CN109580936B - Microsphere reagent and preparation method and application thereof - Google Patents
Microsphere reagent and preparation method and application thereof Download PDFInfo
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- CN109580936B CN109580936B CN201811475594.6A CN201811475594A CN109580936B CN 109580936 B CN109580936 B CN 109580936B CN 201811475594 A CN201811475594 A CN 201811475594A CN 109580936 B CN109580936 B CN 109580936B
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- 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
Abstract
The invention provides a microsphere reagent and a preparation method and application thereof, wherein by optimizing an experimental operation method, microspheres, an antibody and a coupling agent are creatively mixed according to a specific proportion, the concentration and the formula of an auxiliary agent are optimized, the requirements of mixed liquid in each step are screened and matched, the types and the concentrations of a stabilizer, a storage agent and a preservative are optimized, the auxiliary agents are matched with each other, then ultrasonic mixing is carried out, each step is subjected to synergistic interaction under each condition, a reagent system with uniform and stable dispersion is realized, the method is simple and efficient, the production cost is effectively reduced, the production efficiency is improved, and the microsphere reagent has a wide application prospect and a huge market value.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a microsphere reagent and a preparation method and application thereof.
Background
The beta 2-microglobulin is a small-molecule globulin produced by lymphocytes, platelets and polymorphonuclear leukocytes, has the molecular mass of 11.8KD, is a beta chain (light chain) part (a single-chain polypeptide) of a cell surface Human Lymphocyte Antigen (HLA), widely exists in plasma, urine, cerebrospinal fluid, saliva and colostrum, and has very low content. The synthesis rate and release amount of normal human beta 2-microglobulin from cell membrane are quite constant, beta 2-microglobulin can be freely filtered from glomerulus, 99.9% of beta 2-microglobulin is absorbed by proximal tubule, so that the measurement of blood beta 2-microglobulin can be used as an index for reflecting the filtration rate of glomerulus. The beta 2-microglobulin detection is considered as a simple, accurate and sensitive method for measuring mild renal hypofunction of diabetic patients and observing curative effect. Therefore, the determination of the beta 2-microglobulin has various values in clinic.
The currently common methods for measuring beta 2-microglobulin include immunodiffusion, immunoelectrophoresis, radioimmunoassay, enzyme-linked immunosorbent assay, and latex-enhanced immunoturbidimetry. The most applied method is latex enhanced immunotransmission turbidimetry, and the basic principle is as follows: coating the antibody on latex particles, immunoreacting with corresponding antigen to form aggregate particles, and measuring the turbidity generated by the aggregate at a certain wavelength to determine the content of the detected substance in the specimen.
At present, a detection kit for beta 2 microglobulin prepared by an immunoturbidimetric method in the market mostly adopts a two-step method, wherein a coupling agent activates functional groups on the surfaces of microspheres in the first step, and the microspheres after the activation in the second step are combined with antibodies to generate covalent bonds. This method requires filtration and washing several times to remove unbound antibodies and coupling agents, and centrifugation and ultrafiltration are generally used in the filtration and washing. The filtering and cleaning processes are long in time consumption, complex in process, limited by various factors such as equipment, personnel and sites, small in single-batch preparation amount, large in loss and high in production cost.
