 Hydrogen peroxide (H), a two-electron reduction product 2 O 2 ) Hydroxyl radical (OH) of three-electron reduction product, nitric oxide and singlet oxygen (1O) 2 ) Etc.
The term "acceptor particle" as used herein refers to a particle comprising a compound capable of reacting with reactive oxygen species to produce a detectable signal. The donor particles are induced to activate by energy or an active compound and release active oxygen in a high energy state which is captured by the acceptor particles in close proximity, thereby transferring energy to activate the acceptor particles. In some embodiments of the application, the acceptor particle comprises a luminescent composition and a carrier, the luminescent composition being filled in the carrier and/or coated on the surface of the carrier. The "carrier" according to the application is selected from the group consisting of tapes, sheets, rods, tubes, wells, microtiter plates, beads, particles and microspheres, which may be microspheres or microparticles well known to the person skilled in the art, which may be of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which has any density, but preferably has a density close to that of water, preferably floats in water, and is composed of transparent, partially transparent or opaque materials. The carrier may or may not be charged and when charged is preferably negatively charged. The carrier may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.
In the present application, the "light-emitting composition", i.e., a compound called a label, may undergo a chemical reaction to cause light emission, such as by being converted into another compound formed in an electronically excited state. The excited state may be a singlet state or a triplet excited state. The excited state may relax to the ground state to emit light directly or by transferring excitation energy to an emission energy acceptor, thereby restoring itself to the ground state. In this process, the energy acceptor particles will be transitioned to an excited state to emit light.
The term "C.V value of the particle size distribution coefficient of variation" as used herein refers to the coefficient of variation of the particle size in the Gaussian distribution in the result of the detection by the nanoparticle analyzer. The calculation formula of the variation coefficient is as follows: C.V values = (standard deviation SD/Mean) x 100%. The standard deviation (Standard Deviation, SD), also called standard deviation, describes the average of the distances (from mean deviation) of the individual data from the average, which is the square root after the sum of the squares of the deviations, denoted sigma. The standard deviation is the arithmetic square root of the variance. The standard deviation reflects the degree of dispersion of a data set, and the smaller the standard deviation, the less the values deviate from the average and vice versa. The standard deviation sigma is the distance from the inflection point (0.607 times the peak height) on the normal distribution curve to the perpendicular line of the peak height and the time axis, i.e., half the distance between the two inflection points on the normal distribution curve. The half height peak width (Wh/2) refers to the peak width at half the peak height, wh/2=2.355 σ. The intercept at the base line is called the peak width or base line width, w=4σ or w=1.699 Wh/2, by making tangents to the inflection points on both sides of the normal distribution curve.
The term "test sample" as used herein refers to a mixture to be tested that contains or is suspected of containing a target molecule to be tested. Samples to be tested that may be used in the present application include body fluids such as blood (which may be anticoagulated blood as is commonly found in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, and the like. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells from prokaryotes. The sample to be measured can be diluted with a diluent as required before use. For example, in order to avoid the HOOK effect, the sample to be tested may be diluted with a diluent before on-machine testing and then tested on a testing instrument.
The term "target molecule to be detected" as used herein refers to a substance in a sample to be detected during detection. One or more substances having a specific binding affinity for the target molecule to be detected may be used to detect the target molecule. The target molecule to be tested may be a protein, peptide, antibody or hapten which can be conjugated to an antibody. The target molecule to be detected may be a nucleic acid or oligonucleotide that binds to a complementary nucleic acid or oligonucleotide. The target molecule to be tested may be any other substance that can form a specific binding pair member. Examples of other typical target molecules to be measured include: drugs such as steroids, hormones, proteins, glycoproteins, mucins, nucleoproteins, phosphoproteins, drugs of abuse, vitamins, antibacterial agents, antifungal agents, antiviral agents, purines, antitumor agents, amphetamines, heteronitrogen compounds, nucleic acids and prostaglandins, and metabolites of any of these drugs; pesticides and metabolites thereof; and a recipient. Analytes also include cells, viruses, bacteria, and fungi.
