CN111693710A - Troponin I determination kit and preparation method thereof - Google Patents
Troponin I determination kit and preparation method thereof Download PDFInfo
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- CN111693710A CN111693710A CN201910182687.8A CN201910182687A CN111693710A CN 111693710 A CN111693710 A CN 111693710A CN 201910182687 A CN201910182687 A CN 201910182687A CN 111693710 A CN111693710 A CN 111693710A
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- troponin
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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 discloses a troponin I determination kit, which comprises an R2 reagent and an R1 buffer solution, wherein the R2 reagent comprises a conjugate of latex and an antibody. The invention also provides a preparation method of the troponin I determination kit, which comprises the following steps: preparing a conjugate of latex and an antibody, preparing an R2 reagent and preparing an R1 buffer solution; according to the troponin I determination kit provided by the invention, the coupling efficiency of a troponin antibody and latex microspheres is improved through a biotin avidin system, the detection sensitivity is improved, and the coincidence rate of clinical samples is improved when the kit is applied to clinical use; the invention also provides a preparation method of the troponin I determination kit, and during preparation, the particle size of the latex microspheres is selected differently, so that the degree of improving the detection sensitivity is different, and the applicability is wide; meanwhile, the use of the polyethylene glycol with large molecular weight plays a role in promoting polymerization, and the detection sensitivity is also improved.
Description
Technical Field
The invention belongs to the field of troponin I determination, and particularly relates to a troponin I determination kit. Meanwhile, the invention also relates to a preparation method of the troponin I determination kit.
Background
Troponin, consisting of T, C, I tridyads, together with tropomyosin regulates actin and myosin interactions by regulating the activity of calcium ions on striated actin atpase. Troponin complexes are released into the blood after myocardial injury and, after 4-6 hours, begin to rise in the blood, and elevated troponin I can remain in the blood for a long period of 6-10 days. Troponin I has high myocardial specificity and sensitivity, so troponin I has become the most ideal myocardial infarction marker at present.
Troponin I (cTnI I) is used as a Cardiac muscle injury marker, because of its high Cardiac muscle specificity, high sensitivity to Cardiac muscle injury and long window, cTnI has been widely accepted by clinicians and testers, has replaced CK-MB, and has become a "gold standard" for judging Cardiac muscle injury, especially for diagnosing acute myocardial infarction. But also becomes the most suitable marker for judging the risk of myocardial damage of patients with coronary syndrome. Elevation of troponin also serves as strong evidence to support clinicians in making early decisions about anti-thrombosis, anti-platelet aggregation, and interventional therapies.
Immunoturbidimetry (Turbidimetric inhibition assay) is an antigen-antibody binding kinetic assay. The basic principle is as follows: when the antigen and the antibody on the latex react in a buffer solution system and the proportion is proper (generally, the antibody is excessive), the formed soluble immune complex forms particles under the action of a polymerization promoter in the buffer solution system, and the reaction solution generates turbidity. When the antibody concentration is fixed, the amount of the immunocomplex formed increases with the increase in the amount of the antigen in the sample, and the turbidity of the reaction solution also increases. The content of the antigen in the sample can be calculated by measuring the turbidity of the reaction solution and comparing with a series of standard products.
At present, troponin I determination kits (immunoturbidimetry) are composed of two reagents, R1 and R2. R1 is buffer solution, most of which adopts polyethylene glycol as polymerization promoter, and a certain amount of surfactant such as Tween 20 and antiseptic; r2 is a latex reagent, the main component is a conjugate of latex particles and troponin I antibody, and in addition, a certain amount of protein protective agent and preservative are also included. During the test, the full-automatic biochemical analyzer firstly adds R1 and a sample into a cuvette of the biochemical analyzer according to a set program, the sample and R1 are incubated in the cuvette at 37 ℃ for a certain time, then an R2 reagent is added, the absorbance value A1 is tested, the absorbance value A2 is tested after a certain time, and the absorbance difference delta A is calculated to be A2-A1. The relationship between delta A and concentration can be obtained by calibrating the calibrator, and then the quantitative determination of troponin I can be carried out.
