CN114544977A - Antithrombin III determination kit - Google Patents
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- CN114544977A CN114544977A CN202210300272.8A CN202210300272A CN114544977A CN 114544977 A CN114544977 A CN 114544977A CN 202210300272 A CN202210300272 A CN 202210300272A CN 114544977 A CN114544977 A CN 114544977A
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
- G01N2333/8121—Serpins
- G01N2333/8128—Antithrombin III
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Abstract
The invention belongs to the field of in-vitro detection kits, and particularly relates to an antithrombin III determination kit. The antithrombin III determination kit comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises a buffer solution, a surfactant, a reaction regulator, an anti-interference agent and a preservative, and the reagent 2 comprises a buffer solution, salt ions, a protein protective agent, a surfactant, a preservative and an antithrombin III antibody. The invention has the beneficial effects that: by adding the surfactant, the uniformity of the reagent is improved, and the reaction specificity of the reagent for determining a target object is enhanced by adding the anti-interference agent, so that the accuracy of a reagent determination result is improved; the sensitivity of the reagent and the accuracy of the measured result of the sample are enhanced by adding the reaction regulator; by optimizing the formulas of the protein protective agent and the preservative, the stability of the reagent is improved, the reagent can be stored for a long time, and the production cost is reduced.
Description
Technical Field
The invention belongs to the field of in-vitro detection kits, and particularly relates to an antithrombin III determination kit.
Background
Antithrombin III is mainly synthesized in liver cells and endothelial cells, is a single-chain glycoprotein consisting of 432 amino acid residues, and has the relative molecular mass of 58200 KD. Antithrombin iii is a member of the serine protease inhibitor superfamily, exists primarily in both alpha and beta forms in vivo, and contains 3 beta sheets, 9 alpha helices and 1 RCL, with similar three-dimensional structural conformations. An important difference between antithrombin iii and serine protease inhibitors is that antithrombin iii can increase its inhibitory activity by binding to heparin like molecules.
Antithrombin III is an inhibitor of proteases of thrombin and blood coagulation factors XII α, XI α, IX α, X α and the like. It is bound to thrombin by arginine-serine peptide bonds to form an antithrombin-thrombin complex which inactivates thrombin, and heparin accelerates this reaction by more than a thousand fold. Antithrombin III acts as the most important inhibitor of active blood coagulation factors in the blood, controlling blood coagulation and fibrinolysis. The level of antithrombin III in blood varies depending on various diseases and symptoms, and the concentration of antithrombin III is reduced in diseases such as acute myocardial infarction, disseminated intravascular coagulation, liver diseases, and nephrotic syndrome. Therefore, the concentration of antithrombin III is of great significance as an index for the diagnosis and treatment monitoring and prognosis judgment of the diseases.
At present, most of antithrombin III determination kits sold in the market are S-2238 substrate methods, most of antithrombin III determination kits are freeze-dried dosage forms, a plurality of reagents are formed, the production process and operation are complex, and the cost investment is large.
Disclosure of Invention
The invention aims to provide an antithrombin III determination kit.
An antithrombin III assay kit according to an embodiment of the present invention includes a reagent 1, a reagent 2, wherein,
the reagent 1 comprises buffer solution, surfactant, reaction regulator, preservative and anti-interference agent,
reagent 2 includes buffers, surfactants, reaction modifiers, protein protectants, preservatives, and antithrombin iii antibodies.
The volume ratio of the reagent 1 to the reagent 2 is (1-10): 1, or the volume ratio of the reagent 1 to the reagent 2 is (1-5): 1, preferably, the volume ratio of the reagent 1 to the reagent 2 is 4: 1.
According to the antithrombin III determination kit of the embodiment of the invention, the buffer solution of the reagent 1 is selected from Tris-HCl buffer solution and Na2HPO4-NaH2PO4Buffer solution, MES buffer solution, MOPSO-MOPSO/Na buffer solution, PIPES buffer solution, HEPES buffer solution, Na2HPO4-HCl buffer, Na2HPO4-KH2PO4One of the buffer solutions is used as the buffer solution,
the buffer solution of the reagent 2 is selected from Tris-HCl buffer solution and Na2HPO4-NaH2PO4Buffer solution, MES buffer solution, MOPSO-MOPSO/Na buffer solution, PIPES buffer solution, HEPES buffer solution, Na2HPO4-HCl buffer, Na2HPO4-KH2PO4One kind of buffer solution.
