CN110794139A - Aldosterone latex enhanced detection kit and preparation method thereof - Google Patents
Aldosterone latex enhanced detection kit and preparation method thereof Download PDFInfo
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- CN110794139A CN110794139A CN201910868070.1A CN201910868070A CN110794139A CN 110794139 A CN110794139 A CN 110794139A CN 201910868070 A CN201910868070 A CN 201910868070A CN 110794139 A CN110794139 A CN 110794139A
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
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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 relates to the technical field of biology, relates to an aldosterone latex enhanced detection kit, and particularly relates to a preparation method of the aldosterone latex enhanced detection kit, and discloses components and concentrations including a reagent R1 and a reagent R2, wherein the reagent R2 consists of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, the preservation solution consists of glycine, a second stabilizing agent and a second preservative, and the latex microsphere antibody conjugate consists of latex microspheres, an anti-aldosterone monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. The invention has the advantages that: the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision and short detection time, and can be widely applied to various fields of basic and clinical medicine.
Description
Technical Field
The invention relates to the technical field of biology, relates to an aldosterone latex enhancement detection kit, and particularly relates to a preparation method of the aldosterone latex enhancement detection kit.
Background
Aldosterone (Aldosterone) is a steroid hormone (mineralocorticoid family) that acts primarily on the kidneys for the reabsorption of sodium ions and water molecules. Generally, aldosterone, a hormone that enhances the reabsorption of ions and water molecules by the kidney, is part of the renin-angiotensin system, and acts to promote Na + retention in the body while expelling K +, is produced by the adrenal cortico-zona and is regulated by renin, angiotensin peptide, secreted by the kidney, and after entering the epithelial cells of the distal tubule and collecting duct, it binds to intracytoplasmic receptors to form a hormone-receptor complex, which interacts with DNA-specific binding sites in the nucleus via the nuclear membrane to regulate specific mRNA transcription, and finally synthesizes a variety of aldosterone-induced proteins, which in turn increases the permeability of the luminal membrane to Na +, ATP synthesis in the mitochondria and the mobility of the sodium pump on the peritubular membrane. This results in increased reabsorption of Na +, increased reabsorption of water, and increased K + output.
At present, in China, aldosterone detection sensitivity is low, the linear range is narrow, the detection process consumes long time, and the method is only suitable for manual operation and is not beneficial to detection of a full-automatic detection instrument, so that the popularization and the application of the method are limited.
Disclosure of Invention
The purpose of the invention is: aiming at the defects, the aldosterone latex enhanced detection kit and the preparation method thereof are provided, wherein the aldosterone latex enhanced detection kit has the advantages of good stability, high sensitivity, wide linear range, high specificity, good precision and short detection time.
In order to achieve the purpose, the invention adopts the technical scheme that:
the reinforced detection kit of aldosterone latex comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration:
the reagent R2 consists of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate consists of latex microspheres, an anti-aldosterone monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows:
0.01-0.3g/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
The first buffer solution and the second buffer solution in the reagent R1 and the reagent R2 comprise one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution and Tris buffer solution.
The first stabilizer of the reagent R1 and the second stabilizer of the reagent R2 are one or more of casein, mannitol and bovine serum albumin.
The surfactant in the reagent R1 is one or a mixture of more than one of Tween 20, Triton, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.
The first preservative in the reagent R1 and the second preservative in the reagent R2 are respectively one of sodium azide, Proclin-950, Proclin-300 and thimerosal.
The pH value of the reagent R1 is between 6.00 and 8.50, and the pH value of the reagent R2 is between 6.00 and 8.50.
A preparation method of an aldosterone latex enhanced detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a first stabilizer, D-trehalose, a surfactant, polyethylene glycol 6000 and a first preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.00-8.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-8.50, continuously stirring for 5-10 minutes until the solution is clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding glycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-8.50, continuously stirring for 5-10 minutes until all the materials are completely dissolved, enabling the solution to be clear and transparent, enabling the bottom of the liquid preparation tank to have no precipitate, fixing the volume to a final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 0.1-2.0% by using a buffer solution, diluting an anti-aldosterone monoclonal antibody to the concentration of 0.05-1.5g/L by using the buffer solution, uniformly mixing the two, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01-0.3g/L by using the buffer solution, dripping the mixture into the mixed solution while shaking the mixture, reacting the mixture at room temperature for 180 minutes, adding bovine serum albumin for sealing, shaking the mixture for uniformly mixing, reacting the mixture at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking a supernatant, adding a preserving solution, carrying out ultrasonic resuspension, repeating the centrifugal washing for 2 times, centrifuging the supernatant for the last time, adding the preserving solution, carrying out resuspension and ultrasonic treatment, and filling the prepared R2 into a finished product tank for marking.
