CN115078739A - Kit for detecting apolipoprotein A1 and application thereof - Google Patents
Kit for detecting apolipoprotein A1 and application thereof Download PDFInfo
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- CN115078739A CN115078739A CN202211002286.8A CN202211002286A CN115078739A CN 115078739 A CN115078739 A CN 115078739A CN 202211002286 A CN202211002286 A CN 202211002286A CN 115078739 A CN115078739 A CN 115078739A
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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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/775—Apolipopeptides
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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Abstract
The invention relates to the technical field of detection reagents, in particular to a kit for detecting apolipoprotein A1 and application thereof. The Gemini surfactant has stronger selectivity on the active site of the apolipoprotein A1 antibody, and can expose the active site of the apolipoprotein A1 antibody to the maximum extent, thereby improving the activity and specificity of the antibody in the reagent for detecting the apolipoprotein A1, and a kit prepared by the Gemini surfactant can solve the problem that the detection result in the market has false positive or has a high deviation value.
Description
Technical Field
The invention relates to the technical field of detection reagents, in particular to a kit for detecting apolipoprotein A1 and application thereof.
Background
Apolipoprotein a1 is the major structural protein of high density lipoprotein, and can transport cholesterol in cells to liver for processing, and slow down and prevent the occurrence and development of atherosclerosis. Can effectively reduce the risks of diabetes, atherosclerosis, hyperlipoproteinemia, acute and chronic hepatitis B, alcoholic liver disease, fatty liver, liver cirrhosis, liver cancer and other diseases.
The current methods for detecting apolipoprotein A1 mainly comprise a scattering immunoassay method, a transmission immunoturbidimetry method and the like. Among them, the transmission immunoturbidimetry is receiving wide attention due to its high sensitivity, good specificity and wide linear range. However, most of the apolipoprotein A1 reagents in the current market are easily interfered to generate false positive, the detection result is influenced, and the treatment is delayed. The false positive is mainly caused by the fact that human serum contains APOA1-APOC3-APOA4-APOA5, the four antigen gene clusters are located in the region of chromosome 11 q23-q24, and are evolved from the same progenitor gene and have high homology, and further cross reaction can be generated, so that the false positive or the measured value is higher.
Disclosure of Invention
In order to solve the above problems, the present invention provides a kit for detecting apolipoprotein A1 and applications thereof. The invention utilizes the function that the Gemini surfactant can improve the activity and specificity of the apolipoprotein A1 antibody, and solves the problem that the detection result on the market has false positive or a higher measured value.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an application of a Gemini surfactant in improving the activity and/or specificity of an apolipoprotein A1 antibody.
The invention also provides a kit for detecting the apolipoprotein A1, which comprises an R1 reagent and an R2 reagent which are independently packaged, wherein the R2 reagent is a mixed reagent of a mother solution and an apolipoprotein A1 antibody; the composition of the mother liquor is the same as that of the R1 reagent;
the R1 reagent comprises a buffer solution, a sensitizer, an electrolyte, a preservative and an active agent;
the active agent is a Gemini surfactant or a mixture of the Gemini surfactant and other surfactants;
when the active agent is a Gemini surfactant, the mass content of the Gemini surfactant in the R1 reagent is 0.05% -0.10%;
when the active agent is a mixture of a Gemini surfactant and other surfactants, the mass content of the Gemini surfactant in the R1 reagent is 0.02-0.04%, and the mass content of the other surfactants in the R1 reagent is 1-2%.
Preferably, the R1 reagent also comprises a stabilizer; the stabilizer comprises EDTA-2Na and/or glycerol.
Preferably, the mass content of the EDTA-2Na in the R1 reagent is 0.02% -0.04%; the mass content of the glycerol in the R1 reagent is 7% -8%.
Preferably, the buffer comprises: HEPES buffer, MES buffer, or TRIS buffer; the pH value of the buffer solution is 6.5-9.
Preferably, the sensitizer comprises one or more of polyethylene glycol 6000, polyethylene glycol 20000 and polyethylene glycol 35000; the mass content of the sensitizer in the R1 reagent is 2% -5%.