According to the antithrombin III determination kit of the embodiment of the invention, the pH of the buffer solution of the reagent 1 is 6.0-8.0, and the concentration is 10-200 mM; the pH value of the buffer solution of the reagent 2 is 6.0-8.0, and the concentration is 10-200 mM.
According to the antithrombin III determination kit of the embodiment of the invention, in the reagent 1, the surfactant comprises one or more of Tween 20, Tween 80, Triton X-100 and Brij-35, and the surfactant accounts for 0.01-2 wt% of the buffer solution.
According to the antithrombin III determination kit of the embodiment of the invention, in the reagent 2, the surfactant comprises one or more of Tween 20, Tween 80, Triton X-100 and Brij-35, and the surfactant accounts for 0.01-2 wt% of the buffer solution.
According to the antithrombin III determination kit of the embodiment of the invention, in the reagent 1, the reaction regulator comprises a component A and a component B, wherein,
the component A is polyethylene glycol 4000 or polyethylene glycol 6000,
the component B is one or more of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and disodium ethylene diamine tetraacetate;
the reaction regulator accounts for 0.05-40 wt% of the buffer solution.
In the reagent 2, the reaction regulator comprises a component A and a component B, wherein,
the component A is polyethylene glycol 4000 or polyethylene glycol 6000,
the component B is one or more of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and disodium ethylene diamine tetraacetate;
in the reagents 1 and 2, the reaction regulator accounts for 0.05-10 wt% of the buffer solution, the mass ratio of the component A to the component B is 1:0-200, or the mass ratio of the component A to the component B is 1:0.1-200, or the mass ratio of the component A to the component B is 1: 1-100.
According to the antithrombin III determination kit provided by the embodiment of the invention, in the reagent 1, the anti-interference agent is potassium ferrocyanide and a blocking agent, and the anti-interference agent accounts for 0.01-1 wt% of the buffer solution.
Preferably, the blocking agent is an heterophilic antibody blocking agent, and the mass ratio of the potassium ferrocyanide to the blocking agent is 1: 0.05-80.
The potassium ferrocyanide can react with bilirubin to remove interference of bilirubin; the blocking agent is combined with the rheumatoid factor to block the combination of the rheumatoid factor and the antithrombin III antibody, thereby eliminating the interference of the rheumatoid factor on the test.
According to the antithrombin III determination kit of the embodiment of the invention, in the reagent 2, the protein protective agent is one or more of glycerol, bovine serum albumin, glycine, trehalose, mannitol or sucrose; the protein protective agent accounts for 0.05-30 wt% of the buffer solution.
According to the antithrombin III determination kit provided by the embodiment of the invention, in the reagent 1, the preservative is one or more of sodium azide, ProClin300, gentamicin sulfate and thimerosal, and the preservative accounts for 0.01-0.5 wt% of the buffer solution;
in the reagent 2, the preservative is at least 2 of sodium azide, ProClin300, gentamicin sulfate and thimerosal, and accounts for 0.01-0.5 wt% of the buffer solution.
According to an embodiment of the present invention, the antithrombin III antibody includes one or more of goat anti-human antithrombin III antiserum antibody, rabbit anti-human antithrombin III antiserum antibody, mouse anti-human antithrombin III antibody, and the like.
The use method of the kit comprises the following steps:
the test conditions are as follows:
the method comprises the following operation steps:
and on a full-automatic biochemical analyzer, calibrating the reagent according to the test conditions and the operation steps, drawing a concentration-absorbance change value curve, and calculating the concentration of antithrombin III in the sample according to the absorbance change value of the sample when the sample is tested.
The invention has the beneficial effects that:
the antithrombin III determination kit disclosed by the invention has the advantages that the uniformity of the reagent is improved by adding the surfactant; the reaction specificity of the reagent for measuring the target object is enhanced by adding the anti-interference agent, so that the accuracy of the reagent measurement result is improved; the sensitivity of the reagent is enhanced by adding the reaction regulator, and the accuracy of the measurement result of the low-end sample (the low-end sample mainly refers to a sample lower than 100) is improved.
