CN111007254A - Protein-coated amino microsphere, preparation method thereof and angiotensin I detection kit - Google Patents
Protein-coated amino microsphere, preparation method thereof and angiotensin I detection kit Download PDFInfo
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- CN111007254A CN111007254A CN201911117102.0A CN201911117102A CN111007254A CN 111007254 A CN111007254 A CN 111007254A CN 201911117102 A CN201911117102 A CN 201911117102A CN 111007254 A CN111007254 A CN 111007254A
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
- G01N2410/02—Angiotensins; Related peptides
Abstract
An amino microsphere coated with protein, a preparation method and application thereof, and an angiotensin I determination kit, belonging to the technical field of solid phase reagents. Solves the problems of limited effective combination degree between protein and microsphere, low coupling efficiency, complex process and high cost in the existing method for preparing the protein-coated microsphere. The preparation method comprises the following steps: firstly, respectively dissolving the amino microsphere and the activating agent in a buffer solution with the pH value of 4.5; then mixing the obtained buffer solution containing the amino microspheres with the buffer solution containing an activating agent, and activating to obtain a solution containing activated microspheres; then adding protein, and coupling to obtain a solution containing protein coupled activated microspheres; adding buffer solution with pH of 8.5 for reaction, and adding sealant for sealing after the reaction is finished to obtain the protein-coated amino microsphere. The preparation method greatly improves the efficiency of chemical coupling, and simultaneously has simpler operation and lower cost.
Description
Technical Field
The invention belongs to the technical field of angiotensin I detection, and particularly relates to an amino microsphere coated with protein, a preparation method thereof and an angiotensin I detection kit.
Background
Hypertension is the most common cardiovascular disease and is a major public health problem worldwide. Hypertension can cause the pathological changes of cerebral vessels, hearts and kidneys, and is a main disease which harms human health. The existing hypertension patients in China have 2 hundred million people, and the prevalence rate all over the world is as high as 10-20%. With the development of economy and the improvement of living standard of people, hypertension is increasingly becoming a common disease worldwide.
Angiotensin I (Ang I) is an intermediate product in the Angiotensin converting process, is a decapeptide produced by hydrolyzing Angiotensinogen (Angiotensinogen) through the action of Renin (Renin), has weak activity of contracting blood vessels, and has obvious activity only after being converted into Angiotensin ii through the action of pulmonary convertase. Angiotensin II can contract arteriole of whole body to increase blood pressure, and can promote adrenal cortex to secrete aldosterone, which acts on renal tubule to retain sodium, water and potassium, thereby increasing blood volume and blood pressure.
Under normal conditions, the renin is rarely secreted, the angiotensin in blood is also less, and the renin has no obvious effect on blood pressure regulation. However, when excessive blood loss occurs, the blood flow to the kidney decreases due to a significant decrease in arterial blood pressure, and angiotensin production increases, playing an important role in preventing blood pressure from rising due to an excessive decrease in blood pressure. In addition, angiotensin causes strong constriction of blood vessels, but is not normally involved in blood pressure regulation. When the body is in a blood loss condition or the like to reduce the amount of circulating blood, the concentration of the hormone in the blood is significantly increased, and the hormone plays a certain role in maintaining the amount of circulating blood and maintaining arterial blood pressure.
Clinically, angiotensin I has important significance in diagnosis and typing of hypertension, and evaluation of severity and prognosis of coronary heart disease, heart failure and other diseases. Diseases such as essential hypertension, idiopathic or pseudo-aldosteronism, adrenal cancer, adrenocortical insufficiency, hormone synthesis deficiency, etc. can cause the decrease of angiotensin I; and diseases such as secondary aldosteronism, renal hypofunction, nephritis, congestive heart failure, renal hemangioma, rennin-secreting pararenal glomerular hyperplasia, pheochromocytoma, and liver cirrhosis can cause the increase of angiotensin I.