Preferably, the electrolyte comprises sodium chloride and/or potassium chloride; the mass content of the electrolyte in the R1 reagent is 0.6-0.8%.
Preferably, the preservative comprises sodium azide and/or proclin 300; the mass content of the preservative in the mother liquor is 0.1-0.2%.
Preferably, the content of the apolipoprotein A1 antibody in the R2 reagent is 25-40% by volume.
The invention also provides application of the kit in detection of apolipoprotein A1.
Has the advantages that:
the invention provides an application of a Gemini surfactant in improving the activity and/or specificity of an apolipoprotein A1 antibody. The Gemini surfactant has stronger selectivity on the active site of the apolipoprotein A1 antibody, and can expose the active site of the apolipoprotein A1 antibody to the maximum extent, thereby improving the activity and specificity of the antibody in the reagent for detecting the apolipoprotein A1, and a kit prepared by the Gemini surfactant can solve the problem that the detection result in the market has false positive or has a high deviation value.
Detailed Description
The invention provides an application of a Gemini surfactant in improving the activity and/or specificity of an apolipoprotein A1 antibody. The Gemini surfactant has stronger selectivity on the active site of the apolipoprotein A1 antibody, and can expose the active site of the apolipoprotein A1 antibody to the maximum extent, thereby improving the activity and specificity of the antibody in the reagent for detecting the apolipoprotein A1.
The invention also provides a kit for detecting the apolipoprotein A1, which comprises an R1 reagent and an R2 reagent which are independently packaged, and a mixed reagent of the R2 reagent mother liquor and the apolipoprotein A1 antibody; the composition of the mother liquor is the same as that of the R1 reagent;
the R1 reagent comprises a buffer solution, a sensitizer, an electrolyte, a preservative and an active agent.
The present invention does not require any particular source for each component of the kit unless otherwise specified, and commercially available products well known to those skilled in the art may be used.
The R1 reagent comprises a buffer solution. In the present invention, the buffer preferably includes: HEPES buffer, MES buffer, or TRIS buffer, more preferably TRIS buffer; the concentration of the buffer solution is preferably 50-150 mmol/kg, more preferably 70-130 mmol/kg, and even more preferably 100 mmol/kg; the pH value of the buffer solution is preferably 6.5-9, more preferably 7.5-8.5, and even more preferably 8.0.
The R1 reagent provided by the invention comprises a sensitizer. In the invention, the mass percentage of the sensitizer in the R1 reagent is preferably 2-5%, more preferably 3-5%, and even more preferably 5%. In the present invention, the sensitizer preferably includes one or more of polyethylene glycol 6000, polyethylene glycol 20000, and polyethylene glycol 35000, and more preferably polyethylene glycol 6000. The sensitizer of the invention is helpful for improving the reaction sensitivity and linearity of the reagent.
The R1 reagent of the present invention includes an electrolyte. In the invention, the mass percentage of the electrolyte in the R1 reagent is preferably 0.6-0.8%, and more preferably 0.8%. In the present invention, the electrolyte preferably includes sodium chloride and/or potassium chloride, more preferably sodium chloride. The electrolytes of the present invention facilitate the reagents to provide physiological conditions for antigen-antibody reactions.
The R1 reagent of the present invention includes a preservative. In the invention, the mass content of the preservative in the R1 reagent is preferably 0.1-0.2%, and more preferably 0.1%. In the present invention, the preservative preferably comprises sodium azide and/or proclin300, more preferably sodium azide. The preservative disclosed by the invention is beneficial to long-term preservation of the reagent, and avoids the propagation of microorganisms so as to influence the performance of the reagent.
The R1 reagent of the present invention includes an active agent. The active agent is a Gemini surfactant or a mixture of the Gemini surfactant and other surfactants;
when the active agent is a Gemini surfactant, the mass content of the Gemini surfactant in the R1 reagent is 0.05-0.10%, and preferably 0.05%;
when the active agent is a mixture of a Gemini surfactant and other surfactants, the mass content of the Gemini surfactant in the R1 reagent is 0.02-0.04%, preferably 0.02%; the mass content of the other surfactants in the R1 reagent is 1-2%, and the preferred mass content is 1%.