CN108226531A discloses a beta 2 microglobulin detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 is a buffer solution, the reagent R2 comprises a beta 2 microglobulin antibody coupled with polystyrene latex particles and a buffer solution, and the preparation method of the reagent R2 comprises the steps of latex particle activation, beta 2 microglobulin antibody activation and coupling. However, the method needs separate operations of multiple steps of activation, activation and coupling, washing operation, complex steps, complex preparation process and low production efficiency. CN106645753A discloses a rapid detection kit for beta 2 microglobulin and application thereof. The kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 consists of potassium dihydrogen phosphate, dodecyl glucoside, octa (ethylene glycol) dodecyl ether, monododecyl nonaethylene glycol ether, calcium chloride, p-hydroxybenzoate and water; the reagent R2 consists of monododecyl nonaethylene glycol ether, dodecyl glucoside, anti-human beta 2 microglobulin antibody latex, p-hydroxybenzoate and water; the diameter of the anti-human beta 2 microglobulin antibody latex is 130-150 nm. However, the reagent of the invention has various components and higher cost, and is not beneficial to popularization and application because the supernatant needs to be centrifuged. CN102590526B provides a beta 2-microglobulin detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a buffer solution, a preservative, a stabilizer, an electrolyte and a surfactant, the balance is purified water, the reagent R2 comprises beta 2-microglobulin antibody-sensitized polystyrene latex particles, the buffer solution, the preservative and the stabilizer, and the balance is purified water. The beta 2-microglobulin antibody sensitized polystyrene latex particle adopts a directional coupling method, and the preparation steps comprise: activating polystyrene latex particles; oxidation of beta 2-microglobulin antibodies; coupling of beta 2-microglobulin antibodies to polystyrene latex particles. The method is not optimized for activation, oxidation and coupling methods, and the system stability and detection sensitivity of the method need to be further improved.
Therefore, the beta 2-microglobulin detection reagent is optimized, a convenient and quick preparation method of the microglobulin detection reagent is provided, the detection accuracy, the system stability and the production efficiency are improved, and the method has wide application prospect and great market value.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a microsphere reagent and a preparation method and application thereof, the method adopts a one-step method to prepare the microsphere reagent, microspheres, antibodies and a coupling agent are mixed according to specific concentrations, and a stabilizer, a preservative, a storage agent and the like are added after uniform mixing and ultrasonic treatment so as to ensure that the obtained system is stably stored.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing a microspherical reagent, the method comprising the steps of:
(1) uniformly mixing the microsphere solution, the microglobulin antibody solution and the coupling agent solution, and performing ultrasonic dispersion;
(2) adding an auxiliary agent 2 into the solution dispersed in the step (1), uniformly mixing, and performing ultrasonic dispersion;
(3) and (3) adding an auxiliary agent 3 into the solution dispersed in the step (2), and uniformly mixing to obtain the microsphere reagent.
In the invention, the inventor deeply studies the advantages and disadvantages of a detection reagent for preparing beta 2 microglobulin by an immunoturbidimetric method in long-term production practice, creatively mixes microspheres, an antibody and a coupling agent according to a specific proportion and then carries out ultrasonic mixing in order to simplify the production process, save the steps of ultrafiltration and centrifugation and optimize the experimental operation method, and each step has synergistic interaction under each condition, so that a reagent system with uniform and stable dispersion is realized, the method is simple and efficient, the production cost is effectively reduced, and the production efficiency is improved.
Wherein, the concentration of the mixed microspheres in the step (1) is 0.1-0.5%, the concentration of the antibody is 0.1-0.5%, and the concentration of the coupling agent is 0.1-1.0 mg/L.
Wherein the concentration of the microspheres may be, for example, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%.
The concentration of the antibody may be, for example, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.
The concentration of the coupling agent may be, for example, 0.1mg/L, 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L or 1.0 mg/L.
Preferably, the solvent of the microsphere solution, the beta 2 microglobulin antibody solution and the coupling agent solution is an auxiliary agent 1.
Wherein, the auxiliary agent 1 comprises the following components: 10-100mM buffer solution, 1-5% inorganic salt and 0.5% -2.0% suspension agent.
The concentration of the buffer may be, for example, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100 mM.
The concentration of the inorganic salt may be, for example, 1%, 2%, 3%, 4% or 5%.
Preferably, the pH of the adjuvant 1 is 5.0 to 7.5, and may be, for example, 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5.
Preferably, the buffer of the auxiliary 1 comprises any one of or a combination of at least two of MES, MOPSO or PBS.
The inorganic salt of the auxiliary 1 comprises KCl and/or NaCl.
The suspending agent of the auxiliary 1 comprises one or the combination of at least two of trehalose, sucrose or PEG.
Preferably, the time for mixing uniformly in step (1) is 1-72h, for example, 1h, 5h, 10h, 20h, 30h, 40h, 50h, 60h, 70h or 72h, and the rotation speed for mixing uniformly is 20-50 r/min, for example, 20 r/min, 30 r/min, 40 r/min or 50 r/min.
