CN111596052A - Immunoturbidimetry detection reagent and preparation method thereof - Google Patents
Immunoturbidimetry detection reagent and preparation method thereof Download PDFInfo
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- CN111596052A CN111596052A CN202010255431.8A CN202010255431A CN111596052A CN 111596052 A CN111596052 A CN 111596052A CN 202010255431 A CN202010255431 A CN 202010255431A CN 111596052 A CN111596052 A CN 111596052A
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- buffer solution
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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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
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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
- G01N33/54346—Nanoparticles
Abstract
The invention discloses an immunoturbidimetric assay detection reagent and a preparation method thereof, wherein the immunoturbidimetric assay detection reagent comprises latex microspheres, a buffer solution, polylysine, an activator and a preservative, the activator comprises N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the concentration of the activator is 50-300 mg/ml, the pH of the buffer solution is 6.0-8.0, the molar mass of the buffer solution is 20-100 mM, and the mass percent of the preservative is 0.05-0.2%. According to the detection reagent for the immune turbidimetry, the latex microspheres before coupling are treated by optimizing the components and the proportion of the reagent, so that the sensitivity of the latex microspheres is improved, the absorbance of some detection items at a low value is improved, the detection deviation caused by the fact that the real condition of a sample cannot be accurately reflected when the low value is detected is avoided, and the detection accuracy is improved.
Description
Technical Field
The invention relates to the technical field of biochemical analysis, in particular to a detection reagent by an immune turbidimetric method.
Background
The serum immunology examination is a kind of physiological function test that the organism recognizes self and non-self antigens, forms natural immune tolerance to autoantibodies, and generates rejection to non-self antigens. The in vitro diagnostic reagent of the immunization method is to apply the principle and detect the antibody or antigen in serum or body fluid in clinic by using the known antigen or antibody in vitro. Different detection methods in antigen-antibody binding have different characteristics and are used for calculating quantitative parameters in detection, such as absorbance, luminescence values and the like.
The immunolabeling technique is to label a known antigen or antibody on a substance which is easy to display, and to detect the label to calculate the state of antigen-antibody reaction, thereby indirectly determining the quality or quantity of the antibody or antigen to be detected. The immunolabeling technology has the characteristics of rapidness, qualitative or quantitative, and is the most widely applied immunology detection technology at present.
The latex particle enhanced turbidimetry is a relatively stable and accurate body fluid protein homogeneous phase immune turbidimetry detection technology which appears in recent years. The specific principle is that antibodies (monoclonal antibodies or polyclonal antibodies) are crosslinked or physically adsorbed on the surfaces of the polymer latex microspheres, and when the microspheres (generally called Reagent 2 or R2) crosslinked with the antibodies are combined with antigens in a Reagent reaction solution (generally called Reagent 1 or R1), the microspheres can be rapidly aggregated together in a short time, so that the light dispersion performance or the light transmission performance of the reaction solution is changed. The concentration of the antigen to be detected can be reflected within a certain range. Its advantages are direct measurement of absorbance value of reaction liquid after reaction between antigen and antibody, no need of repeated incubation and washing plate by ELISA method, and several minutes to obtain result. In addition, the simplification of the operation steps of the nano immunoturbidimetry correspondingly avoids the interference of a plurality of human operation factors and external factors such as reagents, environment and the like, has better stability and repeatability, and can reflect the content of the substance to be detected more truly.
Point-of-care testing (POCT) refers to clinical testing performed by a patient (bedside testing), and is not always performed by a clinical tester. The method is a new method for analyzing the sample immediately on the sampling site, saving complex processing procedures of the sample during laboratory test and quickly obtaining test results. Its advantage does: 1. the POCT aims to obtain experimental results more quickly; 2. the POCT is simple to use, simple and convenient to operate and easy to use, and the POCT becomes a part of a diagnosis system by virtue of the usability of the POCT; POCT assumes laboratory functions without the need for traditional hospital laboratory equipment. POCT can be done either in the doctor's office or in a car that is driven. POCT can serve patients in all directions within 24 hours without the limitation of time and place; 3. the greatest problem facing the experimenter, in terms of overall cost savings, is the cost of controlling the diagnosis, but the result is always a reduction in the cost of the individual tests, rather than an overall reduction in the cost of the patient's overall hospitalization procedure. POCT is relatively high in terms of "individual inspection cost"; however, in many cases, the use of POCT can not only improve the experimental results but also reduce the resource occupation, the time of patient stay, the sampling time, the occupation time of medical staff, and the like.
In the latex reagent for the immunoturbidimetry in the prior art, when the antibody is labeled in the immunoturbidimetry, the results of absorbance tests of biochemical instruments on various items are different due to different items, and for some items with lower overall absorbance, the detection result deviation is large when the biochemical instruments test a low value, and the detection precision is low, so that the latex reagent needs to be improved.