Compared with a chemiluminescence troponin I reagent, the troponin I determination kit (immunoturbidimetry) has lower detection sensitivity and low clinical compliance rate (the compliance rate of clinical specimens is less than 45 percent) compared with the chemiluminescence method reagent, the chemiluminescence reagent needs special equipment and has higher cost, and the cost is easy to accept by the immunoturbidimetry. To solve this problem, we propose a highly sensitive troponin I assay kit (immunoturbidimetry).
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a troponin I determination kit.
In order to achieve the purpose, the invention provides the following technical scheme:
a troponin I assay kit comprising an R2 reagent and R1 buffer, the R2 reagent comprising a conjugate of latex and an antibody.
The invention also provides a preparation method of the troponin I determination kit, which comprises the following steps:
preparing a conjugate of latex and an antibody:
s1, diluting a certain amount of carboxyl latex microspheres to a certain concentration by using a PBS buffer solution;
s2, adding EDC solution in proportion, and activating for 0.5 hour at room temperature;
s3, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S1 by using PBS buffer solution;
s4, adding avidin according to a proportion, and activating for 10 hours at room temperature;
s5, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S1 by using PBS buffer solution;
s6, diluting the troponin antibody to a certain concentration by using a PBS buffer solution;
s7, adding NHS solution in proportion, and activating for 0.5h at room temperature;
s8, adding biotin in proportion, and activating for 2 hours at room temperature;
s9, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S6 by using PBS buffer solution;
s10, mixing the reagent obtained after S5 and the reagent obtained after S9 in proportion, and activating for 2 hours at room temperature;
s11, centrifuging, removing supernatant, and redissolving the supernatant to the latex microsphere concentration in the S1 by using a Tris buffer solution to obtain a conjugate of latex and an antibody;
preparing an R2 reagent:
s12, weighing purified water with the volume of about 80 percent;
s13, weighing a certain amount of Tris, Tween 20, a protein protective agent and a preservative, sequentially adding into pure water, stirring, dissolving and uniformly mixing;
s14, adjusting the pH value to an allowable range;
s15, supplementing pure water to the preparation volume;
s16, adding a conjugate of latex and an antibody in proportion to obtain an R2 reagent;
preparing R1 buffer solution:
s17, weighing purified water with the volume of about 80 percent;
s18, weighing a certain amount of Tris, magnesium chloride, polyethylene glycol, Tween 20, a protein protective agent and a preservative, sequentially adding into pure water, stirring, dissolving and uniformly mixing;
s19, adjusting the pH value to an allowable range;
and S20, supplementing pure water to the preparation volume to obtain the R1 reagent.
Preferably, the carboxyl latex microspheres mentioned in S1 have a particle size of 150nm to 400 nm.
Preferably, both the Tris buffer mentioned in S11 and the PBS buffer mentioned in S3 may be replaced with MES buffer or CB buffer, and each buffer concentration is in the range of 5mM to 500 mM.
Preferably, the EDC solution mentioned in S2 needs to be ready for use.
Preferably, the Tris buffer mentioned in S11 contains an amount of tween 20, a protein protecting agent and a preservative.
Preferably, the polyethylene glycol mentioned in the S18 can be replaced by polyethylene glycol 8000 or polyethylene glycol 10000 polymerization promoter.
Preferably, the protein protectant mentioned in S13 is a bovine serum albumin protectant.
Preferably, the preservatives mentioned in S13 and S18 are ProClin300, sodium azide or thimerosal preservatives.