The ultrasonic dispersion conditions in the step (1) are as follows: the ultrasonic power is 10-50%, for example 10%, 20%, 30%, 40% or 50%, the ultrasonic on-time is 1-3s, for example 1s, 2s or 3s, the ultrasonic off-time is 3-6s, for example 3s, 4s, 5s or 6s, and the total ultrasonic time is 5-30min, for example 5min, 10min, 15min, 20min, 25min or 30 min.
Preferably, the mixing time in step (2) is 1-5h, such as 1h, 2h, 3h, 4h or 5h, and the mixing speed is 20-50 rpm, such as 20 rpm, 30 rpm, 40 rpm or 50 rpm.
Preferably, the volume ratio of the auxiliary agent 2 in the step (2) to the solution dispersed in the step (1) is 1 (9-11).
The ultrasonic dispersion conditions in the step (2) are as follows: the ultrasonic power is 10-50%, for example 10%, 20%, 30%, 40% or 50%, the ultrasonic on-time is 1-3s, for example 1s, 2s or 3s, the ultrasonic off-time is 3-6s, for example 3s, 4s, 5s or 6s, and the total ultrasonic time is 5-30min, for example 5min, 10min, 15min, 20min, 25min or 30 min.
Preferably, the auxiliary agent 2 in the step (2) comprises 10-100mM buffer solution, 1-5% inorganic salt, 0.5-2.0% suspending agent and 5-10% stabilizing agent.
The concentration of the buffer may be, for example, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100 mM.
The concentration of the inorganic salt may be, for example, 1%, 2%, 3%, 4% or 5%.
The concentration of the suspension may be, for example, 0.5%, 1%, 1.5% or 2%.
The concentration of the stabilizer is 5%, 6%, 7%, 8%, 9% or 10%.
The pH of the auxiliary 2 in the step (2) is 5.0-7.5, and can be 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5.
The buffer solution of the auxiliary agent 2 in the step (2) comprises any one or a combination of at least two of MES, MOPSO or PBS.
The inorganic salt of the auxiliary agent 2 in the step (2) comprises KCl and/or NaCl.
The suspending agent in the step (2) comprises any one or the combination of at least two of trehalose, sucrose or PEG.
The stabilizing agent in the step (2) comprises any one or a combination of at least two of BSA, gelatin or glycerol.
Preferably, the auxiliary 3 in step (3) comprises 10-100mM buffer solution, 1-5% inorganic salt, 0.1-0.5% preservative, 1-5% suspending agent, 1-5% stabilizer and 0.01-1% surfactant.
The concentration of the buffer may be, for example, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100 mM.
The concentration of the inorganic salt may be, for example, 1%, 2%, 3%, 4% or 5%.
The concentration of the preservative may be, for example, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.
The concentration of the suspending agent may be, for example, 1%, 2%, 3%, 4% or 5%.
The concentration of the stabilizer may be, for example, 1%, 2%, 3%, 4% or 5%.
The concentration of the surfactant may be, for example, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.
Preferably, the buffer solution of the auxiliary 3 in the step (3) comprises any one of or a combination of at least two of MOPS, PBS or TAPS.
Preferably, the inorganic salt of the auxiliary 3 in the step (3) comprises KCl and/or NaCl.
Preferably, the suspending agent of the auxiliary 3 in the step (3) comprises any one or a combination of at least two of trehalose, sucrose or PEG.
Preferably, the preservative of the auxiliary 3 in the step (3) comprises Proclin300 and/or NaN3。
Preferably, the stabilizer of the auxiliary 3 in the step (3) comprises any one or a combination of at least two of BSA, gelatin or glycerol.
Preferably, the surfactant of the auxiliary 3 in the step (3) comprises any one or a combination of at least two of Tween-20, Tween-80 or TritonX-100.
Preferably, the rotation speed of the blending in the step (3) is 20-50 r/min, for example, 20 r/min, 30 r/min, 40 r/min or 50 r/min.
Preferably, the volume ratio of the auxiliary agent 3 in the step (3) to the solution after dispersion in the step (2) is 10 (9-11).