Disclosure of Invention
The present invention is directed to the problems of the prior art, such as large deviation of the detection result and low detection precision when the conventional turbidimetric immunoassay test reagent is used for testing low values, and provides a turbidimetric immunoassay test reagent capable of solving the problems.
In order to achieve the purpose, the invention is realized by the following technical scheme: the immunoturbidimetric assay reagent is characterized in that: the emulsion comprises latex microspheres, a buffer solution, -polylysine, an activator and a preservative, wherein the activator comprises N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the concentration of the activator is 50-300 mg/ml, the pH value of the buffer solution is 6.0-8.0, the molar mass of the buffer solution is 20-100 mM, and the mass percent of the preservative is 0.05-0.2%.
Polylysine is a homo-monomer polymer containing 25 to 30 lysine residues.
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EDC, is a water-soluble carbodiimide, and is used as a carboxyl activating reagent in amide synthesis, as well as for activating phosphate groups, crosslinking proteins and nucleic acids, and preparing immune conjugates. The pH range is 4.0-6.0 when in use, and the N-hydroxysuccinimide or N-hydroxythiosuccinimide is often used together to improve the coupling efficiency.
N-hydroxysuccinimide, whose English symbol is NHS, is white to off-white crystal, and is used for synthesizing amino acid protective agent, semi-synthetic kanamycin and medical intermediate.
In a further scheme, the preservative is one of sodium azide, thimerosal, Proclin-300 and Proclin-950.
In a further scheme, the buffer solution is MES buffer solution. MES is an abbreviation of 2- (N-morpholine) ethanesulfonic acid.
Another object of the present invention is to provide a method for preparing a test agent by an immunoturbidimetric assay, which comprises the following steps:
1) adding a certain amount of latex microspheres into a container, adding a buffer solution with the pH value of 6.0-8.0, and fully stirring;
2) adding a certain amount of activating agent into the container for activation, simultaneously adding a certain amount of polylysine, treating the latex microspheres, and fully stirring the solution in the process;
3) after activation, performing centrifugal treatment and water washing on the latex microspheres in the container, then performing heavy suspension by using a PB buffer solution, and performing ultrasonic treatment after full heavy suspension for 1-3 h;
4) stirring for a certain time on a magnetic stirring instrument after the ultrasonic treatment is finished, then adding the antibody, fully stirring, and moving into a refrigerator at 4 ℃ to continue stirring overnight;
5) taking out the container the next day, and removing the stirrer for ultrasonic treatment; after the ultrasonic treatment is finished, adding bovine serum albumin for sealing, stirring at room temperature for 4-6h, and then continuing to stir in a refrigerator at 4 ℃ overnight;
6) after overnight, taking out the stirrer, centrifuging twice, washing once with water, and resuspending with an R2buffer solution; and (4) carrying out ultrasonic treatment after resuspension, and adding a preservative into the mixture after ultrasonic treatment is finished, and diluting the mixture to a required concentration for storage.
The PB buffer solution is a phosphate buffer solution for short.
In a further scheme, the buffer solution in the step 1 is MES buffer solution, and the molar mass of the buffer solution is 20-100 mM.
In a further scheme, the activating agent in the step 2 comprises N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of the activating agent is 50-300 mg/ml.
The further proposal is that the preservative in the step 3 is one of sodium azide, thimerosal, Proclin-300 and Proclin-950, and the mass percent of the preservative is 0.05-0.2%.
The immune turbidimetric assay test agent has the advantages that the latex microspheres before coupling are treated by optimizing the components and the proportion of the reagent, more antibodies are polymerized by the latex microspheres in the process of labeling the antibodies by the latex microspheres by adding polylysine, the sensitivity of the latex microspheres is improved, the absorbance of some detection items at a low value is improved, the detection deviation caused by the fact that the real condition of a sample cannot be accurately reflected when the low value is tested is avoided, and the detection accuracy is improved.
Drawings
FIG. 1 is a graph comparing samples CV at different concentrations tested in A, B groups in a comparative experiment;
Detailed Description
The technical solutions of the present invention are described clearly and completely by the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The immunoturbidimetric assay reagent comprises latex microspheres, a buffer solution, polylysine, an activator and a preservative.
The activating agent comprises N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of the activating agent is 50 mg/ml.
The buffer was MES buffer, the pH of the buffer was 6.0 and the molar mass of the buffer was 20 mM.
The preservative is 0.05 percent by mass and is sodium azide.
Example 2
On the basis of example 1, in contrast to example 1, the concentration of the activator is 100 mg/ml; the pH of the buffer was 8.0 and the molar mass of the buffer was 50 mM; the preservative is 0.1 percent by mass and the preservative is the thimerosal.