In the present invention, the other surfactant preferably comprises tween 20 and/or triton x-100, more preferably tween 20.
The Gemini surfactant can improve the activity and specificity of the antibody in the reagent for detecting the apolipoprotein A1, thereby solving the problem that the detection result on the market has false positive or a higher measured value; considering that the cost of the Gemini surfactant is high, the cost can be reduced under the condition of ensuring the detection accuracy by compounding a proper amount of the Gemini surfactant and other surfactants with low prices.
The R1 reagent of the invention preferably further comprises a stabilizer. In the present invention, the stabilizer preferably includes EDTA-2Na and/or glycerin; more preferably a mixture of EDTA-2Na and glycerol. In the invention, the mass content of EDTA-2Na in the R1 reagent is preferably 0.02-0.04%, and more preferably 0.02%; the mass content of the glycerol in the R1 reagent is 7-8%, and more preferably 8%.
According to the invention, by adding a proper reagent as a stabilizer into the R1 reagent, the viscosity can be increased, intermolecular collision can be reduced, protein aggregation can be effectively prevented from causing precipitation, the effect of eliminating the precipitation can be achieved without centrifugation, and the problem that the use effect is influenced because the existing detection reagent is easy to precipitate in different degrees is solved.
The R2 reagent is a mixed reagent of mother liquor and an apolipoprotein A1 antibody; the composition of the mother liquor is the same as that of the R1 reagent, and the details are not repeated. In the invention, the volume percentage content of the apolipoprotein A1 antibody in the R2 reagent is preferably 25-40%, more preferably 30-38%, and more preferably 35%.
In the present invention, the mass ratio of the R1 reagent and the R2 reagent packaged separately in the kit is preferably 5: 1.
the R1 solution is directly used as the dilution mother solution of the R2 solution, so that the production operation steps can be greatly simplified, and the method has important significance for the quantification of production.
The invention preferably also provides a preparation method of the kit, which comprises the following steps:
mixing the components of the R1 reagent, adjusting the pH, and filtering the mixture by using a 0.4 mu m filter membrane to obtain the R1 reagent;
mixing a part of R1 reagent and apolipoprotein A1 antibody to obtain the R2 reagent;
and subpackaging the R1 reagent and the R2 reagent according to a proportion to obtain the kit.
In the invention, the step-by-step mixing sequence has no special requirements, and only the used materials are completely dissolved. In the present invention, the method for mixing the components of the R1 reagent preferably comprises: mixing water with a preparation raw material of a buffer solution to obtain the buffer solution;
mixing the buffer solution with a sensitizer to obtain a first mixed solution;
mixing the first mixed solution with electrolyte to obtain a second mixed solution;
mixing the second mixed solution with a preservative to obtain a third mixed solution;
mixing the third mixed solution with an active agent to obtain a fourth mixed solution;
and mixing the fourth mixed solution with a stabilizing agent to obtain a primary R1 reagent.
In the present invention, the water preferably includes purified water.
In the present invention, the mass ratio of the partial R1 reagent to the prepared R1 reagent is preferably 0.13: 1.13.
Compared with the prior art, the kit provided by the invention has the characteristics of high sensitivity, strong specificity and good stability, and has better clinical prospect. The kit is simple to prepare, is easy for industrial production, and can efficiently detect the content of APOA1 in blood.
The invention also provides application of the kit in detection of apolipoprotein A1.
The kit for detecting apolipoprotein A1 according to the present invention may be used for non-diagnostic purposes in order to obtain an intermediate value of apolipoprotein A1.
To further illustrate the present invention, the following examples are given to describe in detail a kit for detecting apolipoprotein A1 and its application, which should not be construed as limiting the scope of the present invention.