The preparation method of the auxiliary agent 1-3 comprises the following steps: the respective substances were weighed out separately and dissolved in purified water.
In the invention, the inventor artificially realizes the uniformity and stability of a mixed system of the microsphere, the antibody and the coupling agent, optimizes the concentration and formula of the auxiliary agent, screens the auxiliary agents 1-3 with three different formulas, matches the requirements of mixed liquid in each step, preferably selects the types and concentrations of the stabilizer, the storage agent and the preservative, and mutually matches the auxiliary agents to realize synergistic interaction, thereby promoting the stable and efficient preparation of the microsphere reagent to the maximum extent.
As a preferred technical scheme, the preparation method of the microsphere reagent specifically comprises the following steps:
(1) uniformly mixing the microsphere solution, the beta 2 microglobulin antibody solution and the coupling agent solution for 1-72 hours at the rotating speed of 20-50 revolutions/min, wherein the concentration of the microspheres after mixing is 0.1-0.5%, the concentration of the antibody is 0.1-0.5%, the concentration of the coupling agent is 0.1-1.0mg/L, ultrasonically dispersing, the ultrasonic power is 10-50%, the ultrasonic opening time is 1-3s, the ultrasonic closing time is 3-6s, and the total ultrasonic time is 5-30 min;
(2) adding an auxiliary agent 2 into the solution dispersed in the step (1), wherein the volume ratio of the auxiliary agent 2 in the step (2) to the solution dispersed in the step (1) is 1 (9-11), and after the solution is uniformly mixed for 1-5h at 20-50 r/min, ultrasonic dispersion is carried out, the ultrasonic power is 10-50%, the ultrasonic on-time is 1-3s, the ultrasonic off-time is 3-6s, and the total ultrasonic time is 5-30 min;
(3) and (3) adding an auxiliary agent 3 into the solution dispersed in the step (2), wherein the volume ratio of the auxiliary agent 3 in the step (3) to the solution dispersed in the step (2) is 10 (9-11), and the microsphere reagent is obtained after uniformly mixing at the rotating speed of 20-50 r/min.
In a second aspect, the present invention provides a microspherical reagent prepared by the method of the first aspect.
In a third aspect, the present invention provides a kit comprising reagent R1 and the microsphere reagent of the second aspect;
wherein the components of the reagent R1 comprise 10-100mM buffer solution, 1-5% inorganic salt, 0.01-0.1% preservative and 0.01-0.2% surfactant;
preferably, the pH of the reagent R1 is 6.0-8.0.
Wherein the buffer solution comprises any one or the combination of at least two of MES, MOPSO or PBS; the inorganic salt comprises KCl and/or NaCl; the preservative is preferably Proclin300 and/or NaN3(ii) a The surfactant comprises any one or the combination of at least two of Tween-20, Tween-80 or TritonX-100.
In a fourth aspect, the present invention provides a use of the microsphere reagent according to the second aspect or the kit according to the third aspect for detecting β 2 microglobulin.
Compared with the prior art, the invention has the following beneficial effects:
the detection reagent for preparing the beta 2 microglobulin by the one-step method only needs to mix microspheres, an antibody and a coupling agent, uniformly mix and ultrasonically process, and then add a stabilizer, a preservative, a storage agent and the like to ensure that the obtained system is stably stored, and each step has synergistic effect under the synergistic effect of each condition.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following embodiments further illustrate the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments.
Example 1
1) Dissolving the microspheres in the aid 1, and marking as a mixed solution A;
2) dissolving the beta 2 microglobulin antibody in an auxiliary agent 1, and marking as a mixed solution B;
3) dissolving a coupling agent in the aid 1, and marking as a mixed solution C;
4) adding the mixed solution A, B, C into a container, uniformly mixing for 36h at the rotating speed of 35 r/min, the concentration of the mixed microspheres is 0.3%, the concentration of the antibody is 0.3%, the concentration of the coupling agent is 0.5mg/L, ultrasonically dispersing, wherein the ultrasonic power is 35%, the ultrasonic opening time is 2s, the ultrasonic closing time is 5s, and the total ultrasonic time is 20 min;
5) adding one tenth of the volume of the auxiliary agent 2, continuously mixing for 3 hours at the rotating speed of 35 revolutions per minute, performing ultrasonic dispersion, wherein the ultrasonic power is 35%, the ultrasonic opening time is 2s, the ultrasonic closing time is 5s, and the total ultrasonic time is 20 min;
6) adding the auxiliary agent 3 with the same volume, and uniformly mixing at the rotating speed of 30R/min to obtain the beta 2 microglobulin detection kit R2.