The term "antibody" as used herein is used in its broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody may be further conjugated to other moieties, such as a member of a specific binding pair member, e.g., biotin or avidin (a member of a biotin-avidin specific binding pair member), and the like.
The term "antigen" as used herein refers to a substance that stimulates the body to produce an immune response and binds to antibodies and sensitized lymphocytes, which are the products of the immune response, in vivo and in vitro, resulting in an immune effect.
The term "binding" as used herein refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.
The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, and from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants. Under the technical ideas disclosed in the present application, the detection method of the specific binding reaction includes, but is not limited to: a diabody sandwich method, a competition method, a neutralization competition method, an indirect method or a capture method.
II. Detailed description of the preferred embodiments
The present application will be described in more detail with reference to examples.
The inventors of the present application controlled the particle size distribution of the receptor particles in the receptor reagent, thereby controlling the amount of surface reporter molecules (e.g., antibodies/antigens) per receptor particle (the specific surface area of small particle size microspheres is large, the amount of surface reporter molecules per unit mass of microspheres is large, the specific surface area of large particle size microspheres is small, and the amount of surface reporter molecules per unit mass of microspheres is small). The larger the variation coefficient of the particle size distribution of the receptor particles in the receptor reagent is, the higher the degree of non-uniformity is, which is equivalent to the existence of the receptor particles with different sizes in the system, thereby having higher sensitivity and wider detection range.
In the technical field of photoexcitation chemiluminescence, donor particles and acceptor particles form a pair of double-ball systems together, and the two particles are combined with each other by virtue of antigen-antibody to realize the transfer of singlet oxygen and induce a photoexcitation chemiluminescence process, so that the separation-free homogeneous immunoassay is realized. The double spheres are nano microspheres which complement each other, interact, cooperate and influence each other in a light-activated chemiluminescence system, and are indispensable. The two nano-microspheres have good suspension characteristics in a liquid phase, and the microspheres meet antigen or antibody in the liquid phase to completely meet the liquid dynamic characteristics. Under 680nm laser irradiation, the photosensitizer of the donor particle is responsible for exciting oxygen in the surrounding environment into singlet oxygen molecules. When singlet oxygen molecules diffuse into the acceptor particles, a series of chemiluminescent reactions with the chemiluminescent composition in the acceptor particles occur, thereby producing an emission wavelength optical signal of 610nm to 620 nm.
Acceptor reagents are an indispensable and important component of a photoexcited chemiluminescent system, and luminescent substances in acceptor particles contained in acceptor reagents can react with singlet oxygen to generate detection signals. The final detection result is directly affected by the acceptor particle preparation process, the particle size distribution of the acceptor particles, the choice of luminescent substances, the surface treatment of the acceptor particles, and the like. The inventor finds that, on one hand, the sugar content in the acceptor particles is strictly controlled within a proper range, and on the other hand, the C.V value of the particle size distribution variation coefficient of the acceptor particles in the acceptor reagent is regulated, so that the acceptor reagent with low cost, qualified quality and stable performance in mass production can be effectively solved.
The inventors of the present application found that the value of the coefficient C.V of variation in the particle size distribution of the acceptor particles in the liquid phase directly influences the detection result of the photoexcitation chemiluminescence. If commercial application of the photo-excitation chemiluminescence system in clinical immunodiagnosis is desired, a large-scale acceptor reagent with low production cost, qualified quality and stable performance is required, and the C.V value of the particle size distribution of the donor particles in the donor reagent must be strictly controlled within a proper range. It should be noted that, unlike the particle size distribution of conventional blank polystyrene microspheres (i.e., the carrier of the present application, which is not internally filled with functional substances, and which is not surface modified with specific binding partners or polysaccharides), the C.V values of the present application refer to the value of the coefficient of variation C.V of the particle size distribution of the acceptor particles in the acceptor reagent. Since the value of the variation coefficient C.V of the particle size distribution of the receptor particles is continuously changed from the carrier in the preparation process of the receptor particles, particularly after the polysaccharide or the biological molecule or the specific pairing conjugate is coated, the variation of the value C.V of the particle size distribution of the nano-microsphere is more obvious and unstable, and the requirements of in-vitro diagnostic reagents for registration of medical instrument products and clinical application are difficult to meet. Therefore, the applicant has made extensive experimental studies and has gradually searched and found that if it is desired to realize commercial application of the receptor reagent in clinical immunodiagnosis, it is necessary to strictly control C.V value of particle size distribution of the receptor microspheres in the receptor reagent to be in a proper range, so that the receptor reagent with low cost, qualified quality and stable performance can be mass-produced.