Compared with the prior art, the invention achieves the technical effects that: the invention realizes the detection of the content of aldosterone on a biochemical analyzer for the first time, and the detection range can reach 5-1000 pg/mL;
compared with other detection methods, the method has the characteristics of simple, accurate and rapid operation, and can realize high-throughput detection by batch analysis on a full-automatic biochemical analyzer;
the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision and short detection time, and can be widely applied to various fields of basic and clinical medicine.
Drawings
FIG. 1 is a graph of the calibration curve for the indirect coupling of latex microspheres to aldosterone in example 4 of the present invention. Wherein the X-axis represents standard concentration and the Y-axis represents absorbance.
FIG. 2 is a graph showing the measurement of the linear range in example 4 of the present invention.
Detailed Description
The invention is further described with reference to the following figures and examples:
the first embodiment is as follows:
the invention discloses an aldosterone latex enhanced detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration: 6.0g/L of tris (hydroxymethyl) aminomethane as a first buffer, 0.5g/L, D-trehalose as a first stabilizer, 5g/L of triton as a surfactant at 1ml/L, 60g/L of polyethylene glycol 6000 as a first preservative at 0.8 ml/L;
the reagent R2 consists of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of glycine, bovine serum albumin serving as a second stabilizing agent and Proclin-950 serving as a second preservative, the latex microsphere antibody conjugate consists of latex microspheres, an anti-aldosterone monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows: the content of 2- (N-morpholine) ethanesulfonic acid monohydrate is 10.00g/L, the content of glycine is 1.26g/L, the content of bovine serum albumin is 0.5g/L, Proclin-950, the content of bovine serum albumin is 0.8ml/L, the content of latex microspheres is 0.1%, the content of aldosterone resistant monoclonal antibody is 0.05g/L, and the content of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.01 g/L.
The pH of the reagent R1 is 6.00, and the pH of the reagent R2 is 6.00.
A preparation method of an aldosterone latex enhanced detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding tris (hydroxymethyl) aminomethane, bovine serum albumin, D-trehalose, triton, polyethylene glycol 6000 and Proclin-950 while stirring, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.00-8.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring for 20 minutes until the material is completely dissolved, adjusting the pH value to 6.00, continuously stirring for 5 minutes until the solution is clear and transparent, keeping no precipitate at the bottom of the liquid preparation tank, fixing the volume to the final volume, and storing the solution for later use for marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding glycine, bovine serum albumin and Proclin-950 while stirring, stirring for 20 minutes until the materials are completely dissolved, adjusting the pH value to 6.00, continuing stirring for 5 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 0.1% by using a buffer solution, diluting an anti-aldosterone monoclonal antibody to the concentration of 0.05g/L by using the buffer solution, uniformly mixing the latex microspheres and the buffer solution, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01g/L by using the buffer solution, shaking uniformly and dripping into the mixed solution while stirring, reacting for 180 minutes at room temperature, adding bovine serum albumin for sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking off supernatant, adding a preserving solution, carrying out ultrasonic resuspension, repeating the centrifugal cleaning for 2 times, centrifuging the supernatant for the last time, adding the preserving solution, carrying out ultrasonic resuspension, filling prepared R2 into a finished product tank, and marking.