The reaction regulator can regulate the reactivity of the reagent test samples, namely absorbance change values, for example, when different formulas are used for testing samples with the same concentration of about 75, the reactivity (absorbance change value delta A) of one reagent is 0.1200, the reactivity of one reagent is 0.0900, and when the reagent with the reactivity of 0.1200 is used for testing samples with the concentration of less than 75, the accuracy is higher, because the reactivity divided into each concentration unit is larger (0.0016).
According to the invention, glycerol/bovine serum albumin, saccharides and a preservative are added together, so that the antibody protein structure can be protected, the degradation of the antibody protein structure can be prevented, the microbial propagation can be inhibited, and a better broad-spectrum antibacterial effect can be achieved compared with a single preservative, so that the glycerol/bovine serum albumin, the saccharides and the preservative have a synergistic effect, and the stability of the reagent can be better improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The antithrombin III determination kit comprises a reagent 1 and a reagent 2, wherein
Reagent 1:
10-200 mM buffer (pH6.0-8.0): Tris-HCl buffer, Na2HPO4-NaH2PO4Buffer solution, MES buffer solution, MOPSO-MOPSO/Na buffer solution, PIPES buffer solution, HEPES buffer solution, Na2HPO4-HCl buffer or Na2HPO4-KH2PO4A buffer solution;
surfactant (b): one or more of Tween 20, Tween 80, Triton X-100 and Brij-35; the buffer solution accounts for 0.01-2 wt% of the buffer solution;
reaction regulator: the component A is polyethylene glycol 4000 and polyethylene glycol 6000
The component B is one or more of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and disodium ethylene diamine tetraacetate;
the reaction regulator accounts for 0.05-5 wt% of the buffer solution;
anti-interference agent: comprises potassium ferrocyanide and a blocking agent, wherein the anti-interference agent accounts for 0.01-1 wt% of the buffer solution
Preservative: one or more of sodium azide, ProClin300, gentamicin sulfate and thimerosal, wherein the preservative accounts for 0.01-0.5 wt% of the buffer solution.
Reagent 2:
10-200 mM buffer (pH6.0-8.0): Tris-HCl buffer, Na2HPO4-NaH2PO4Buffer, MES buffer, MOPSO-MOPSO/Na buffer, PIPES buffer, HEPES buffer, Na2HPO4-HCl buffer, or Na2HPO4-KH2PO4A buffer solution;
surfactant (b): one or more of Tween 20, Tween 80, Triton X-100 and Brij-35; the buffer solution accounts for 0.01-2 wt% of the buffer solution;
reaction regulator: the component A is polyethylene glycol 4000 and polyethylene glycol 6000
The component B is one or more of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and disodium ethylene diamine tetraacetate;
the reaction regulator accounts for 0.05-5 wt% of the buffer solution;
protein protective agent: one or more of glycerol, bovine serum albumin, glycine, trehalose, mannitol or sucrose, wherein the protein protective agent accounts for 0.05-30 wt% of the buffer solution
Preservative: one or more of sodium azide, ProClin300, gentamicin sulfate and thimerosal, wherein the preservative accounts for 0.01-0.5 wt% of the buffer solution;
an anti-thrombin III antibody.
Example 1
1.1 the antithrombin III assay kit of the invention, comprising:
reagent 1:
the buffer was HEPES buffer, pH 6.5, buffer concentration 100 mM.
The surfactant comprises Tween 20 and Triton X-100, wherein the Tween 20 accounts for 0.02 wt% of the buffer solution, and the Triton X-100 accounts for 0.10 wt% of the buffer solution;
the reaction regulator is polyethylene glycol 6000, potassium chloride and sodium sulfate, wherein the polyethylene glycol 6000 accounts for 2.00 wt% of the buffer solution, the potassium chloride accounts for 0.05 wt% of the buffer solution, and the sodium sulfate accounts for 0.60 wt% of the buffer solution;
the anti-interference agent is potassium ferrocyanide and an isophilic antibody blocking agent, the potassium ferrocyanide accounts for 0.5 wt% of the buffer solution, and the isophilic antibody blocking agent accounts for 0.02 wt% of the buffer solution;
the preservative is gentamicin sulfate and sodium azide, the gentamicin sulfate accounts for 0.01 wt% of the buffer solution, and the sodium azide accounts for 0.2 wt% of the buffer solution.