In the prior art, angiotensin I is typically detected by latex-enhanced immunoturbidimetry. In the latex enhanced immunoturbidimetry, a physical or chemical method is needed to adsorb or couple protein to the surface of latex microspheres, the physical adsorption method is not ideal in stability, the protein is easy to fall off from particles and is easily influenced by interfering substances in a sample, and the test result is inaccurate; the chemical coupling method can couple the protein to the microsphere through bridging of various chemical substances, but the method has the defects of limited effective binding degree between the protein and the microsphere, low coupling efficiency, complex process and high reagent cost.
Disclosure of Invention
In view of the above, the present invention provides protein-coated amino microspheres, and a preparation method and applications thereof, in order to solve the technical problems of limited effective binding degree between protein and microspheres, low coupling efficiency, complex process and high cost in the method for preparing protein-coated microspheres in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows.
The invention provides a preparation method of protein-coated amino microspheres, which comprises the following steps:
step one, respectively dissolving the amino microspheres and the activating agent in a buffer solution with the pH of 4.5 to obtain a buffer solution containing the microspheres and a buffer solution containing the activating agent;
step two, mixing the buffer solution containing the amino microspheres with the buffer solution containing an activating agent, and activating to obtain a solution containing activated microspheres;
adding protein into the solution containing the activated microspheres, and coupling for 60-90min to obtain a solution containing protein coupled activated microspheres;
and step four, adding a buffer solution with the pH value of 8.5 into the solution containing the protein coupling activated microspheres for reaction, adding a sealant after the reaction is finished, and sealing for more than 30min to obtain the protein-coated amino microspheres.
Preferably, in the first step, the mass ratio of the amino microspheres to the activator is (40-100): 1.
Preferably, in the first step, the amino microsphere is an amino magnetic bead, and the activating agent is glutaraldehyde.
Preferably, in the second step, the activation time is 30-90 min.
Preferably, in the third step, the mass ratio of the protein to the activated microspheres is (1-2): 1, and the protein is an angiotensin I antibody.
Preferably, in the fourth step, the reaction time is 60min or more.
Preferably, in the fourth step, the blocking agent is 1% bovine serum albumin or 1% gelatin.
Preferably, in the fourth step, a buffer solution with the pH value of 8 is added into the protein-coated amino microsphere, the pH value is adjusted to 7.5 by using a NaOH solution, and then a protective agent is added to obtain a protein-coated amino microsphere solution.
More preferably, the protectant is 0.2% sucrose or 0.2% polyvinylpyrrolidone.
The invention also provides the protein-coated amino microsphere prepared by the preparation method of the protein-coated amino microsphere.
The invention also provides an angiotensin I determination kit, which comprises an R1 reagent and an R2 reagent;
the R1 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L, Tween-201-5 g/L of bovine serum albumin, 5-30 g/L, PC-3001 g/L of polyethylene glycol and 0.05-0.1% of amino microspheres coated with protein, wherein the protein in the amino microspheres coated with protein is angiotensin I antibody;
the R2 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L of bovine serum albumin, 10-20 g/L, PC-3001 g/L of sucrose and 0.05-0.2 mu g/mL of conjugate of acridine ester labeled angiotensin I and protein, wherein the protein in the conjugate is bovine serum albumin.
Compared with the prior art, the invention has the beneficial effects that:
according to the preparation method of the protein-coated amino microsphere, the protein and the amino microsphere are chemically coupled by adopting gradient pH, the protein and the amino microsphere are effectively connected, the effective combination between the protein and the microsphere is improved, the chemical coupling efficiency is greatly improved, the operation is simpler, and the cost is lower.
The angiotensin I determination kit has obvious detection effect.