Example 1
A kit for detecting apolipoprotein A1, which is prepared by the following method:
preparation of reagent R1:
taking 800g of purified water, sequentially adding 90.4g of glycerol, 0.226g of EDTA-2Na, 13.6888g of TRIS, 56.5g of PEG6000, 0.226g of Gemini surfactant, 9.04g of NaCl, 1.13g of sodium azide and 1.13g of Tween 20, stirring the materials added each time until the materials are completely dissolved, adjusting the pH value to 8.0, weighing to 1.13kg by using the purified water, and filtering by using a 0.40 mu m filter membrane;
preparation of reagent R2:
taking 130g of R1 reagent, adding 70g of APOA1 antibody, and stirring uniformly;
the kit comprises the following components: mixing the prepared R1 reagent and R2 reagent according to the mass ratio of R1: and (3) packaging R2=5:1 into a bottle to form a kit, wherein the kit specification is as follows: r1 was 50 g/4 per vial and R2 was 20 g/2 per vial.
Example 2
A kit similar to example 1, except that 90.4g of glycerol and 0.226g of EDTA-2Na were not added to the reagent R1.
Example 3
A kit similar to that of example 1, except that the amount of Gemini surfactant was replaced with 0.452 g.
Example 4
A kit similar to example 1, except that 1.13g of tween 20 was not added to the reagent R1, and the amount of Gemini surfactant was replaced with 0.565 g.
Example 5
A kit similar to example 1, except that 90.4g of glycerol was not added to reagent R1.
Example 6
A kit similar to example 1, except that 0.226g of EDTA-2Na was not added to the reagent R1.
Example 7
A kit similar to example 1, except that tween 20 was replaced with triton x-100.
Comparative example 1
A kit similar to example 1 except that no Gemini surfactant was added to reagent R1.
Comparative example 2
A kit similar to that of example 2 except that no Gemini surfactant was added to the reagent R1.
Comparative example 3
A kit similar to example 1, except that the amount of Gemini surfactant was replaced with 0.1 g.
Comparative example 4
A kit similar to that of example 1, except that the amount of Gemini surfactant was replaced with 20 g.
Application example 1
Verifying the effect of the Gemini surfactant on reducing false positive of the reagent
Collecting clinical samples measured by hospital gold standards, and measuring the concentration of apolipoprotein A1 in the clinical samples by using an AU480 full-automatic biochemical analyzer in combination with the kits prepared in examples 1-7 and comparative examples 1-4, wherein the detection parameters are shown in table 1, and the detection results are shown in table 2.
TABLE 1 measurement of parameters
Note: in the table, s is the sample addition amount.
TABLE 2 detection results (mg/dL) of different kits
Note: the sample concentration values in the table are the concentration values of apolipoprotein A1 in clinical samples determined by gold standards.
As can be seen from Table 2: the comparison result of the embodiment added with the Gemini surfactant is basically consistent with that of the sample of the gold standard, and the detection result is lower than that of the embodiment not added when a high-value sample is tested, which shows that the Gemini surfactant with proper concentration can reduce the interference of other homologous apolipoprotein heteroantibodies and improve the accuracy. And as demonstrated by the data of example 1, comparative example 3 and comparative example 4, the concentration of the Gemini surfactant in the reagent R1 should not be too high or too low, and too low concentration reduces the binding force between the antibody and the antigen, thereby making the measured value of the sample lower; too high concentration can affect the activity of the antigen-antibody binding site of APOA2-APOC3-APOA4-APOA5 and the like which have a homology gene cluster with APOA1 antigen in human serum, so that the APOA1 antibody is wrongly bound, and the measured value of the sample is higher.
Application example 2
The kits prepared in examples 1 to 7 and comparative examples 1 to 4 were left at 2 to 8 ℃ and observed for the presence of precipitation of the reagent R2 after 2, 5, 8 and 12 months, the results of which are shown in Table 3.
TABLE 3 results of different kits at different standing times
The experimental data show that: the addition of the composite stabilizer has a positive effect on eliminating reagent precipitation.
As is clear from Table 3, the addition of an appropriate stabilizer can avoid precipitation of a precipitate and improve the stability of the reagent compared with the reagent R2 in which no stabilizer is added.
Application example 3
Evaluation of the Performance of the kit prepared in example 1
Appearance evaluation
Under visual inspection under natural light, R1 is a colorless clear solution, and R2 is a colorless clear or yellowish solution.
Blank absorbance evaluation
And (3) detecting by taking distilled water as a sample, wherein the blank absorbance of the reagent obtained after the detection is 0.31.