In one aspect, the application provides a receptor reagent, wherein receptor particles combined with biological molecules are suspended in a first buffer solution, and the variation coefficient C.V value of the particle size distribution of the receptor particles in the first buffer solution is more than or equal to 5% and less than or equal to 20%; while the sugar content in the acceptor particle per milligram mass is not higher than 40 mug.
The variation coefficient C.V value of the particle size distribution of the receptor particles in the receptor reagent is more than or equal to 8%; preferably, the variation coefficient C.V value of the particle size distribution is less than or equal to 15%; further preferably, the variation coefficient C.V value of the particle size distribution is 20% or less.
The receptor particles may have a coefficient of variation C.V in particle size distribution in the receptor reagent of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.
The acceptor particle comprises a carrier, wherein the inside of the carrier is filled with a luminous composition, and the surface of the carrier is combined with biomolecules. In some preferred embodiments of the application, the particle size distribution of the receptor particles after attachment to the biomolecule exhibits polydispersibility. The biomolecule may be capable of specifically binding to the analyte molecule or may be a member of a specific binding pair member.
And the surface of the receptor particle is coated with polysaccharide, the polysaccharide reacts with the biomolecules, the biomolecules are indirectly connected to the surface of the carrier, and the polysaccharide can reduce the nonspecific adsorption of the particle. However, polysaccharides can also present other problems, such as: the cost is higher, the process is complex, and the detection signal is reduced. In particular, in the field of in vitro diagnosis, since the composition of body fluids is complex and contains many unknown components, it has been found that the use of a polysaccharide-coated receptor reagent for in vitro diagnosis tends to have a large influence on the detection signal. In order to solve these problems in combination, the present inventors have found that the sugar content on the receptor particles is strictly controlled, and the sugar content in the receptor particles per milligram by mass is not higher than 40. Mu.g, which is excellent. The inventors have found that further increasing the sugar content on the acceptor particle is a preferred embodiment, and that the sugar content in the acceptor particle may be not higher than 20 μg per mg mass. It is also possible that the sugar content in the acceptor particle is not higher than 10. Mu.g per milligram mass.
In some embodiments of the application, the polysaccharide is selected from carbohydrates containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose. The polysaccharide is preferably selected from dextran and derivatives of dextran. As a still further preferred embodiment, the polysaccharide is Dextran, chinese name Dextran.
The acceptor reagent of the present application has a sugar content of not less than 0.010g and not more than 0.30g per liter of the volume of the first buffer solution. More preferably, the sugar content in the first buffer solution per liter of volume is not less than 0.015g and not more than 0.20g.
The sugar content on the above acceptor particle and the sugar content in the first buffer solution were detected by the anthrone method.
The application also provides a chemiluminescent detection kit, which comprises the acceptor reagent.
The kit comprises a plurality of reagent strips, wherein each reagent strip is provided with a plurality of reagent hole slots for containing reagents, and at least one reagent hole slot is used for containing the acceptor reagents.
The application also provides application of the receptor reagent or the kit to a chemiluminescent analyzer.
The application also provides application of the receptor reagent or the kit in POCT detection. POCT refers to a rapid test on site or a clinical test performed beside a patient.
III. Examples
Example 1 preparation of acceptor particle a
1.1 preparation of the Carrier and characterization procedure
1. A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced 2 30min；
2. 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step 1, and continuing to introduce N 2 30min；
3. Heating the reaction system to 70 ℃ for reaction for 15 hours;
4. the emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, diluting with water, and preserving in an emulsion form;
5. the average particle diameter of the Gaussian distribution of the latex microsphere particle diameter at this time was 202.2nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=4.60%.