Reagent 2:
the buffer solution is Tris-HCl buffer solution, the pH value is 8.0, and the concentration of the buffer solution is 20 mM;
the surfactant comprises Tween 20 and Triton X-100, wherein the Tween 20 accounts for 0.02 wt% of the buffer solution, and the Triton X-100 accounts for 0.10 wt% of the buffer solution;
the reaction regulator comprises potassium chloride and sodium sulfate, wherein the potassium chloride accounts for 0.05 wt% of the buffer solution, and the sodium sulfate accounts for 0.60 wt% of the buffer solution;
the protein protective agent comprises glycine, glycerol and bovine serum albumin, wherein the glycine accounts for 5.00 wt% of the buffer solution, the glycerol accounts for 10.00 wt% of the buffer solution, and the bovine serum albumin accounts for 0.80 wt% of the buffer solution;
the preservative is gentamicin sulfate and sodium azide, the gentamicin sulfate accounts for 0.01 wt% of the buffer solution, and the sodium azide accounts for 0.20 wt% of the buffer solution;
the antithrombin III antibody is a goat anti-human antithrombin III antiserum antibody, and accounts for 15.00 wt% of the buffer solution.
1.2 the antithrombin III determination kit of the invention, including reagent 1 and reagent 2:
reagent 1:
the buffer solution is Na2HPO4-NaH2PO4Buffer, pH 7.0, buffer concentration 50 mM.
The surfactant comprises Tween 80 and Brij-35, wherein the Tween 80 accounts for 0.01 wt% of the buffer, and the Brij-35 accounts for 0.30 wt% of the buffer;
the reaction regulator comprises polyethylene glycol 4000, sodium chloride and potassium bicarbonate, wherein the polyethylene glycol 4000 accounts for 4.00 wt% of the buffer solution, the sodium chloride accounts for 2.00 wt% of the buffer solution, and the potassium bicarbonate accounts for 0.20 wt% of the buffer solution;
the anti-interference agent is potassium ferrocyanide and an isophilic antibody blocker, the potassium ferrocyanide accounts for 0.3 wt% of the buffer solution, and the isophilic antibody blocker accounts for 0.05 wt% of the buffer solution;
the preservative is ProClin300 and sodium azide, the ProClin300 accounts for 0.10 wt% of the buffer, and the sodium azide accounts for 0.10 wt% of the buffer.
Reagent 2:
the buffer solution is Na2HPO4-NaH2PO4Buffer, pH 7.0, buffer concentration 50 mM.
The surfactant comprises Tween 80 and Brij-35, wherein the Tween 80 accounts for 0.01 wt% of the buffer, and the Brij-35 accounts for 0.30 wt% of the buffer;
the reaction regulator is sodium chloride and accounts for 2.00 wt% of the buffer solution;
the protein protective agent is glycerol and sucrose, wherein the glycerol accounts for 25.00 wt% of the buffer solution, and the sucrose accounts for 2.00 wt% of the buffer solution;
the preservative is ProClin300 and sodium azide, the ProClin300 accounts for 0.10 wt% of the buffer solution, and the sodium azide accounts for 0.10 wt% of the buffer solution;
the antithrombin III antibody is a goat anti-human antithrombin III antiserum antibody, and accounts for 20.00 wt% of the buffer solution.
1.3 the antithrombin III determination kit of the invention, including reagent 1 and reagent 2:
reagent 1:
the buffer solution is MOPSO-MOPSO/Na buffer solution, the pH value is 7.0, and the concentration of the buffer solution is 100 mM;
the surfactant comprises Triton X-100, Brij-35 and Tween 80, wherein the Triton X-100 accounts for 1.50 wt% of the buffer, the Brij-35 accounts for 0.10 wt% of the buffer, and the Tween 80 accounts for 0.02 wt% of the buffer;
the reaction regulator is polyethylene glycol 6000 and disodium ethylene diamine tetraacetate, the polyethylene glycol 6000 accounts for 8.00 wt% of the buffer solution, and the disodium ethylene diamine tetraacetate accounts for 0.25 wt% of the buffer solution;
the anti-interference agent is potassium ferrocyanide and an isophilic antibody blocking agent, the potassium ferrocyanide accounts for 0.10 wt% of the buffer solution, and the isophilic antibody blocking agent accounts for 0.01 wt% of the buffer solution;
the preservative is ProClin300 and thimerosal, the ProClin300 accounts for 0.02 wt% of the buffer solution, and the thimerosal accounts for 0.01 wt% of the buffer solution.