Example 3
On the basis of example 1, in contrast to example 1, the concentration of the activator is 200 mg/ml; the pH of the buffer was 7.0 and the molar mass of the buffer was 100 mM; the preservative is 0.15 percent by mass and the preservative is Proclin-300.
Example 4
On the basis of example 1, in contrast to example 1, the concentration of the activator is 300 mg/ml; the pH of the buffer solution is 8.0, and the molar mass of the buffer solution is 80 mM; the preservative is 0.2 percent by mass and the preservative is Proclin-950.
The preparation method of the immunoturbidimetric assay reagent comprises the following steps:
1) adding a certain amount of latex microspheres into a container, adding a buffer solution with the pH value of 6.0-8.0, and fully stirring;
2) adding a certain amount of activating agent into the container for activation, simultaneously adding a certain amount of polylysine, treating the latex microspheres, and fully stirring the solution in the process;
3) after activation, performing centrifugal treatment and water washing on the latex microspheres in the container, then performing heavy suspension by using a PB buffer solution, and performing ultrasonic treatment after full heavy suspension for 1-3 h;
4) stirring for a certain time on a magnetic stirring instrument after the ultrasonic treatment is finished, then adding the antibody, fully stirring, and moving into a refrigerator at 4 ℃ to continue stirring overnight;
5) taking out the container the next day, and removing the stirrer for ultrasonic treatment; after the ultrasonic treatment is finished, adding bovine serum albumin for sealing, stirring at room temperature for 4-6h, and then continuing to stir in a refrigerator at 4 ℃ overnight;
6) after overnight, taking out the stirrer, centrifuging twice, washing once with water, and resuspending with an R2buffer solution; and (4) carrying out ultrasonic treatment after resuspension, and adding a preservative into the mixture after ultrasonic treatment is finished, and diluting the mixture to a required concentration for storage.
The buffer solution of R2 comprises a buffer solution, a preservative and a stabilizer, wherein the buffer solution is MES buffer solution 50mM, the preservative is 0.01% of methylchloroisothiazolinone, and the stabilizer is 1% of sucrose.
The following describes the performance of the immunoturbidimetric assay of the present invention and the performance of the immunoturbidimetric assay of the present invention in general. The specific test process is as follows:
1) to two glass vials were added 5ml of 0.2M, pH ═ 6.5 MES buffer and 1.2ml of 80nm latex microspheres simultaneously.
2) 0.1g of NHS activator and 0.2g of EDC activator were weighed out, and 0.07g of polylysine was weighed out, and 1.0ml, 3.0ml and 0.7ml of pure water were added thereto, respectively, and vortexed to dissolve them sufficiently.
3) And (3) dropwise adding the dissolved NHS and EDC solutions into two glass bottles in the step 1 by using a pipette, adding the polylysine liquid in the step 2 into one glass bottle, adding a proper amount of pure water, keeping the liquid amount in the two glass bottles consistent, adding a clean stirring bar, and stirring at room temperature for 60 min.
4) After the activation, the mixture was centrifuged twice and washed with water, resuspended with 0.1M PB, and then sonicated for 60min after sufficient resuspension.
5) Stirring on a magnetic stirring instrument for 30min after the ultrasound is finished, adding 0.5ml of antibody into two glass bottles, quickly adding the antibody, stirring for 60min, and then moving into a refrigerator at 4 ℃ to continue stirring overnight.
6) The next day, the stirrer was removed and subjected to ultrasound for 60 min.
7) After the sonication was completed, 5ml of 1% bovine serum albumin was added for blocking, and the mixture was stirred at room temperature for 5 hours, and then stirred in a refrigerator at 4 ℃ overnight.
8) After overnight the stirrer was removed and centrifuged twice, where it was washed once with water and resuspended in R2 buffer.
9) After resuspension, ultrasonic treatment is carried out for 90min, and the suspension is diluted to the required concentration for storage.
10) Two labeled antibodies (named A, B) were tested on a Hitachi 7020 Biochemical apparatus using a PCT test kit (latex enhanced immunoturbidimetry).
11) Setting parameters to be R1:180 μ l by Hitachi 7020 biochemical analyzer; 60 mu l of R2; sample preparation: 20 mu l of the mixture; reading spot: 21-34.
12) The test results were as follows:
A. b comparison of results of two antibody tests on the same sample (A is no addition-polylysine, B is addition-polylysine)
As can be seen from the table, the CV of group A exceeds 10% at about the detection limit and is less than 10% at a concentration of 0.5 ng/ml; whereas, the CV of group B reached 10% or less at around the detection limit. Thus, the CV values for group B with polylysine added are better than for group A without polylysine.
