CN112710824A - Buffer solution for preserving superparamagnetic particles and protein connectors thereof and preparation method thereof - Google Patents
Buffer solution for preserving superparamagnetic particles and protein connectors thereof and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of protein preservation, and particularly relates to a buffer solution for preserving superparamagnetic particles and protein connectors thereof and a preparation method thereof. The buffer solution for preserving the superparamagnetic particles and the protein connectors thereof comprises the following components: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, xanthan gum, sodium alginate and gelatin. The buffer provided by the invention can store the micron-sized superparamagnetic particles and the connecting substances of the superparamagnetic particles and the specific protein for a long time, so that the micron-sized superparamagnetic particles and the connecting substances of the superparamagnetic particles and the specific protein cannot lose effectiveness due to physical actions (such as sedimentation and hardening); is a special buffer solution used for long-term storage of the superparamagnetic particles and the protein connectors thereof.
Description
Technical Field
The invention belongs to the technical field of protein preservation, and particularly relates to a buffer solution for preserving superparamagnetic particles and protein connectors thereof and a preparation method thereof.
Background
The magnetic particle chemiluminescence method is an advanced immunological detection method and can be used for in vitro detection of specific protein components in blood. The method can effectively assist doctors in auxiliary diagnosis of the illness state of the patients. Protein linkers of micron-sized superparamagnetic microparticles play an important role in this methodology.
However, protein conjugates of superparamagnetic particles settle very easily and, due to poor hydrophilicity, gradually harden into a sheet-like or block-like structure after settling. The hardened magnetic particle-protein connector is very unstable, which can cause the whole reaction system to lose efficacy, and thus the product fails. At present, the following methods are the main methods for solving the problem of hardening.
Firstly, the surface structure of the magnetic particles is modified, and the hydrophilicity is increased. The method can effectively improve the hardening phenomenon of the magnetic particles. But the overall performance of the reaction system is affected, the process is more complicated, and the production cost is increased more. The cost performance is not high.
And secondly, the ionic strength concentration of the buffer solution is changed, and the cleaning strength in the reaction process is enhanced. The method adopts a pure physical mode, breaks a hardened structure and has a common effect. For fully automated equipment, implementation is difficult (mainly time-dependent), and higher ion concentrations can have a great impact on the performance of the kit under conditions of incomplete washing.
And thirdly, buffer solution components are improved, so that the hardened magnetic particles become looser and are easier to mix uniformly. The method is a common method and has good effect. However, for reaction consumables with special shapes (such as V-bottom reaction cups), the method is not easy to mix the magnetic particles uniformly, and large repeatability deviation is caused.
Therefore, it is of great significance to research a storage buffer solution capable of storing the linker of superparamagnetic particles and specific proteins for a long time, so that the linker will not lose effectiveness due to physical effects.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a buffer solution for preserving superparamagnetic particles and protein connectors thereof and a preparation method thereof. The buffer prepared by the invention can store the micron-sized superparamagnetic particles and the connecting substances of the superparamagnetic particles and the specific protein for a long time, and has a good preservation effect.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a buffer solution for preserving superparamagnetic particles and protein conjugates thereof comprises the following components:
disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, xanthan gum, sodium alginate and gelatin.
Preferably, the buffer solution comprises the following components in parts by weight:
4-8 parts of disodium hydrogen phosphate dodecahydrate, 0.5-1 part of sodium dihydrogen phosphate, 5-10 parts of sodium chloride, 1-50 parts of bovine serum albumin, 60-150 parts of sucrose, 0.1-5 parts of xanthan gum, 0.1-5 parts of sodium alginate and 1-15 parts of gelatin.
Preferably, the buffer solution comprises the following components in parts by weight:
5.6 to 5.9 parts of disodium hydrogen phosphate dodecahydrate, 0.55 to 0.60 part of sodium dihydrogen phosphate, 5 to 9 parts of sodium chloride, 1 to 10 parts of bovine serum albumin, 70 to 135 parts of sucrose, 0.2 to 2 parts of xanthan gum, 0.5 to 5 parts of sodium alginate and 5 to 15 parts of gelatin.
Preferably, the mass ratio of the xanthan gum to the gelatin is 1:10-1: 50; the mass ratio of the sodium alginate to the gelatin is 1:10-1: 30.
Preferably, the buffer solution further comprises an emulsifier.
Preferably, the emulsifier is tween-20, tween-60 or tween-80.
Preferably, the volume of the emulsifier is 0.1-0.3% of the total volume of the buffer.
The invention also aims to provide a preparation method of the buffer solution for preserving the superparamagnetic particles and the protein connectors thereof, which comprises the following steps:
(1) weighing purified water, and adding disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose and an emulsifier; stirring and dissolving; preparing a basic buffer solution;
(2) adding xanthan gum, sodium alginate and gelatin into the basic buffer solution in sequence, stirring and heating, adjusting the pH value of the solution, adding purified water to a constant volume, and filtering to obtain the buffer solution.