Linear range evaluation (detection method same as application example 1)
The high value samples near the upper limit of the linear range are diluted with 0.9% NaCl to at least 5 concentrations in a proportion where the low value samples must be near the lower limit of the linear range. The test was repeated 3 times for each concentration of sample, and the test was averaged. The results are shown in Table 4.
TABLE 4 Linear Range assessment results
As can be seen from Table 4, the highest detection range of the kit provided by the invention can reach 290mg/dL, R 2 ≥0.99。
Evaluation of repeatability (detection method same as application example 1)
The reproducibility of the kit was evaluated using 3 quality controls of different concentrations, 15 measurements per concentration. The straw is shown in Table 5.
TABLE 5 results of evaluation of repeatability
As can be seen from Table 5, the in-batch reproducibility of the kit was 1.27%, 0.61%, and 1.55%, respectively.
Accuracy evaluation (detection method same as application example 1)
The test was carried out using commercially available quality control products for Landau lipid as samples, and each sample was tested 3 times, and the test results are shown in Table 6 below.
TABLE 6 accurate evaluation results
As can be seen from Table 6, the detection deviations of the kit for different quality control products are-0.56%, 0.55% and-0.32%, respectively, which are within the allowable range.
In conclusion, the kit disclosed by the invention is excellent in performance, simple to prepare, beneficial to enterprise quantitative production and practical in popularization. In the invention, R1 is directly used as the dilution mother liquor of R2, and the compound stabilizer is used, so that centrifugal filtration is avoided, and the preparation and production steps are greatly simplified. The Gemini surfactant is added to both R1 and R2, so that the active site of the apolipoprotein A1 antibody can be exposed to the maximum extent under the condition of extremely small dosage, the interference of homologous hybrid antibodies of other apolipoproteins is reduced, the false positive of a reagent is effectively reduced, and the accuracy of reagent detection is improved.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
- The application of the Gemini surfactant in improving the activity and/or specificity of an apolipoprotein A1 antibody.
- 2. A kit for detecting apolipoprotein A1 comprises an R1 reagent and an R2 reagent which are independently packaged, and is characterized in that the R2 reagent is a mixed reagent of mother liquor and apolipoprotein A1 antibody; the composition of the mother liquor is the same as that of the R1 reagent;the R1 reagent comprises a buffer solution, a sensitizer, an electrolyte, a preservative and an active agent;the active agent is a Gemini surfactant or a mixture of the Gemini surfactant and other surfactants;when the active agent is a Gemini surfactant, the mass content of the Gemini surfactant in the R1 reagent is 0.05% -0.10%;when the active agent is a mixture of a Gemini surfactant and other surfactants, the mass content of the Gemini surfactant in the R1 reagent is 0.02-0.04%, and the mass content of the other surfactants in the R1 reagent is 1-2%.
- 3. The kit of claim 2, wherein the R1 reagent further comprises a stabilizer; the stabilizer comprises EDTA-2Na and/or glycerol.
- 4. The kit according to claim 3, wherein the mass content of EDTA-2Na in the R1 reagent is 0.02% -0.04%; the mass content of the glycerol in the R1 reagent is 7% -8%.
- 5. The kit of claim 2 or 3, wherein the buffer comprises: HEPES buffer, MES buffer, or TRIS buffer; the pH value of the buffer solution is 6.5-9.
- 6. The kit according to claim 2 or 3, wherein the sensitizer comprises one or more of polyethylene glycol 6000, polyethylene glycol 20000 and polyethylene glycol 35000; the mass content of the sensitizer in the R1 reagent is 2% -5%.
- 7. A kit according to claim 2 or 3, wherein the electrolyte comprises sodium chloride and/or potassium chloride; the mass content of the electrolyte in the R1 reagent is 0.6-0.8%.
- 8. The kit of claim 2 or 3, wherein the preservative comprises sodium azide and/or proclin 300; the mass content of the preservative in the mother liquor is 0.1-0.2%.
- 9. The kit of claim 2 or 3, wherein the apolipoprotein A1 antibody is present in the R2 reagent in an amount of 25% to 40% by volume.
- 10. Use of the kit according to any one of claims 2 to 9 for the detection of apolipoprotein a 1.
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