Drawings
FIG. 1 is a graph showing the coupling efficiency of protein (angiotensin I) to amino microspheres at different pH conditions in comparative example 1 and example 1 of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The invention provides a preparation method of protein-coated amino microspheres, which comprises the following steps:
step one, respectively dissolving the amino microspheres and the activating agent in a buffer solution with the pH of 4.5 to obtain a buffer solution containing the microspheres and a buffer solution containing the activating agent;
step two, mixing a buffer solution containing the amino microspheres with a buffer solution containing an activating agent, and activating for 30-90 min to obtain a solution containing activated microspheres;
adding protein into the solution containing the activated microspheres, and coupling for 60-90min to obtain a solution containing protein coupled activated microspheres;
and step four, adding a buffer solution with the pH value of 8.5 into the solution containing the protein coupling activated microspheres, reacting for more than 60min, and adding a sealant to seal for more than 30min after the reaction is finished, thus obtaining the protein-coated amino microspheres.
In the technical scheme, in the first step, the mass ratio of the microspheres to the activating agent is preferably (40-100): 1. The amino microspheres are preferably amino magnetic beads, and are available in a manner well known in the art, such as commercially available (Thermo manufacturers). The activator is preferably glutaraldehyde. The buffer is not particularly limited and mainly plays a role of dissolution, and is preferably a carbonate buffer. The concentration of the amino microspheres in the buffer solution containing the microspheres is preferably 10-50 mg/mL, and the concentration of the activator in the buffer solution containing the activator is preferably 20 wt%.
According to the technical scheme, in the second step, the activation time has no influence on the effect, and considering the production efficiency, the activation time is preferably 30-90 min, and more preferably 30 min.
In the technical scheme, in the third step, the protein is an angiotensin I antibody, the mass ratio of the protein to the activated microspheres is (1-2): 1, and the coupling time is preferably 60 min.
In the above technical scheme, in the fourth step, the buffer solution is preferably potassium dihydrogen phosphate-sodium hydroxide buffer solution, the blocking solution is a reagent commonly used in the art, the blocking agent is preferably 1% bovine serum albumin or 1% gelatin, and the reaction time is preferably 60 min.
In the fourth step of the technical scheme, a buffer solution with the pH value of 8 is added into the amino microsphere coated with the protein, the pH value is adjusted to 7.5 by using a NaOH solution (40%), and then a protective agent is added to obtain the amino microsphere solution coated with the protein. The protective agent is a commonly used agent in the art, and is preferably 0.2% sucrose or 0.2% polyvinylpyrrolidone.
The invention also provides the protein-coated amino microsphere prepared by the preparation method of the protein-coated amino microsphere.
The angiotensin I determination kit comprises an R1 reagent and an R2 reagent;
the R1 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L, Tween-201-5 g/L of bovine serum albumin, 5-30 g/L, PC-3001 g/L of polyethylene glycol and 0.05-0.1% of amino microspheres coated with protein;
wherein, the protein-coated amino microspheres are added in the form of a protein-coated amino microsphere solution, the solvent is phosphate buffer solution with pH of 8.0, and the protein in the protein-coated amino microspheres is angiotensin I antibody;
the R2 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L of bovine serum albumin, 10-20 g/L, PC-3001 g/L of sucrose and 0.05-0.2 mu g/mL of acridine ester labeled angiotensin I and protein conjugate (purchased from bang organisms, having a product number of BQ 052054);
wherein the conjugate of acridinium ester labeled angiotensin I and protein is added in the form of solution containing the conjugate of acridinium ester labeled angiotensin I and protein, the solvent is phosphate buffer solution with pH of 6.0, and the conjugate protein is bovine serum albumin.
The present invention is further illustrated by the following examples.