Example 2
1) Dissolving the microspheres in an auxiliary agent 1, diluting to a concentration of 0.1%, and marking as a mixed solution A;
2) dissolving the beta 2 microglobulin antibody in an auxiliary agent 1, diluting to the concentration of 0.1%, and marking as a mixed solution B;
3) dissolving a coupling agent in an auxiliary agent 1, diluting to a concentration of 1mg/mL, and marking as a mixed solution C;
4) adding the mixed solution A, B, C into a container, uniformly mixing for 1h at the rotation speed of 20 r/min, the concentration of the mixed microspheres is 0.1%, the concentration of the antibody is 0.1%, the concentration of the coupling agent is 0.1mg/L, performing ultrasonic dispersion, wherein the ultrasonic power is 10%, the ultrasonic on-time is 3s, the ultrasonic off-time is 3s, and the total ultrasonic time is 5 min;
5) adding an auxiliary agent 2 with one ninth volume, continuously mixing for 1h at the rotating speed of 20 revolutions per minute, performing ultrasonic dispersion, wherein the ultrasonic power is 10%, the ultrasonic opening time is 3s, the ultrasonic closing time is 3s, and the total ultrasonic time is 5 min;
6) adding ten percent of the volume of the auxiliary agent 3, and uniformly mixing at the rotating speed of 20R/min to obtain the beta 2 microglobulin detection kit R2.
Example 3
1) Dissolving the microspheres in the aid 1, and marking as a mixed solution A;
2) dissolving the beta 2 microglobulin antibody in an auxiliary agent 1, and marking as a mixed solution B;
3) dissolving a coupling agent in the aid 1, and marking as a mixed solution C;
4) adding the mixed solution A, B, C into a container, uniformly mixing for 72h at the rotation speed of 50 r/min, the concentration of the mixed microspheres is 0.5%, the concentration of the antibody is 0.5%, the concentration of the coupling agent is 1.0mg/L, ultrasonically dispersing, the ultrasonic power is 50%, the ultrasonic opening time is 1s, the ultrasonic closing time is 6s, and the total ultrasonic time is 30 min;
5) adding the assistant 2 with one tenth volume, continuously mixing for 5 hours at the rotating speed of 50 revolutions per minute, performing ultrasonic dispersion, wherein the ultrasonic power is 50%, the ultrasonic opening time is 1s, the ultrasonic closing time is 6s, and the total ultrasonic time is 30 min;
6) adding ten volume eleven times of the auxiliary agent 3, and uniformly mixing at the rotating speed of 50R/min to obtain the beta 2 microglobulin detection kit R2.
Comparative example 1
Compared with example 1, the conditions were the same as example 1 except that the coupling agent was used to activate the microspheres and the antibody was added for covalent binding.
Comparative example 2
The conditions were the same as in example 1 except that the concentration of microspheres was changed to 1% as compared with example 1.
Comparative example 3
The conditions were the same as in example 1 except that the concentration of the antibody was changed to 1% as compared with example 1.
Comparative example 4
The conditions were the same as in example 1 except that the concentration of the coupling agent was changed to 2mg/mL, as compared with example 1.
Comparative example 5
The conditions were the same as in example 1 except that ultrasonic dispersion was not used, as compared with example 1.
Comparative example 6
The conditions were the same as in example 1 except that the pH of the assistant 1 was changed to 3.0 as compared with example 1.
Comparative example 7
The conditions were the same as in example 1 except that the concentration of the stabilizer in the auxiliary 2 was changed to 1% as compared with example 1.
Comparative example 8
The same conditions as in example 1 were followed except that the auxiliary 3 was changed to contain no surfactant, as compared with example 1.