Reagent 2:
the buffer solution is MOPSO-MOPSO/Na buffer solution, the pH value is 7.0, and the concentration of the buffer solution is 100 mM;
the surfactant comprises Triton X-100, Brij-35 and Tween 80, wherein the Triton X-100 accounts for 1.50 wt% of the buffer solution, the Brij-35 accounts for 0.10 wt% of the buffer solution, and the Tween 80 accounts for 0.02 wt% of the buffer solution;
the reaction regulator comprises disodium ethylene diamine tetraacetate and potassium chloride, wherein the disodium ethylene diamine tetraacetate accounts for 0.10 wt% of the buffer solution, and the potassium chloride accounts for 5.00 wt% of the buffer solution;
the protein protective agent is trehalose and glycerol, wherein the trehalose accounts for 3.00 wt% of the buffer solution, and the glycerol accounts for 20.00 wt% of the buffer solution;
the preservative is ProClin300 and thimerosal, the ProClin300 accounts for 0.02 wt% of the buffer solution, and the thimerosal accounts for 0.01 wt% of the buffer solution;
the antithrombin III antibody is a goat anti-human antithrombin III antiserum antibody and accounts for 30.00 wt% of the buffer solution.
Example 2 verification of the efficacy of the kits of the invention
2.1 homogeneity test
Reagent preparation was performed according to 3 formulations (1.1, 1.2 and 1.3) in example 1 of the present invention and comparative examples 1, 2 and 3, and the produced reagents were tested simultaneously with commercial reagents on Hitachi 3500 full-automatic biochemical analyzer for two samples of serum at levels (level 1 being normal (180-
Value (x), Standard Deviation (SD), and Coefficient of Variation (CV).
Comparative example 1: the other ingredients were the same as in example 1.1, except that no surfactant was added.
Comparative example 2: the surfactant was sodium dodecylbenzenesulfonate, concentration 0.10 wt%, other ingredients were the same as in example 1.1.
Comparative example 3: the surfactant was polyoxyethylene tridecyl ether with a concentration of 0.10 wt%, and the other ingredients were the same as in example 1.1.
The uniformity measurement results are shown in table 1.
Table 1 repeatability test results comparison table
As can be seen from Table 1, the Coefficient of Variation (CV) values of the reproducibility test results of the reagent of the present invention were not more than 1.36%, which was smaller than those of the comparative examples 1-3 and the commercially available reagents, indicating that the reagents of the present invention had better reproducibility.
2.2 anti-interference test
According to the invention, 3 formulas (1.1, 1.2 and 1.3) in the example 1 and the formula in the comparative example 2 are used for preparing and producing the reagent, and the produced reagent is subjected to an anti-interference test on a Hitachi 3500 full-automatic biochemical analyzer.
Comparative example 4: the components are the same as those in example 1.1 except that no anti-interference agent is added.
Comparative example 5: the anti-interference agent is potassium ferrocyanide with the concentration of 0.50 wt%, and other components are the same as those in example 1.1.
Comparative example 6: the anti-interference agent is heterophilic antibody blocking agent with concentration of 0.02 wt%, and other components are the same as in example 1.1.
The test method comprises the following steps: and collecting a mixed plasma sample, fully mixing the mixed plasma sample, and averagely dividing the mixed plasma sample into three parts.
Control samples: the ratio of the serum to the interferent is 19:1, and the interferent comprises 0.1mol/L NaOH solution, 1200mg/dL bilirubin and 4000IU/mL rheumatoid factor;
BIL interference sample: 60mg/dL Bilirubin (BIL);
RF interference samples: rheumatoid Factor (RF) at 200 IU/mL.
The prepared samples were tested 3 times with 3 formulations from example 1 (1.1, 1.2 and 1.3) and comparative examples 4, 5 and 6, respectively. And comparing the relative deviation of the measurement results of the control sample and the interference sample, and evaluating the anti-interference performance of the reagent.
The anti-interference test results are shown in table 2.