Comparative example 1
Step one, respectively dissolving 50mg of amino microspheres (manufactured by Thermo) and 1mg of glutaraldehyde in a carbonate buffer solution with the pH value of A to obtain a buffer solution containing the amino microspheres and a buffer solution containing the glutaraldehyde;
step two, mixing the buffer solution containing the amino microspheres and the buffer solution containing glutaraldehyde, and activating for 30min to activate the amino groups on the surfaces of the amino microspheres to obtain a solution containing activated amino microspheres;
adding 100 mu g of protein (angiotensin I antibody) into the solution containing the activated amino microspheres, coupling the protein with the activated amino microspheres for 60min to obtain the solution containing the protein coupled microspheres;
step four, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with the pH value of A into a solution containing the protein coupled microspheres, and reacting for 60 min;
step five, adding 1% bovine serum albumin after the reaction is finished, and sealing for 30 min;
and step six, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with the pH value of A after the sealing is finished, adjusting the pH value to 7.5 by using a NaOH solution (40%), adding 0.2% of cane sugar serving as a protective agent to obtain a solution of the protein-coated amino microspheres, and storing.
Comparative example 1 the test was carried out using buffers of different pH, respectively, and the specific pH (value of A) is shown in Table 1.
And detecting the standard substance, drawing a standard curve, and calculating the coupling efficiency, wherein the coupling efficiency is (absorbance of the protein solution before coupling-absorbance of the protein solution after coupling)/absorbance of the protein solution before coupling, and the result is shown in table 1 and fig. 1.
Example 1
Step one, respectively dissolving 50mg of amino microspheres (manufactured by Thermo) and 1mg of glutaraldehyde in a carbonate buffer solution with the pH value of 4.5 to obtain a buffer solution containing the amino microspheres and a buffer solution containing the glutaraldehyde;
step two, mixing the buffer solution containing the amino microspheres and the buffer solution containing glutaraldehyde, and activating for 30min to activate the amino groups on the surfaces of the amino microspheres to obtain a solution containing activated amino microspheres;
adding 100 mu g of protein (angiotensin I antibody) into the solution containing the activated amino microspheres, coupling the protein with the activated amino microspheres for 60min to obtain the solution containing the protein coupled microspheres;
step four, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with the pH value of 8.5 into the solution containing the protein coupled microspheres, and reacting for 60 min;
step five, adding 1% bovine serum albumin after the reaction is finished, and sealing for 30 min;
and step six, adding a potassium dihydrogen phosphate-sodium hydroxide buffer solution with the pH value of 8.0 after the sealing is finished, adjusting the pH value to 7.5 by using a NaOH solution (40%), adding 0.2% of cane sugar serving as a protective agent to obtain a solution of the protein-coated amino microspheres, and storing.
And detecting the standard substance, drawing a standard curve, and calculating the coupling efficiency, wherein the coupling efficiency is (absorbance of the protein solution before coupling-absorbance of the protein solution after coupling)/absorbance of the protein solution before coupling, and the result is shown in table 1 and fig. 1.
TABLE 1 calibration curve values after coating at different pH
As can be seen from Table 1, the coating was linear and more fold better in the manner of pH gradient coating in example 1. As can be seen from FIG. 1, the pH gradient coating mode adopted in example 1 can greatly increase the coupling efficiency, and the effect is very remarkable.
Example 2
Example 2 the same procedure as in example 1 was followed, except that the activation time in step two was adjusted to 30min, 60min and 90 min.
Example 2 gives the values of the calibration curve after activation coating under different activation time conditions, and the results are shown in table 2.
TABLE 2 values of calibration curves for different activation times
As can be seen from table 2, the activation time had no significant effect on the coupling efficiency between the protein and the microspheres.
Example 3
Example 3 the same procedure as in example 1 was followed, except that the coupling times in step three were adjusted to 30min, 60min and 90 min.
Example 3 gives the values of the calibration curve for different coupling times and the results are shown in table 3.
TABLE 3 values of calibration curves for different coupling time conditions
As can be seen from Table 3, the coupling time in step three has an influence on the reactivity of the reagents, and the coupling time is preferably 60min to 90min, and more preferably 60min to save the working time.