1.2 landfill Process and characterization of luminescent compositions
1. A25 ml round-bottomed flask was prepared and charged with 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) 3+ ) 10ml of 95% ethanol is magnetically stirred, and the temperature of the water bath is raised to 70 ℃ to obtain a complex solution;
2. preparing a 100ml three-neck flask, adding 10ml 95% ethanol, 10ml water and 10ml aldehyde polystyrene latex microspheres with concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;
3. slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling;
4. centrifuging the emulsion for 1 hour, 30000G, and removing supernatant after centrifuging to obtain aldehyde polystyrene microspheres filled with luminous composition;
5. the average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 204.9nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=5.00%.
1.3 surface coating of receptor particles with dextran
1. 50mg of aminodextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and the solution was stirred at 30 ℃ in the absence of light;
2. taking 100mg of prepared aldehyde polystyrene microspheres filled with the luminous composition, adding the prepared aldehyde polystyrene microspheres into an aminodextran solution, and stirring for 2 hours;
3.10mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;
4. after the reaction mixture was centrifuged at 30000G for 45min, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugal washing three times, the volume is fixed by 50 mM/pH=10 carbonate buffer solution to make the final concentration of the solution be 20mg/ml;
5. 100mg of aldehyde dextran solid was taken in a 20mL round bottom flask, 5mL of 50 mm/ph=10 carbonate buffer was added, and stirred at 30 ℃ in the absence of light for dissolution;
6. adding the microsphere into an aldehyde dextran solution, and stirring for 2 hours;
7.15mg of sodium borohydride was dissolved in 0.5ml of 50 mM/pH=10 carbonate buffer, and then added dropwise to the above reaction solution, followed by reaction at 30℃overnight in the absence of light;
8. after the reaction mixture was centrifuged at 30000G for 45min, the supernatant was discarded, and 50 mM/ph=10 carbonate buffer was added for ultrasonic dispersion. After repeating the centrifugation washing three times, the final concentration was set to 20mg/ml by constant volume with 50 mM/pH=10 carbonate buffer.
9. The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 241.6nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=12.90% (as shown in fig. 1).
1.4 conjugation procedure for anti-HBsAg antibodies
1. HBsAg antibody i was dialyzed to 50 mcb buffer at ph=10 to give a concentration of 1mg/ml.
2. The receptor particles obtained in 0.5ml1.3, 0.5ml and the pairing antibody I were added to a 2ml centrifuge tube, and after mixing, 100. Mu.l of 10mg/ml NaBH was added 4 The solution (50 mM CB buffer) was reacted at 2-8℃for 4 hours.
3. After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50 mM B buffer) was added thereto, and the reaction was carried out at 2-8℃for 2 hours.
4. After the reaction, the mixture was centrifuged for 45min at 30000G, and the supernatant was discarded after centrifugation and resuspended in 50mM HES buffer. The centrifugation washing was repeated four times and diluted with the first buffer solution to a final concentration of 100. Mu.g/ml to obtain a solution of the receptor particles conjugated with the anti-HBsAg antibody I. The first buffer solution comprises the following components: 0.1mol tris/l, 0.3mol NaCl, 25mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15ppm ProClin-300, pH 8.00.
5. The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 253.5nm as measured by a nanoparticle analyzer, and the coefficient of variation (C.V value) =12.60% (as shown in fig. 2).
Example 2 preparation of acceptor particle b
2.1 preparation of the Carrier and characterization procedure
1. A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein and 10ml of water were added thereto, and after stirring for 10 minutes, N was introduced 2 30min；
2. 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water to prepare an aqueous solution. Adding the aqueous solution into the reaction system of the step 1, and continuing to introduce N 2 30min；
3. Heating the reaction system to 70 ℃ for reaction for 15 hours;
4. the emulsion after completion of the reaction was cooled to room temperature and filtered through a suitable filter cloth. Washing the obtained emulsion by centrifugal sedimentation with deionized water until the conductivity of the supernatant fluid at the beginning of centrifugation is close to that of the deionized water, diluting with water, and preserving in an emulsion form;
5. the average particle diameter of the Gaussian distribution of the latex microsphere particle diameter at this time was 202.2nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=4.60%.