Comparative example 9
The detection was carried out using a commercially available kit (beta 2-microglobulin assay kit from Peking Lindman).
Example 4
Preparation of reagent R1: 10-100mM buffer solution, inorganic salt, preservative and surfactant, wherein the pH value is 6.0-8.0; wherein the buffer solution is preferably MES, MOPSO or PBS; the inorganic salt is preferably KCl or NaCl; the preservative is preferably Proclin300, NaN3(ii) a The surfactant is preferably Tween-20, Tween-80 or TritonX-100.
The preparation method of R1 comprises the following steps: weighing each substance respectively, dissolving in purified water, and stirring.
Experimental detection
The microsphere reagents prepared in examples 1-3 and comparative examples 1-8 are respectively assembled with the reagent R1 prepared in example 4 to form a kit, and the commercial kit of comparative example 9 is added for experimental detection;
reagent detection method (taking Hitachi 7170 as an example): two-point rate method; the detection wavelength is 546 nm; 18-26 light measuring points; serum sample size/R1/R2 ═ 3/240/60; the reaction direction was upward.
TABLE 1
As can be seen from Table 1, in examples 1-3, the microsphere reagent prepared according to the technical scheme provided by the invention, the kit obtained by assembly has accurate detection result, which is basically consistent with the result of the kit sold in the market, but the preparation method is simpler; the operation sequence of the application is not adopted in the comparative example 1 and the comparative example 5, the formula of the microsphere reagents in the comparative examples 2 to 4 exceeds the range, the formula of the auxiliary agents in the comparative examples 6 to 8 exceeds the range, the detection result of the kit obtained by assembling the components is obviously reduced or increased, and is contrary to the standard result of a commercially available kit, so that the preparation method of the application shows that the formulas of the components are synergistic and can not be matched, the specific experiment step sequence is matched, the stability of the system and the accuracy of the detection result are jointly realized, the production cost is reduced, and the production efficiency is improved.
In summary, according to the microsphere reagent and the preparation method and application thereof provided by the invention, the microsphere reagent is prepared by adopting a one-step method, the experimental operation method is optimized, the microspheres, the antibody and the coupling agent are creatively mixed according to a specific proportion and then ultrasonically mixed uniformly, each step and each condition synergize, a uniformly and stably dispersed reagent system is realized, the method is simple and efficient, the production cost is effectively reduced, and the production efficiency is improved.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (20)
1. A method for preparing a microsphere reagent, which is characterized by comprising the following steps:
(1) uniformly mixing the microsphere solution, the beta 2 microglobulin antibody solution and the coupling agent solution for 1-72 hours at the rotating speed of 20-50 revolutions/min, wherein the concentration of the microspheres after mixing is 0.1-0.5%, the concentration of the antibody is 0.1-0.5%, the concentration of the coupling agent is 0.1-1.0mg/L, ultrasonically dispersing, the ultrasonic power is 10-50%, the ultrasonic opening time is 1-3s, the ultrasonic closing time is 3-6s, and the total ultrasonic time is 5-30 min;
(2) adding an auxiliary agent 2 into the solution dispersed in the step (1), wherein the volume ratio of the auxiliary agent 2 in the step (2) to the solution dispersed in the step (1) is 1 (9-11), and after the solution is uniformly mixed for 1-5h at 20-50 r/min, ultrasonic dispersion is carried out, the ultrasonic power is 10-50%, the ultrasonic on-time is 1-3s, the ultrasonic off-time is 3-6s, and the total ultrasonic time is 5-30 min;
(3) adding an auxiliary agent 3 into the solution dispersed in the step (2), wherein the volume ratio of the auxiliary agent 3 in the step (3) to the solution dispersed in the step (2) is 10 (9-11), and the microsphere reagent is obtained after uniformly mixing at the rotating speed of 20-50 r/min;
the solvents of the microsphere solution, the beta 2 microglobulin antibody solution and the coupling agent solution are auxiliary agents 1;
the auxiliary agent 1 comprises the following components: 10-100mM buffer solution, 1-5% inorganic salt and 0.5-2.0% suspension agent;
the pH value of the auxiliary agent 1 is 5.0-7.5;
the auxiliary agent 2 in the step (2) comprises 10-100mM buffer solution, 1-5% inorganic salt, 0.5-2.0% suspending agent and 5-10% stabilizing agent;
the auxiliary agent 3 in the step (3) comprises 10-100mM buffer solution, 1-5% inorganic salt, 0.1-0.5% preservative, 1-5% suspending agent, 1-5% stabilizer and 0.01-1% surfactant.