FIG. 1 is a graph comparing CV values of A, B samples tested at different concentrations, and it can be determined that the CV values of the B group with polylysine added thereto are more accurate than those of the A group when testing low value samples.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. An immune turbidimetric assay reagent, which is characterized in that: the emulsion comprises latex microspheres, a buffer solution, -polylysine, an activator and a preservative, wherein the activator comprises N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the concentration of the activator is 50-300 mg/ml, the pH value of the buffer solution is 6.0-8.0, the molar mass of the buffer solution is 20-100 mM, and the mass percent of the preservative is 0.05-0.2%.
2. The immunoturbidimetric assay kit according to claim 1, wherein: the preservative is one of sodium azide, thimerosal, Proclin-300 and Proclin-950.
3. The immunoturbidimetric assay kit according to claim 1, wherein: the buffer solution is MES buffer solution.
4. The preparation method of the immunoturbidimetric assay reagent is characterized by comprising the following steps: which comprises the following steps:
1) adding a certain amount of latex microspheres into a container, adding a buffer solution with the pH value of 6.0-8.0, and fully stirring;
2) adding a certain amount of activating agent into the container for activation, simultaneously adding a certain amount of polylysine, treating the latex microspheres, and fully stirring the solution in the process;
3) after activation, performing centrifugal treatment and water washing on the latex microspheres in the container, then cleaning and resuspending the latex microspheres by using a PB buffer solution, and performing ultrasonic treatment after full resuspension for 1-3 h;
4) stirring for a certain time on a magnetic stirring instrument after the ultrasonic treatment is finished, then adding the antibody, fully stirring, and moving into a refrigerator at 4 ℃ to continue stirring overnight;
5) taking out the container the next day, and removing the stirrer for ultrasonic treatment; after the ultrasonic treatment is finished, adding bovine serum albumin for sealing, stirring at room temperature for 4-6h, and then continuing to stir in a refrigerator at 4 ℃ overnight;
6) after overnight, taking out the stirrer, centrifuging twice, washing once with water, and resuspending with an R2buffer solution; and (4) carrying out ultrasonic treatment after resuspension, and adding a preservative into the mixture after ultrasonic treatment is finished, and diluting the mixture to a required concentration for storage.
5. The method for preparing a test agent for immunoturbidimetric assay according to claim 4, wherein: the buffer solution in the step 1 is MES buffer solution, and the molar mass of the buffer solution is 20-100 mM.
6. The method for preparing a test agent for immunoturbidimetric assay according to claim 4, wherein: the activating agent in the step 2 comprises N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of the activating agent is 50-300 mg/ml.
7. The method for preparing a test agent for immunoturbidimetric assay according to claim 4, wherein: the preservative in the step 3 is one of sodium azide, thimerosal, Proclin-300 and Proclin-950, and the mass percentage of the preservative is 0.05-0.2%.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0348174A2 (en) * | 1988-06-23 | 1989-12-27 | Bio-Rad Laboratories, Inc. | Sperm antibody test |
| CN101981451A (en) * | 2008-04-02 | 2011-02-23 | 维瓦克塔有限公司 | A method for sensing a chemical |
| CN108008130A (en) * | 2017-11-24 | 2018-05-08 | 海格德生物科技（深圳）有限公司 | Kit based on latex enhancing immune turbidimetry for Determination Lp-PLA2 and preparation method thereof |
| CN109633172A (en) * | 2019-01-03 | 2019-04-16 | 迪瑞医疗科技股份有限公司 | Human urine immunoglobulin G detection reagent box based on Immunoturbidimetry |
| CN110007092A (en) * | 2019-04-08 | 2019-07-12 | 杭州博谱医药科技有限公司 | The kit of CMPF content in a kind of latex microsphere-albumen-CMPF conjugate and preparation method thereof and measurement serum |
-
2020
- 2020-04-02 CN CN202010255431.8A patent/CN111596052A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0348174A2 (en) * | 1988-06-23 | 1989-12-27 | Bio-Rad Laboratories, Inc. | Sperm antibody test |
| CN101981451A (en) * | 2008-04-02 | 2011-02-23 | 维瓦克塔有限公司 | A method for sensing a chemical |
| CN108008130A (en) * | 2017-11-24 | 2018-05-08 | 海格德生物科技（深圳）有限公司 | Kit based on latex enhancing immune turbidimetry for Determination Lp-PLA2 and preparation method thereof |
| CN109633172A (en) * | 2019-01-03 | 2019-04-16 | 迪瑞医疗科技股份有限公司 | Human urine immunoglobulin G detection reagent box based on Immunoturbidimetry |
| CN110007092A (en) * | 2019-04-08 | 2019-07-12 | 杭州博谱医药科技有限公司 | The kit of CMPF content in a kind of latex microsphere-albumen-CMPF conjugate and preparation method thereof and measurement serum |
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