2.2 landfill Process and characterization of luminescent compositions
1. A25 ml round-bottomed flask was prepared and charged with 0.1g of a dimethylthiophene derivative and 0.1g of europium (III) complex (MTTA-EU) 3+ ) 10ml of 95% ethanol is magnetically stirred, and the temperature of the water bath is raised to 70 ℃ to obtain a complex solution;
2. preparing a 100ml three-neck flask, adding 10ml 95% ethanol, 10ml water and 10ml aldehyde polystyrene latex microspheres with concentration of 10% obtained in the step 1.1, magnetically stirring, and heating to 70 ℃ in a water bath;
3. slowly dripping the complex solution in the step 1) into the three-neck flask in the step 2), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling;
4. centrifuging the emulsion for 1 hour, 30000G, and discarding supernatant after centrifuging to obtain aldehyde polystyrene microsphere filled with luminous composition.
5. The average particle diameter of the Gaussian distribution of the particle diameters of the microspheres at this time was 204.9nm as measured by a nanoparticle analyzer, and the coefficient of variation (c.v.) was=5.00%.
2.3 coupling procedure of antibodies
1. HBsAg antibody i was dialyzed to 50 mcb buffer at ph=10 to give a concentration of 1mg/ml.
2. 0.5ml of the receptor particles obtained in step 2.3 and 0.5ml of HBsAg antibody I were added to a 2ml centrifuge tube, and 100. Mu.l of 10mg/ml NaBH was added after mixing 4 The solution (50 mM CB buffer) was reacted at 2-8℃for 4 hours.
3. After completion of the reaction, 0.5ml of 100mg/ml BSA solution (50 mM B buffer) was added thereto, and the reaction was carried out at 2-8℃for 2 hours.
4. After the reaction, the mixture was centrifuged for 45min at 30000G, and the supernatant was discarded after centrifugation and resuspended in 50mM HES buffer. The centrifugation washing was repeated four times and diluted with a first buffer solution having a composition comprising: 0.1mol tris/l, 0.3mol NaCl, 25mmol EDTA, 0.1% dextran, 0.01% gentamicin and 15ppm ProClin-300, pH 8.00.
5. The average particle diameter value of the Gaussian distribution of the particle diameters of the microspheres at this time was 223.1nm as measured by a nanoparticle analyzer, and the coefficient of variation of the particle diameter distribution (C.V value) =9.60%.
Example 3: detection of sugar content of microspheres by anthrone method
1. Pretreatment of microsphere samples:
separately, donor reagent A containing 1mg of donor microsphere a in example 1 and donor reagent B containing 1mg of donor microsphere B in example 2 were taken, centrifuged for 40min at 20000g, the supernatant was removed, and then sonicated with purified water, and after repeating the centrifugation and dispersion three times, the volume was fixed to 1mg/mL with purified water.
2. Preparing a glucose standard solution:
the standard solution curves of 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.075mg/mL, 0.10mg/mL, 0.15mg/mL were prepared from 1mg/mL glucose stock solution using purified water, as shown in FIG. 1.
3. Configuration of anthrone solution: 2mg/mL of the solution (stable at room temperature for 24h, ready-to-use) was prepared with 80% sulfuric acid solution.
4. To the centrifuge tube, 0.1mL of glucose standard solution and microsphere sample at each concentration were added, and 1mL of anthrone test solution was added to each tube.
Incubation at 5.85℃for 30min.
6. The sample reaction tube was centrifuged at 15000g for 40min, and the pipette tip suctioned clear liquid from the bottom of the tube to measure absorbance, avoiding suctioning out the upper suspension. 7. The temperature was returned to room temperature and absorbance at 620nm was measured (preferably within 2 h).
8. And (3) carrying out primary linear regression by taking the concentration of the standard substance as an X value and the absorbance as a Y value to obtain the sugar concentration of the microsphere sample.