2. The method according to claim 1, wherein the buffer of aid 1 comprises any one or a combination of at least two of MES, MOPSO or PBS.
3. The process according to claim 1, characterized in that the inorganic salt of adjuvant 1 comprises KCl and/or NaCl.
4. The method according to claim 1, wherein the suspending agent of the auxiliary 1 comprises any one or a combination of at least two of trehalose, sucrose or PEG.
5. The method according to claim 1, wherein the pH of the auxiliary 2 in the step (2) is 5.0 to 7.5.
6. The method according to claim 1, wherein the buffer of the auxiliary 2 in step (2) comprises any one or a combination of at least two of MES, MOPSO or PBS.
7. The method according to claim 1, wherein the inorganic salt of the auxiliary 2 in step (2) comprises KCl and/or NaCl.
8. The method according to claim 1, wherein the suspending agent of the auxiliary agent 2 in the step (2) comprises any one or a combination of at least two of trehalose, sucrose or PEG.
9. The method according to claim 1, wherein the stabilizer of the auxiliary 2 in step (2) comprises any one or a combination of at least two of BSA, gelatin or glycerol.
10. The method according to claim 1, wherein the buffer of the auxiliary 3 in step (3) comprises any one of MOPS, PBS or TAPS or a combination of at least two thereof.
11. The method according to claim 1, wherein the inorganic salt of the auxiliary 3 in step (3) comprises KCl and/or NaCl.
12. The method according to claim 1, wherein the suspending agent of the auxiliary 3 in the step (3) comprises any one or a combination of at least two of trehalose, sucrose or PEG.
13. The method according to claim 1, wherein the preservative of the auxiliary 3 in step (3) comprises Proclin300 and/or NaN3。
14. The method according to claim 1, wherein the stabilizer of the auxiliary 3 in step (3) comprises any one or a combination of at least two of BSA, gelatin or glycerol.
15. The method as claimed in claim 1, wherein the surfactant of the adjuvant 3 in step (3) comprises any one of Tween-20, Tween-80 or triton x-100 or a combination of at least two thereof.
16. A microspheroidal reagent prepared by the method of any one of claims 1 to 15.
17. A kit comprising a reagent R1 and the microsphere reagent of claim 16;
wherein the components of the reagent R1 comprise 10-100mM buffer solution, 1-5% inorganic salt, 0.01-0.1% preservative and 0.01-0.2% surfactant.
18. The kit according to claim 17, wherein the reagent R1 has a pH of 6.0 to 8.0.
19. Use of the microsphere reagent according to claim 16 for the detection of β 2 microglobulin.
20. Use of a kit according to claim 17 or 18 for the detection of β 2 microglobulin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811475594.6A CN109580936B (en) | 2018-12-04 | 2018-12-04 | Microsphere reagent and preparation method and application thereof |
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| CN102590526A (en) * | 2012-01-13 | 2012-07-18 | 宁波美康生物科技股份有限公司 | Beta 2-microglobulin detection kit |
| CN104897904A (en) * | 2015-05-29 | 2015-09-09 | 中生北控生物科技股份有限公司 | Method for marking protein by carboxyl microsphere |
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| CN102590526A (en) * | 2012-01-13 | 2012-07-18 | 宁波美康生物科技股份有限公司 | Beta 2-microglobulin detection kit |
| CN104897904A (en) * | 2015-05-29 | 2015-09-09 | 中生北控生物科技股份有限公司 | Method for marking protein by carboxyl microsphere |
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| CN108761089A (en) * | 2018-06-29 | 2018-11-06 | 迈克生物股份有限公司 | Preparation method for the reagent for detecting β2-microglobulin |
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