Preferably, in step (2), the pH value is 7.0-7.6.
Preferably, in step (2), the filtration is performed using a 0.2-0.5 μm filter membrane; preferably 0.22 μm;
preferably, in the step (2), the stirring and heating are carried out at 30-42 ℃ for 12-16 h.
The invention also aims to provide application of the buffer solution for preserving the superparamagnetic particles and the protein connectors thereof in a preservative for the superparamagnetic particles and the protein connectors thereof.
Compared with the prior art, the invention has the technical advantages that:
(1) the buffer solution provided by the invention has two completely different states of the magnetic particle-protein connector under the conditions of 2-8 ℃ and 33-40 ℃. The magnetic particle-protein connector can not cause the hardening problem. The invention makes the buffer system present a gel state at 2-8 ℃, under which the magnetic particle-protein connector can be evenly suspended in the buffer system without sedimentation. At this time, the viscosity coefficient of the buffer system is large, and the buffer system cannot participate in normal reaction. At the temperature of 33-40 ℃, the invention leads the buffer system to present a flowing state, under the state, the viscosity coefficient of the buffer system is very small, the magnetic particles-protein connecting substances can be easily mixed by a manual or automatic instrument, and the integral effect of the reaction system is not influenced.
(2) The buffer provided by the invention can store the micron-sized superparamagnetic particles and the connecting substances of the superparamagnetic particles and the specific protein for a long time, so that the micron-sized superparamagnetic particles and the connecting substances of the superparamagnetic particles and the specific protein cannot lose effectiveness due to physical actions (such as sedimentation and hardening); is a special buffer solution used for long-term storage of the superparamagnetic particles and the protein connectors thereof.
(3) The proportion of xanthan gum, sodium alginate and gelatin in the invention is the key point of different forms under different temperature conditions.
(4) The bovine serum albumin and the sucrose are protein stabilizers and provide structural stability for the magnetic particle-protein connector.
(5) The invention improves the buffer system of the magnetic particle carrier in the magnetic particle luminescence method. The magnetic particles are particles with a certain mass and mainly comprise FeO and Fe2O3With a diameter of 1-4 μmThe magnetic particle antibody conjugate is connected with protein, so that the magnetic particle antibody conjugate has certain hydrophilicity. Due to gravity, the magnetic particle antibody conjugate will sink rapidly in an aqueous medium. After a certain time, the mixture can be hardened even, and the difficulty of mixing uniformly again after hardening is high.
(6) The buffer solution provided by the invention ensures that the magnetic particle-protein connector does not sink visible to naked eyes within 7 days in a normal temperature environment; meanwhile, the magnetic particle-protein connector keeps good fluidity in the environment of 33-40 ℃. The absorption quantity can not be influenced when the instrument absorbs the operation, and the wall-hanging residue can not appear in the solution when the solution is uniformly mixed. The magnetic particle antibody conjugate can be changed into gel at the temperature of 2-8 ℃, and the suspension property of the magnetic particle antibody conjugate can be maintained within 9 months; the magnetic particle antibody conjugate was still very well mixed after 13 months of testing.
Detailed Description
The present invention will be described below with reference to specific examples to make the technical aspects of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 5.6g of disodium hydrogen phosphate dodecahydrate, 0.55g of sodium dihydrogen phosphate, 9g of sodium chloride, 30g of bovine serum albumin, 70g of sucrose and 3ml of tween-20; stirring and dissolving; preparing a basic buffer solution;
(2) 0.3g of xanthan gum, 0.5g of sodium alginate and 15g of gelatin are sequentially added into the basic buffer solution, stirred and heated for 12 hours at the temperature of 35 ℃, the pH value of the solution is adjusted to 7.0, purified water is added to the solution to be constant volume of 1L, and a filter membrane with the diameter of 0.22 mu m is used for filtering, thus obtaining the buffer solution.
Example 2
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 5.9g of disodium hydrogen phosphate dodecahydrate, 0.60g of sodium dihydrogen phosphate, 5g of sodium chloride, 10g of bovine serum albumin, 135g of sucrose and 1ml of tween-60; stirring and dissolving; preparing a basic buffer solution;
(2) sequentially adding 0.5g of xanthan gum, 0.5g of sodium alginate and 5g of gelatin into the basic buffer solution, stirring and heating at 42 ℃ for 16h, adjusting the pH value of the solution to 7.6, adding purified water to a constant volume of 1L, and filtering with a 0.2-micron filter membrane to obtain the buffer solution.