Table 1 standard sugar content determination table
The sugar content of the two acceptor particles prepared in example 1 and example 2 were as follows:
sugar content per mg of acceptor microsphere a in example 1: 38.6 μg
Sugar content per mg of example 2 receptor microsphere b: 8.40 mug
Example 4:
according to the method described in example 2, solutions of acceptor particles of conjugated antibody I with different coefficients of variation in the particle size distribution were obtained, specifically:
receptor particle 1: the mean particle size of the Gaussian distribution is 221.8nm, and the variation coefficient C.V value of the particle size distribution=3.9%; nicomp distribution is unimodal.
Receptor particle 2: the mean particle size of the Gaussian distribution is 220.4nm, and the variation coefficient C.V value of the particle size distribution=5.0%; nicomp distribution is unimodal.
Receptor particles 3: the mean particle size of the Gaussian distribution is 218.1nm, and the variation coefficient C.V value of the particle size distribution=7.9%; nicomp distribution is unimodal.
Receptor particles 4: the mean particle size of the Gaussian distribution is 222.3nm, and the variation coefficient C.V value of the particle size distribution=10.3%; nicomp distribution is unimodal.
Receptor particles 5: the mean particle size of the Gaussian distribution is 226.0nm, and the variation coefficient C.V value of the particle size distribution=18.8%; nicomp distribution is unimodal.
Receptor particles 6: the mean particle size of the Gaussian distribution is 222.2nm, and the variation coefficient C.V value of the particle size distribution=24.5%; the Nicomp distribution was bimodal.
Example 5:
the sensitivity point is defined as when the signal of concentration Cx is higher than the signal of double concentration C0, i.e. RLU (Cx) >2RLU (C0), the corresponding detection reagent sensitivity is Cx. The upper detection limit point is defined as the upper range limit determined using the method in NCCLS EP-6 file.
(1) The HBsAg antigen was diluted to a series of concentrations of 0.03IU/mL, 0.04IU/mL, 0.05IU/mL, 0.06IU/mL, 0.07IU/mL, 0.10IU/mL, 1IU/mL, 10IU/mL, 50IU/mL, 125IU/mL, 200IU/mL, 250IU/mL, 300IU/mL, using the acceptor reagent comprising the acceptor particle conjugated to HBsAg antibody I prepared in example 3, respectively, and then the HBsAg antigen was detected in the series of concentrations as described above with the same biotin-labeled HBsAg monoclonal antibody 2 (diluted to 1 ug/mL) and universal solution (reagent containing donor particle), and the detection sensitivity and upper limit of detection were as shown in Table 2 using a photo-excitation chemiluminescence analysis system developed by Boyang biotechnology (Shanghai).
TABLE 2
As can be seen from table 2, when the coefficient of variation of the particle size distribution of the receptor particles is 5% or more and not more than 20%, the kit containing the receptor particles has both a relatively suitable sensitivity and a wide detection range.
Example 6:
acceptor reagents C to F comprising a series of acceptor microspheres of different sugar contents were prepared using the acceptor particle and acceptor reagent preparation method described in example 1 above, and the sugar content in the acceptor microspheres was measured using the anthrone method given in example 3.
And then under the same condition, analyzing the chemiluminescent detection effects of different receptor reagents, wherein the chemiluminescent detection process is completed on a full-automatic light-activated chemiluminescent analysis system developed by Boyang biotechnology (Shanghai) limited company, and outputting detection results. The specific experimental steps are as follows: 1. preparing biotin-coupled receptor microspheres into 30 mug/mL, and preparing receptor microspheres with different sugar contents into 25 mug/mL receptor reagent; 2. adding 75 mu L of a reagent prepared by biotin-coupled receptor microspheres into the reaction hole; 3. 175. Mu.L of LiCA stock solution was added; 4. after 15min of reaction, the signal values were read on a photoexcitation chemiluminescent assay system.
TABLE 3 Table 3
As can be seen from Table 3, when the sugar content of the acceptor microsphere in the acceptor reagent is not higher than 40. Mu.g/mg, the photo-excitation chemiluminescent detection signal at this time is higher.