Example 4
An angiotensin I assay kit comprising an R1 reagent and an R2 reagent;
the R1 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L, Tween-201-5 g/L of bovine serum albumin, 5-30 g/L, PC-3001 g/L of polyethylene glycol and 0.1% of the amino microsphere coated with the protein in example 1;
the R2 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L of bovine serum albumin, 10-20 g/L, PC-3001 g/L of sucrose and 0.2 mu g/mL of acridine ester labeled angiotensin I and protein conjugate, wherein the protein in the conjugate is bovine serum albumin.
The angiotensin I assay kit of embodiment 4 of the present invention is compared with the angiotensin I assay kit in the prior art (shenzhen new industry), the detection method is a magnetic particle chemiluminescence method, and the detection results are shown in table 4.
TABLE 4 comparison of Performance with control kit
As can be seen from Table 4, the angiotensin I determination kit has the same performance as that of Shenzhen New Production angiotensin I determination kit, and has very obvious effect.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The preparation method of the protein-coated amino microsphere is characterized by comprising the following steps:
step one, respectively dissolving the amino microspheres and the activating agent in a buffer solution with the pH of 4.5 to obtain a buffer solution containing the microspheres and a buffer solution containing the activating agent;
step two, mixing the buffer solution containing the amino microspheres with the buffer solution containing an activating agent, and activating to obtain a solution containing activated microspheres;
adding protein into the solution containing the activated microspheres, and coupling for 60-90min to obtain a solution containing protein coupled activated microspheres;
and step four, adding a buffer solution with the pH value of 8.5 into the solution containing the protein coupling activated microspheres for reaction, adding a sealant after the reaction is finished, and sealing for more than 30min to obtain the protein-coated amino microspheres.
2. The method for preparing protein-coated amino microspheres according to claim 1, wherein in the first step, the mass ratio of the amino microspheres to the activator is (40-100): 1.
3. The method according to claim 1, wherein the amino microsphere is an amino magnetic bead and the activator is glutaraldehyde.
4. The method for preparing protein-coated amino microspheres according to claim 1, wherein the activation time in the second step is 30-90 min.
5. The method for preparing protein-coated amino microspheres according to claim 1, wherein in the third step, the mass ratio of the protein to the activated microspheres is (1-2): 1, and the protein is angiotensin I antibody.
6. The method according to claim 1, wherein the reaction time in the fourth step is 60min or more.
7. The method of claim 1, wherein the blocking agent is 1% bovine serum albumin or 1% gelatin.
8. The method for preparing protein-coated amino microspheres according to claim 1, wherein in step four, a buffer solution with pH of 8 is added to the protein-coated amino microspheres, the pH is adjusted to 7.5 by using NaOH solution, and then a protective agent is added to obtain a protein-coated amino microspheres solution;
the protective agent is 0.2% of sucrose or 0.2% of polyvinylpyrrolidone.
9. Protein-coated amino microspheres produced by the method for producing protein-coated amino microspheres of claims 1 to 7.
10. The kit for measuring angiotensin I comprising the protein-coated amino microsphere according to claim 9, which comprises R1 reagent and R2 reagent;
the R1 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L, Tween-201-5 g/L of bovine serum albumin, 5-30 g/L, PC-3001 g/L of polyethylene glycol and 0.05-0.1% of amino microspheres coated with protein, wherein the protein in the amino microspheres coated with protein is angiotensin I antibody;
the R2 reagent includes: 20-100 mmol/L of phosphate buffer solution, 2-10 g/L of bovine serum albumin, 10-20 g/L, PC-3001 g/L of sucrose and 0.05-0.2 mu g/mL of conjugate of acridine ester labeled angiotensin I and protein, wherein the protein in the conjugate is bovine serum albumin.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113985036A (en) * | 2021-11-04 | 2022-01-28 | 南京岚煜生物科技有限公司 | Angiotensin I detection kit and preparation method thereof |
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Application publication date: 20200414 |