Example 3
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 4g of disodium hydrogen phosphate dodecahydrate, 1g of sodium dihydrogen phosphate, 5g of sodium chloride, 1g of bovine serum albumin, 150g of sucrose and 1ml of tween-80; stirring and dissolving; preparing a basic buffer solution;
(2) sequentially adding 0.1g of xanthan gum, 0.1g of sodium alginate and 1g of gelatin into the basic buffer solution, stirring and heating at 30 ℃ for 16h, adjusting the pH value of the solution to 7.2, adding purified water to a constant volume of 1L, and filtering with a 0.5-micron filter membrane to obtain the buffer solution.
Example 4
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 8g of disodium hydrogen phosphate dodecahydrate, 0.5g of sodium dihydrogen phosphate, 10g of sodium chloride, 50g of bovine serum albumin, 60g of sucrose and 2ml of tween-20; stirring and dissolving; preparing a basic buffer solution;
(2) sequentially adding 1g of xanthan gum, 1g of sodium alginate and 10g of gelatin into the basic buffer solution, stirring and heating at 30 ℃ for 12h, adjusting the pH value of the solution to 7.5, adding purified water to a constant volume of 1L, and filtering by using a filter membrane of 0.22 mu m to obtain the buffer solution.
Comparative example 1
The difference compared to example 1 is the difference in pH.
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 5.6g of disodium hydrogen phosphate dodecahydrate, 0.55g of sodium dihydrogen phosphate, 9g of sodium chloride, 30g of bovine serum albumin, 70g of sucrose and 3ml of tween-20; stirring and dissolving; preparing a basic buffer solution;
(2) 0.3g of xanthan gum, 0.5g of sodium alginate and 15g of gelatin are sequentially added into the basic buffer solution, stirred and heated for 12 hours at the temperature of 35 ℃, the pH value of the solution is adjusted to 6.5, purified water is added to the solution to be constant volume of 1L, and a filter membrane with the diameter of 0.22 mu m is used for filtering, thus obtaining the buffer solution.
Comparative example 2
The difference compared to example 1 is the difference in pH.
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 5.6g of disodium hydrogen phosphate dodecahydrate, 0.55g of sodium dihydrogen phosphate, 9g of sodium chloride, 30g of bovine serum albumin, 70g of sucrose and 3ml of tween-20; stirring and dissolving; preparing a basic buffer solution;
(2) 0.3g of xanthan gum, 0.5g of sodium alginate and 15g of gelatin are sequentially added into the basic buffer solution, stirred and heated for 12 hours at the temperature of 35 ℃, the pH value of the solution is adjusted to 8.2, purified water is added to fix the volume to 1L, and a filter membrane with the diameter of 0.22 mu m is used for filtering, thus obtaining the buffer solution.
Comparative example 3
Compared with example 1, the difference is that the mass ratio of xanthan gum, sodium alginate and gelatin is different.
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 5.6g of disodium hydrogen phosphate dodecahydrate, 0.55g of sodium dihydrogen phosphate, 9g of sodium chloride, 30g of bovine serum albumin, 70g of sucrose and 3ml of tween-20; stirring and dissolving; preparing a basic buffer solution;
(2) sequentially adding 2.3g of xanthan gum, 3.5g of sodium alginate and 10g of gelatin into the basic buffer solution, stirring and heating at 35 ℃ for 12h, adjusting the pH value of the solution to 7.0, adding purified water to a constant volume of 1L, and filtering with a 0.22-micron filter membrane to obtain the buffer solution.
Comparative example 4
The difference compared to example 1 is the amount of emulsifier used.
A buffer solution for preserving superparamagnetic particles and protein conjugates thereof, comprising the steps of:
(1) weighing 500ml of purified water, and adding 5.6g of disodium hydrogen phosphate dodecahydrate, 0.55g of sodium dihydrogen phosphate, 9g of sodium chloride, 30g of bovine serum albumin, 70g of sucrose and 6ml of tween-20; stirring and dissolving; preparing a basic buffer solution;
(2) 0.3g of xanthan gum, 0.5g of sodium alginate and 15g of gelatin are sequentially added into the basic buffer solution, stirred and heated for 12 hours at the temperature of 35 ℃, the pH value of the solution is adjusted to 7.0, purified water is added to the solution to be constant volume of 1L, and a filter membrane with the diameter of 0.22 mu m is used for filtering, thus obtaining the buffer solution.
Examples of effects
Experimental methods
1. Suspension effect
An OD value test was performed on 40ug/ml of the magnetic particle reagent (solute is magnetic particle-protein conjugate, and solvent is buffer prepared in examples 1 to 4 of the present invention and comparative examples 1 to 4) using an ultraviolet spectrophotometer at three temperatures of 2 to 8 ℃, room temperature, and 33 to 40 ℃. The ultraviolet light wavelength is 280 nm. Each condition was tested 10 times and the mean OD value, the repetitive CV, was calculated. The results are shown in Table 1.