TABLE 2 anti-interference test results of the reagents of the invention
As shown in Table 2, the relative deviation of the test result of the interference sample and the control sample in the anti-interference test of the reagent is less than or equal to 1.98 percent, while the relative deviation of the test result of the interference sample and the control sample in the anti-interference test of the reagents of comparative examples (4, 5 and 6) is more than or equal to 4.03 percent, and the anti-interference performance is far lower than that of the reagent of the invention. Therefore, the kit can effectively improve the anti-interference capability of the reagent by adding the anti-interference agent.
2.3 comparison of the stability of the detection kit of the present invention with that of the comparative example and commercial products
Accelerated stability: 3 formulations (1.1, 1.2 and 1.3), comparative example 7, comparative example 8, comparative example 9, comparative example 10 and a commercially available reagent in example 1 were simultaneously subjected to accelerated treatment at 37 ℃, a part of samples were taken at intervals to perform quality control tests, the test results were averaged, the variation tendency of the average of the multiple test results was compared, and accelerated stability of the kit, comparative example and commercially available reagent of the present invention was analyzed.
The shelf life stability is as follows: 3 formulations (1.1, 1.2 and 1.3) in example 1 and comparative examples 5-8 are stored at 2-8 ℃ at the same time, a part of the reagents are taken out at intervals for quality control test, the average value of the test results is calculated, the change trend of the average value of the multiple test results is compared, and the shelf life stability of the kit, the comparative examples and the commercially available reagents of the invention is analyzed.
Comparative example 7: the protein protectant is bovine serum albumin with a concentration of 8.00 wt%, the preservative is ProClin300 with a concentration of 0.10 wt%, and the other ingredients are the same as in example 1.2.
Comparative example 8: the protein protective agent is glycine with the concentration of 5.00 wt%, the preservative is gentamicin sulfate with the concentration of 0.01 wt%, and other components are the same as in example 1.2.
Comparative example 9: the preservative was ProClin300 with a concentration of 0.10 wt% without addition of a protein protectant, and the other ingredients were the same as in example 1.2.
Comparative example 10: the protein protectant is glycerol with a concentration of 30.00 wt%, no preservative is added, and other ingredients are the same as in example 1.2.
The stability results are shown in table 3.
TABLE 3 comparison of the stability of the reagents according to the invention with that of the commercially available reagents
As shown in table 3, it can be seen from the accelerated stability data that the quality control test result of the comparative example shows a continuously decreasing trend, the commercially available reagent has a decreasing trend from day 7, and the change trend is very significant after day 7; the quality control test results of the reagent of the invention after accelerated treatment for 10 days are all better, and the change trend is not obvious. As can be seen from the stability of the expiration date, the quality control results of the stability tests of different comparative examples have different changes after 12 months of storage, while the quality control measurement results of the reagent of the invention after 12 months of storage have insignificant changes, which indicates that the reagent of the invention has good stability. Thus, it is demonstrated that the combination of a protein protectant and a preservative can improve the stability of the agents of the invention.
2.4 testing of specimens with the test kit of the present invention
Clinical specimens were simultaneously measured for 3 formulations (1.1, 1.2 and 1.3), comparative example 11, comparative example 12 and commercially available reagents in example 1, and the samples of the reagents of the present invention, comparative examples (9-10) and commercially available products were compared for consistency.
Comparative example 11: the reaction regulator was polyethylene glycol 6000 at a concentration of 8.00 wt%, and the other ingredients were the same as in example 1.3.
Comparative example 12: the reaction modifier was potassium chloride at a concentration of 5.00 wt%, and the other ingredients were the same as in example 1.3.
The results of the clinical sample measurements are shown in table 4.
TABLE 4 results of the measurement of clinical specimens of the inventive and comparative examples and commercial products
As shown in Table 4, the relative deviation of the result of the antithrombin III of the sample measured by the reagent of the invention and the result of the antithrombin III of the sample measured by the commercial product is between-4.38% and 4.64%, and the relative deviation of the result of the antithrombin III of the sample measured by the reagent of the comparative example 11 and the result of the reagent of the comparative example 12 and the result of the antithrombin III of the sample measured by the commercial product is between-10.10% and 14.92% and-24.28% and 22.86%, respectively, the conformity of the measurement result of the reagent of the invention is obviously better than that of the sample measured by the comparative example, which shows that the addition of a reaction regulator (a combination of a surfactant and a salt ion) can regulate the result of the reagent test sample, correct abnormal data and enable the result of the clinical sample tested by the reagent formula to be more accurate.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. An antithrombin III determination kit, which is characterized by comprising a reagent 1 and a reagent 2, wherein,
the reagent 1 comprises buffer solution, surfactant, reaction regulator, preservative and anti-interference agent,
reagent 2 includes buffers, surfactants, reaction modifiers, protein protectants, preservatives, and antithrombin iii antibodies.