Example 7: detection of HBsAg marker levels in samples of normal humans and patients suspected of having hepatitis B Virus
In this example, 40 clinical samples were tested using an HBsAg quantitative assay kit (light activated chemiluminescence method) consisting of reagent 1 (R1 ') comprising a first anti-HBsAg antibody coated acceptor particle, reagent 2 (R2 ') comprising a biotin-labeled second anti-HBsAg antibody, and further comprising a universal solution (R3 ') comprising donor particles. Among them, reagent 1 was a receptor reagent prepared by using the receptor particle b (the variation coefficient C.V value=9.60% of the particle size distribution, and the sugar content per mg of the receptor particle was 8.4. Mu.g) in example 2. The quantitative determination of HBsAg belongs to POCT detection.
The detection process is completed on a full-automatic photo-excitation chemiluminescence analysis system developed by Boyang biotechnology (Shanghai) limited company and outputs a detection result, and the specific experimental steps are as follows:
1. selecting 40 clinical samples, balancing to room temperature, and uniformly mixing;
2. adding the uniformly mixed sample, the prepared R1 'and R2' into an 8X 12 white board respectively;
3. placing the white board with the sample into a LiCA HT instrument for reaction in the following reaction mode;
(1) Mixing 25ul of sample, 25ul of R1 'and 25ul of R2' uniformly;
(2) Incubation at 37℃for 17min;
(3) 175ul of the universal solution (R3') was added;
(4) Incubation at 37℃for 15min;
(5) Excitation readings, specific detection results are shown in tables 4 and 5 below.
TABLE 4 Table 4
TABLE 5
By data comparison, the yin-yang coincidence rate of the measured value of the yapei and the measured value of the embodiment is 100%, the sensitivity reaches 0.05IU/mL, and the measured value of the yapei is basically consistent with the measured value of the yapei sample and accords with the expected target.
Claims (10)
1. A receptor reagent comprising a first buffer solution and, suspended therein, receptor particles capable of reacting with reactive oxygen species to produce a chemiluminescent signal, the receptor particles having a surface to which biomolecules are bound, characterized in that: the surface of the carrier is not coated with sugar molecules but directly bonded with biological molecules, and the sugar content in the receptor particles is not higher than 40 mug per milligram of mass; the particle size distribution of the acceptor particles exhibits polydispersity.
2. Acceptor reagent according to claim 1, characterized in that the sugar content per milligram of mass of the acceptor particle is not higher than 20 μg, preferably not higher than 10 μg.
3. Acceptor reagent according to claim 1 or 2, characterized in that the acceptor particle has a coefficient of variation C.V of the particle size distribution in the acceptor reagent of not more than 20%, preferably not more than 15%; and/or the receptor particles have a coefficient of variation C.V in the particle size distribution in the receptor agent of not less than 5%, preferably not less than 8%.
4. A acceptor reagent according to any one of claims 1 to 3 wherein the sugar content in the first buffer solution per litre of volume is not less than 0.010g and not more than 0.30g; preferably, the sugar content in the first buffer solution per liter of volume is not less than 0.015g and not more than 0.20g.
5. The acceptor reagent according to any one of claims 1 to 4 wherein the acceptor particle comprises a carrier, the interior of which is filled with a luminescent composition, the surface of which is bound with a biomolecule.
6. The acceptor reagent according to claim 5, wherein the carrier has a bonding functional group on a surface thereof for directly chemically bonding a biomolecule to the surface of the carrier, the bonding functional group being at least one selected from the group consisting of an amine group, an amide group, a hydroxyl group, an aldehyde group, a carboxyl group, an epoxy group, a maleimide group and a mercapto group; preferably selected from aldehyde groups, carboxyl groups, epoxy groups and maleimide groups.
7. The acceptor reagent according to any one of claims 1 to 6 wherein the sugar is a carbohydrate containing three or more unmodified or modified monosaccharide units; preferably selected from the group consisting of dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran and aminodextran; more preferably selected from the group consisting of dextran, starch, glycogen and polyribose, most preferably dextran or dextran derivatives.
8. A homogeneous chemiluminescent assay kit comprising the acceptor reagent of any one of claims 1-7.
9. Use of the acceptor reagent of any one of claims 1 to 7 or the kit of claim 8 in a chemiluminescent analyzer.
10. Use of the receptor reagent of any one of claims 1-7 or the kit of claim 8 in POCT assays.
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