Example two:
the invention discloses an aldosterone latex enhanced detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration: tris (hydroxymethyl) aminomethane as a first buffer solution with a content of 9g/L, mannitol as a first stabilizer with a content of 1.2g/L, D-trehalose with a content of 12g/L, tween 20 as a surfactant with a content of 3ml/L, polyethylene glycol 6000 with a content of 90g/L, Proclin-300 as a first preservative with a content of 1.4 ml/L;
the reagent R2 consists of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of glycine, mannitol serving as a second stabilizing agent and Proclin-300 serving as a second preservative, the latex microsphere antibody conjugate consists of latex microspheres, an anti-aldosterone monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows: the content of 2- (N-morpholine) ethanesulfonic acid monohydrate is 18g/L, the content of glycine is 5.5g/L, the content of mannitol is 1.2g/L, Proclin-300, the content of mannitol is 1.4ml/L, the content of latex microspheres is 1.0%, the content of anti-aldosterone monoclonal antibody is 0.8g/L, and the content of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.1 g/L.
The pH of the reagent R1 is 7.50, and the pH of the reagent R2 is 7.50.
A preparation method of an aldosterone latex enhanced detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding tris (hydroxymethyl) aminomethane, mannitol, D-trehalose, Tween 20, polyethylene glycol 6000 and Proclin-300 while stirring, stirring for 25 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 7.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring for 25 minutes until the material is completely dissolved, adjusting the pH value to 7.50, continuously stirring for 7 minutes until the solution is clear and transparent, keeping no precipitate at the bottom of the liquid preparation tank, fixing the volume to the final volume, and storing the solution for later use for marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding glycine, mannitol and Proclin-300 while stirring, stirring for 25 minutes until the materials are completely dissolved, adjusting the pH value to 7.50, continuously stirring for 7 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 1.0% by using a buffer solution, diluting an anti-aldosterone monoclonal antibody to the concentration of 0.8g/L by using the buffer solution, uniformly mixing the latex microspheres and the buffer solution, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.1g/L by using the buffer solution, shaking uniformly and dripping into the mixed solution while stirring, reacting for 180 minutes at room temperature, adding bovine serum albumin for sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking off supernatant, adding a preserving solution, carrying out ultrasonic resuspension, repeating centrifugal cleaning for 2 times, centrifuging the supernatant for the last time, adding the preserving solution, carrying out ultrasonic resuspension, filling prepared R2 into a finished product tank, and marking.
Example three:
the invention discloses an aldosterone latex enhanced detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration: 12.2g/L of tris (hydroxymethyl) aminomethane as a first buffer, 2g/L, D-trehalose as a first stabilizer, 20g/L of polyvinylpyrrolidone as a surfactant at a concentration of 5ml/L, 120g/L of polyethylene glycol 6000 as a first preservative at a concentration of 2.0ml/L of sodium azide;
the reagent R2 consists of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of glycine, casein serving as a second stabilizing agent and sodium azide serving as a second preservative, the latex microsphere antibody conjugate consists of latex microspheres, an anti-aldosterone monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows: the content of 2- (N-morpholine) ethanesulfonic acid monohydrate is 25.30g/L, the content of glycine is 9.83g/L, the content of casein is 2.0g/L, the content of sodium azide is 2.0ml/L, the content of latex microspheres is 2.0%, the content of aldosterone monoclonal antibody is 1.5g/L, and the content of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 0.3 g/L.
The pH of the reagent R1 is 8.50, and the pH of the reagent R2 is 8.50.
A preparation method of an aldosterone latex enhanced detection kit comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding tris (hydroxymethyl) aminomethane, casein, D-trehalose, polyvinylpyrrolidone, polyethylene glycol 6000 and sodium azide while stirring, stirring for 30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 8.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring for 30 minutes until the material is completely dissolved, adjusting the pH value to 8.50, continuously stirring for 10 minutes until the solution is clear and transparent, keeping no precipitate at the bottom of the liquid preparation tank, fixing the volume to the final volume, and storing the solution for later use for marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding glycine, casein and sodium azide while stirring, stirring for 30 minutes until the materials are completely dissolved, adjusting the pH value to 8.50, continuously stirring for 10 minutes until all the materials are completely dissolved, keeping the solution clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 2.0% by using a buffer solution, diluting an anti-aldosterone monoclonal antibody to the concentration of 1.5g/L by using the buffer solution, uniformly mixing the latex microspheres and the buffer solution, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.3g/L by using the buffer solution, shaking uniformly and dripping into the mixed solution while stirring, reacting for 180 minutes at room temperature, adding bovine serum albumin for sealing, shaking uniformly, reacting for 60 minutes at room temperature, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking off supernatant, adding a preserving solution, carrying out ultrasonic resuspension, repeating centrifugal cleaning for 2 times, centrifuging the supernatant for the last time, adding the preserving solution, carrying out ultrasonic resuspension, filling prepared R2 into a finished product tank, and marking.