The invention has the technical effects and advantages that: according to the troponin I determination kit provided by the invention, the coupling efficiency of a troponin antibody and latex microspheres is improved through a biotin avidin system, the detection sensitivity is improved, and the coincidence rate of clinical samples is improved when the kit is applied to clinical use; the invention also provides a preparation method of the troponin I determination kit, and during preparation, the particle size of the latex microspheres is selected differently, so that the degree of improving the detection sensitivity is different, and the applicability is wide; meanwhile, the use of the polyethylene glycol with large molecular weight plays a role in promoting polymerization, and the detection sensitivity is also improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. 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
A troponin I assay kit comprising an R2 reagent and R1 buffer, the R2 reagent comprising a conjugate of latex and an antibody. When the troponin I determination kit is prepared, the method comprises the following steps:
preparing a conjugate of latex and an antibody:
s1, diluting a certain amount of carboxyl latex microspheres to a certain concentration by using a PBS buffer solution, wherein the particle size of the carboxyl latex microspheres is 150nm-400 nm;
s2, adding an EDC solution in proportion, and activating at room temperature for 0.5 hour, wherein the EDC solution needs to be prepared for use;
s3, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S1 by using PBS buffer solution;
s4, adding avidin according to a proportion, and activating for 10 hours at room temperature;
s5, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S1 by using PBS buffer solution;
s6, diluting the troponin antibody to a certain concentration by using a PBS buffer solution;
s7, adding NHS solution in proportion, and activating for 0.5h at room temperature;
s8, adding biotin in proportion, and activating for 2 hours at room temperature;
s9, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S6 by using PBS buffer solution;
s10, mixing the reagent obtained after S5 and the reagent obtained after S9 in proportion, and activating for 2 hours at room temperature;
s11, centrifuging, discarding supernatant, and redissolving with Tris buffer solution to the latex microsphere concentration in S1 to obtain a conjugate of latex and an antibody, wherein the Tris buffer solution and the PBS buffer solution can be replaced by MES buffer solution or CB buffer solution, the concentration of each buffer solution is within the range of 5mM to 500mM, and the Tris buffer solution contains a certain amount of Tween 20, a protein protective agent and a preservative;
preparing an R2 reagent:
s12, weighing purified water with the volume of about 80 percent;
s13, weighing a certain amount of Tris, Tween 20, a protein protective agent and a preservative, sequentially adding into pure water, stirring, dissolving and uniformly mixing, wherein the protein protective agent is a bovine serum albumin protective agent;
s14, adjusting the pH value to an allowable range;
s15, supplementing pure water to the preparation volume;
s16, adding a conjugate of latex and an antibody in proportion to obtain an R2 reagent;
preparing R1 buffer solution:
s17, weighing purified water with the volume of about 80 percent;
s18, weighing a certain amount of Tris, magnesium chloride, polyethylene glycol, Tween 20, a protein protective agent and a preservative, sequentially adding into pure water, stirring, dissolving and uniformly mixing, wherein polyethylene glycol can be replaced by polyethylene glycol 8000 or polyethylene glycol 10000 polymerization promoter, and the preservative is ProClin300, sodium azide or thimerosal preservative;
s19, adjusting the pH value to an allowable range;
and S20, supplementing pure water to the preparation volume to obtain the R1 reagent.
In summary, the following steps: according to the troponin I determination kit provided by the invention, the coupling efficiency of a troponin antibody and latex microspheres is improved through a biotin avidin system, the detection sensitivity is improved, and the coincidence rate of clinical samples is improved when the kit is applied to clinical use; the invention also provides a preparation method of the troponin I determination kit, and during preparation, the particle size of the latex microspheres is selected differently, so that the degree of improving the detection sensitivity is different, and the applicability is wide; meanwhile, the use of the polyethylene glycol with large molecular weight plays a role in promoting polymerization, and the detection sensitivity is also improved.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (9)
1. A troponin I assay kit, characterized in that: comprises an R2 reagent and an R1 buffer solution, and the R2 reagent comprises a conjugate of latex and an antibody.
2. A method for preparing the troponin I assay kit according to claim 1, characterized in that: the method comprises the following steps:
preparing a conjugate of latex and an antibody:
s1, diluting a certain amount of carboxyl latex microspheres to a certain concentration by using a PBS buffer solution;
s2, adding EDC solution in proportion, and activating for 0.5 hour at room temperature;
s3, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S1 by using PBS buffer solution;
s4, adding avidin according to a proportion, and activating for 10 hours at room temperature;
s5, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S1 by using PBS buffer solution;
s6, diluting the troponin antibody to a certain concentration by using a PBS buffer solution;
s7, adding NHS solution in proportion, and activating for 0.5h at room temperature;
s8, adding biotin in proportion, and activating for 2 hours at room temperature;
s9, centrifuging, removing supernatant, and re-dissolving the precipitate to a certain concentration mentioned in the S6 by using PBS buffer solution;
s10, mixing the reagent obtained after S5 and the reagent obtained after S9 in proportion, and activating for 2 hours at room temperature;
s11, centrifuging, removing supernatant, and redissolving the supernatant to the latex microsphere concentration in the S1 by using a Tris buffer solution to obtain a conjugate of latex and an antibody;
preparing an R2 reagent:
s12, weighing purified water with the volume of about 80 percent;
s13, weighing a certain amount of Tris, Tween 20, a protein protective agent and a preservative, sequentially adding into pure water, stirring, dissolving and uniformly mixing;
s14, adjusting the pH value to an allowable range;
s15, supplementing pure water to the preparation volume;
s16, adding a conjugate of latex and an antibody in proportion to obtain an R2 reagent;
preparing R1 buffer solution:
s17, weighing purified water with the volume of about 80 percent;
s18, weighing a certain amount of Tris, magnesium chloride, polyethylene glycol, Tween 20, a protein protective agent and a preservative, sequentially adding into pure water, stirring, dissolving and uniformly mixing;
s19, adjusting the pH value to an allowable range;
and S20, supplementing pure water to the preparation volume to obtain the R1 reagent.