TABLE 1 suspension effect experiment
2. Settling effect
After 40ug/ml of magnetic particle reagent (solute is magnetic particle-protein conjugate, solvent is buffer prepared in examples 1-4 of the present invention and comparative examples 1-4) was stored at 2-8 ℃ for 13 months, OD value was measured using an ultraviolet spectrophotometer at three temperatures of 2-8 ℃, room temperature, and 33-40 ℃. The ultraviolet light wavelength is 280 nm. Each condition was tested 10 times and the mean OD value, the repetitive CV, was calculated. The results are shown in Table 2.
TABLE 2 sedimentation Effect test
3. Effect of mixing
40ug/ml of magnetic microparticle reagent (solute is magnetic microparticle-protein conjugate, solvent is buffer prepared in examples 1-4 of the present invention and comparative examples 1-4) was mixed well for 2 minutes at 33-40 deg.C by both manual pipetting and automatic pipetting and tested for OD using UV spectrophotometer. The ultraviolet light wavelength is 280 nm. Each condition was tested 10 times and the mean OD value, the repetitive CV, was calculated. The results are shown in Table 3.
TABLE 3 experiment of the blending effect
Therefore, the buffer solution provided by the invention has a good preservation effect on the storage of the magnetic particle-protein connector; and the solution condition of the buffer solution and the proportion of the components in the preparation process have great influence on the effect.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. A buffer solution for preserving superparamagnetic particles and protein conjugates thereof comprises the following components:
disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose, xanthan gum, sodium alginate and gelatin.
2. The buffer solution according to claim 1, comprising the following components in parts by weight:
4-8 parts of disodium hydrogen phosphate dodecahydrate, 0.5-1 part of sodium dihydrogen phosphate, 5-10 parts of sodium chloride, 1-50 parts of bovine serum albumin, 60-150 parts of sucrose, 0.1-5 parts of xanthan gum, 0.1-5 parts of sodium alginate and 1-15 parts of gelatin.
3. The buffer of claim 1, comprising, in parts by weight:
5.6 to 5.9 parts of disodium hydrogen phosphate dodecahydrate, 0.55 to 0.60 part of sodium dihydrogen phosphate, 5 to 9 parts of sodium chloride, 1 to 10 parts of bovine serum albumin, 70 to 135 parts of sucrose, 0.2 to 2 parts of xanthan gum, 0.5 to 5 parts of sodium alginate and 5 to 15 parts of gelatin.
4. The buffer solution according to claim 1, wherein the mass ratio of the xanthan gum to the gelatin is 1:10 to 1: 50; the mass ratio of the sodium alginate to the gelatin is 1:10-1: 30.
5. The buffer according to any one of claims 1 to 4, further comprising an emulsifier in parts by weight; the emulsifier is tween-20 or tween-60 or tween-80.
6. The buffer of claim 5, wherein the emulsifier is present in an amount of 0.1 to 0.3% by weight based on the total volume of the buffer.
7. The method of preparing a buffer according to any of claims 1 to 6, comprising the steps of:
(1) weighing purified water, and adding disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, bovine serum albumin, sucrose and an emulsifier; stirring and dissolving; preparing a basic buffer solution;
(2) adding xanthan gum, sodium alginate and gelatin into the basic buffer solution in sequence, stirring and heating, adjusting the pH value of the solution, adding purified water to a constant volume, and filtering to obtain the buffer solution.
8. The method according to claim 7, wherein the pH in step (2) is 7.0 to 7.6.
9. The method for preparing a buffer according to claim 7, wherein in the step (2), the filtration is performed using a filter of 0.2 to 0.5 μm; preferably 0.22 μm; the stirring and heating are carried out for 12-16h at the temperature of 30-42 ℃.
10. Use of a buffer according to any of claims 1 to 6 or a buffer prepared by a method according to any of claims 7 to 9 in a preservative for superparamagnetic particles and protein conjugates thereof.
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| CN114384239B (en) * | 2021-12-09 | 2022-10-18 | 深圳君和生物科技有限公司 | Immunomagnetic bead storage system and immunomagnetic bead system |
| CN114705868A (en) * | 2022-05-31 | 2022-07-05 | 深圳市帝迈生物技术有限公司 | Kit for determining thrombomodulin content and preparation method thereof |
| CN114689875A (en) * | 2022-06-02 | 2022-07-01 | 深圳市帝迈生物技术有限公司 | Kit for determining TAT content and preparation method thereof |
| CN114689875B (en) * | 2022-06-02 | 2022-08-23 | 深圳市帝迈生物技术有限公司 | Kit for measuring TAT content and preparation method thereof |
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Inventor after: Wang Falong Inventor after: Sun Jia Inventor after: Liu Cong Inventor after: Li Bofei Inventor before: Wang Falong Inventor before: Sun Jia Inventor before: Liu Cong Inventor before: Li Bofei |