Example four:
latex microspheres were indirectly coupled to aldosterone and calibrated using standards, the results are shown in Table 1 and FIG. 1
Table 1: the calibration result of the indirect coupling of the latex microspheres with aldosterone is as follows:
selecting a sample to be detected to dilute the sample by using physiological saline, and generating a sample close to the low-limit concentration level of the linear range of the method, wherein the concentration levels are generally 4, and the concentration levels are respectively as follows: 4.0pg/mL, 4.5 pg/mL, 5.0pg/mL, and 5.5 pg/mL. Each concentration level sample is repeatedly measured for 10 times, CV is less than or equal to 8 percent and is taken as an acceptable threshold, and the lowest concentration level with the CV value being equal to or less than the acceptable threshold is selected from the data and is taken as the lower limit of the detection range, and the result is shown in Table 2.
Selecting a sample to be detected to dilute the sample by using physiological saline, and generating a sample close to the high-limit concentration level of the linear range of the method, wherein the concentration levels are generally 4, and the concentration levels are respectively as follows: 800pg/mL, 1000pg/mL, 1200pg/mL, and 1500 pg/mL. Each concentration level sample is repeatedly measured for 10 times, CV is less than or equal to 8 percent as an acceptable threshold value, the highest concentration level with the CV value being equal to or less than the acceptable threshold value is selected from the data as the upper limit of the detection range, and the result is shown in Table 3.
Table 2:
CV > 8% when the sample concentration is below 5.0 pg/mL; CV < 8% when the sample concentration is above 5.0 pg/mL; therefore, the linear range is limited to 5.0 pg/mL.
Table 3:
CV > 8% when the sample concentration is above 1000 pg/mL; CV < 8% when the sample concentration is below 1000 pg/mL; therefore, the linear range is limited to 1000 pg/mL.
In summary, the linear range of the present invention can be made to 5-1000 pg/mL.
Example five:
and (3) precision CV detection:
the latex microspheres are indirectly coupled with aldosterone, the detection is repeated for 10 times on four samples, and the detection results are shown in table 4:
precision CV was small, within 8%.
The prior aldosterone reagents were tested for relevance to the present invention:
the prior reagent and the kit of the invention are used for simultaneously detecting 20 cases of clinical serum according to respective parameters, and correlation analysis is carried out on the determination results, and the results are shown in the figure, wherein R2 is more than 0.95, and the clinical requirements are met.
In summary, the following steps: compared with the prior art, the invention achieves the technical effects that: the invention realizes the detection of the content of aldosterone on a biochemical analyzer for the first time, and the detection range can reach 5-1000 pg/mL;
compared with other detection methods, the method has the characteristics of simple, accurate and rapid operation, and can realize high-throughput detection by batch analysis on a full-automatic biochemical analyzer;
the kit has the characteristics of good stability, high sensitivity, wide linear range, high specificity, good precision and short detection time, and can be widely applied to various fields of basic and clinical medicine. The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (7)
1. An aldosterone latex enhanced detection kit, characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein each component and concentration of the reagent R1 comprises:
the reagent R2 consists of a second buffer solution, a preservation solution and a latex microsphere antibody conjugate, wherein the preservation solution consists of glycine, a second stabilizing agent and a second preservative, the latex microsphere antibody conjugate consists of latex microspheres, an anti-aldosterone monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the concentration of each component is as follows:
2. the aldosterone latex enhanced detection kit of claim 1, wherein: the first buffer solution and the second buffer solution in the reagent R1 and the reagent R2 comprise one or more of PBS buffer solution, HEPES buffer solution, glycine buffer system, MES buffer solution and Tris buffer solution.
3. The aldosterone latex enhanced detection kit of claim 1, wherein: the first stabilizer of the reagent R1 and the second stabilizer of the reagent R2 are one or more of casein, mannitol and bovine serum albumin.
4. The aldosterone latex enhanced detection kit of claim 1, wherein: the surfactant in the reagent R1 is one or a mixture of more than one of Tween 20, Triton, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.