2. The antithrombin III assay kit according to claim 1, wherein the buffer of reagent 1 and the buffer of reagent 2 are each independently selected from Tris-HCl buffer, Na2HPO4-NaH2PO4Buffer solution, MES buffer solution, MOPSO-MOPSO/Na buffer solution, PIPES buffer solution, HEPES buffer solution, Na2HPO4-HCl buffer, Na2HPO4-KH2PO4One kind of buffer solution.
3. The antithrombin III assay kit according to claim 1 or 2, wherein the buffer solution of the reagent 1 has a pH of 6.0 to 8.0 and a concentration of 10 to 200 mM; the pH value of the buffer solution of the reagent 2 is 6.0-8.0, and the concentration is 10-200 mM.
4. The antithrombin III assay kit according to claim 1, wherein in the reagent 1, the surfactant comprises one or more of Tween 20, Tween 80, Triton X-100, Brij-35, and the surfactant accounts for 0.01 to 2 wt% of the buffer.
5. The antithrombin III assay kit according to claim 1, wherein in the reagent 2, the surfactant comprises one or more of Tween 20, Tween 80, Triton X-100, Brij-35, and the surfactant accounts for 0.01 to 2 wt% of the buffer.
6. The antithrombin III assay kit according to claim 1, wherein in the reagent 1, the reaction modifier comprises an A component and a B component, wherein,
the component A is polyethylene glycol 4000 or polyethylene glycol 6000,
the component B is one or more of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and disodium ethylene diamine tetraacetate;
the reaction regulator accounts for 0.05-10 wt% of the buffer solution.
7. The antithrombin III assay kit according to claim 1, wherein in the reagent 2, the reaction modifier comprises an A component and a B component, wherein,
the component A is polyethylene glycol 4000 or polyethylene glycol 6000,
the component B is one or more of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate and disodium ethylene diamine tetraacetate;
the reaction regulator accounts for 0.05-40 wt% of the buffer solution.
8. The antithrombin III assay kit according to claim 1, wherein in the reagent 1, the anti-interference agent is potassium ferrocyanide and a blocking agent, and the anti-interference agent accounts for 0.01 to 1 wt% of the buffer solution.
9. The antithrombin III assay kit according to claim 1, wherein in reagent 2, the protein protectant is one or more of glycerol, bovine serum albumin, glycine, trehalose, mannitol, or sucrose; the protein protective agent accounts for 0.05-30 wt% of the buffer solution.
10. The antithrombin III assay kit according to claim 1, wherein in reagent 1, the preservative is at least 2 of sodium azide, ProClin300, gentamicin sulfate and thimerosal, and the preservative accounts for 0.01 to 0.5 wt% of the buffer; in the reagent 2, the preservative is at least 2 of sodium azide, ProClin300, gentamicin sulfate and thimerosal, and accounts for 0.01-0.5 wt% of the buffer solution.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210300272.8A CN114544977A (en) | 2022-03-25 | 2022-03-25 | Antithrombin III determination kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210300272.8A CN114544977A (en) | 2022-03-25 | 2022-03-25 | Antithrombin III determination kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703252A (en) * | 2022-06-02 | 2022-07-05 | 深圳市帝迈生物技术有限公司 | Kit for detecting content of hirudin, bivalirudin and dabigatran in blood plasma |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703252A (en) * | 2022-06-02 | 2022-07-05 | 深圳市帝迈生物技术有限公司 | Kit for detecting content of hirudin, bivalirudin and dabigatran in blood plasma |
| CN114703252B (en) * | 2022-06-02 | 2022-11-04 | 深圳市帝迈生物技术有限公司 | Kit for detecting content of hirudin, bivalirudin and dabigatran in blood plasma |
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