3. The method for preparing a troponin I assay kit according to claim 2, wherein: the particle size of the carboxyl latex microsphere mentioned in S1 is 150nm-400 nm.
4. The method for preparing a troponin I assay kit according to claim 2, wherein: both the Tris buffer mentioned in S11 and the PBS buffer mentioned in S3 may be replaced with MES buffer or CB buffer, and each buffer concentration is in the range of 5mM to 500 mM.
5. The method for preparing a troponin I assay kit according to claim 2, wherein: the EDC solution mentioned in S2 needs to be ready for use.
6. The method for preparing a troponin I assay kit according to claim 2, wherein: the Tris buffer mentioned in S11 contains a certain amount of Tween 20, a protein protective agent and a preservative.
7. The method for preparing a troponin I assay kit according to claim 2, wherein: the polyethylene glycol mentioned in the S18 can be replaced by polyethylene glycol 8000 or polyethylene glycol 10000 polymerization promoter.
8. The method for preparing a troponin I assay kit according to claim 2, wherein: the protein protectant mentioned in S13 is bovine serum albumin protectant.
9. The method for preparing a troponin I assay kit according to claim 2, wherein: the preservatives mentioned in S13 and S18 are ProClin300, sodium azide or thimerosal preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910182687.8A CN111693710A (en) | 2019-03-12 | 2019-03-12 | Troponin I determination kit and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910182687.8A CN111693710A (en) | 2019-03-12 | 2019-03-12 | Troponin I determination kit and preparation method thereof |
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| CN111693710A true CN111693710A (en) | 2020-09-22 |
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| CN201910182687.8A Pending CN111693710A (en) | 2019-03-12 | 2019-03-12 | Troponin I determination kit and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189343A (en) * | 2021-03-23 | 2021-07-30 | 北京丹大生物技术有限公司 | Kit for simultaneously detecting retinol binding protein in serum and urine |
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| US20050164317A1 (en) * | 1995-04-18 | 2005-07-28 | Biosite, Inc. | Novel methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays |
| CN104198696A (en) * | 2014-09-04 | 2014-12-10 | 山东博科生物产业有限公司 | Preparation method of sensitization microsphere of troponin I |
| CN106093418A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring Troponin I and preparation method thereof |
| CN107422129A (en) * | 2017-01-15 | 2017-12-01 | 北京科跃中楷生物技术有限公司 | A kind of super quick cardiac muscle troponin I magnetic microsphere immunoturbidimetry detection method and detection kit |
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2019
- 2019-03-12 CN CN201910182687.8A patent/CN111693710A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050164317A1 (en) * | 1995-04-18 | 2005-07-28 | Biosite, Inc. | Novel methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays |
| US20040023309A1 (en) * | 2001-11-27 | 2004-02-05 | Franz Noll | Immunoassay and kit for an early and simultaneous detection of biochemical markers in a patient's sample |
| CN104198696A (en) * | 2014-09-04 | 2014-12-10 | 山东博科生物产业有限公司 | Preparation method of sensitization microsphere of troponin I |
| CN106093418A (en) * | 2016-05-26 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring Troponin I and preparation method thereof |
| CN107422129A (en) * | 2017-01-15 | 2017-12-01 | 北京科跃中楷生物技术有限公司 | A kind of super quick cardiac muscle troponin I magnetic microsphere immunoturbidimetry detection method and detection kit |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189343A (en) * | 2021-03-23 | 2021-07-30 | 北京丹大生物技术有限公司 | Kit for simultaneously detecting retinol binding protein in serum and urine |
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Application publication date: 20200922 |