5. The aldosterone latex enhanced detection kit of claim 1, wherein: the first preservative in the reagent R1 and the second preservative in the reagent R2 are respectively one of sodium azide, Proclin-950, Proclin-300 and thimerosal.
6. The aldosterone latex enhanced detection kit of claim 1, wherein: the pH value of the reagent R1 is between 6.00 and 8.50, and the pH value of the reagent R2 is between 6.00 and 8.50.
7. The method for preparing the aldosterone latex enhanced detection kit according to any one of claims 1-6, characterized in that: the method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a first buffer solution, a first stabilizer, D-trehalose, a surfactant, polyethylene glycol 6000 and a first preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.00-8.50, finally fixing the volume to the final volume, storing in a finished product tank, and marking;
B) reagent R2 preparation:
preparation of B1 buffer: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding a second buffer solution while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-8.50, continuously stirring for 5-10 minutes until the solution is clear and transparent, keeping the bottom of the liquid preparation tank free of precipitate, fixing the volume to the final volume, storing the solution for later use, and marking;
preparation of B2 preservative solution: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting a stirrer to a medium-speed rotating speed to keep the solution in a medium-speed rotating state, adding glycine, a second stabilizer and a second preservative while stirring, stirring for 20-30 minutes until the materials are completely dissolved, adjusting the pH value to 6.00-8.50, continuously stirring for 5-10 minutes until all the materials are completely dissolved, enabling the solution to be clear and transparent, enabling the bottom of the liquid preparation tank to have no precipitate, fixing the volume to a final volume, storing the solution for later use, and marking;
b3 preparation of latex microsphere antibody conjugate: diluting latex microspheres to the concentration of 0.1-2.0% by using a buffer solution, diluting an anti-aldosterone monoclonal antibody to the concentration of 0.05-1.5g/L by using the buffer solution, uniformly mixing the two, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the concentration of 0.01-0.3g/L by using the buffer solution, dripping the mixture into the mixed solution while shaking the mixture, reacting the mixture at room temperature for 180 minutes, adding bovine serum albumin for sealing, shaking the mixture for uniformly mixing, reacting the mixture at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14800rpm for 30 minutes, sucking a supernatant, adding a preserving solution, carrying out ultrasonic resuspension, repeating the centrifugal washing for 2 times, centrifuging the supernatant for the last time, adding the preserving solution, carrying out resuspension and ultrasonic treatment, and filling the prepared R2 into a finished product tank for marking.
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| CN (1) | CN110794139A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114137229A (en) * | 2021-12-03 | 2022-03-04 | 苏州普瑞斯生物科技有限公司 | Adiponectin detection reagent production process adopting latex enhanced immunoturbidimetry |
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| CN107589266A (en) * | 2017-08-09 | 2018-01-16 | 上海原科实业发展有限公司 | A kind of VEGF latex enhancing immune is than turbid kit and its application |
| WO2018129885A1 (en) * | 2017-01-13 | 2018-07-19 | 深圳开立生物医疗科技股份有限公司 | Detection kit for whole blood c-reactive protein |
| CN109187997A (en) * | 2018-09-20 | 2019-01-11 | 苏州普瑞斯生物科技有限公司 | A kind of kit and preparation method of the concentration measuring Remnant lipoprotein cholesterol |
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| WO2004047721A2 (en) * | 2002-11-26 | 2004-06-10 | Defense Research & Development Organisation | A process for preparation of an agglutination reagent for rapid detection of typhoid |
| WO2018129885A1 (en) * | 2017-01-13 | 2018-07-19 | 深圳开立生物医疗科技股份有限公司 | Detection kit for whole blood c-reactive protein |
| CN107589266A (en) * | 2017-08-09 | 2018-01-16 | 上海原科实业发展有限公司 | A kind of VEGF latex enhancing immune is than turbid kit and its application |
| CN109187997A (en) * | 2018-09-20 | 2019-01-11 | 苏州普瑞斯生物科技有限公司 | A kind of kit and preparation method of the concentration measuring Remnant lipoprotein cholesterol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114137229A (en) * | 2021-12-03 | 2022-03-04 | 苏州普瑞斯生物科技有限公司 | Adiponectin detection reagent production process adopting latex enhanced immunoturbidimetry |
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