CN117705549A - Protein protecting agent, diluent and application - Google Patents
Protein protecting agent, diluent and application Download PDFInfo
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- CN117705549A CN117705549A CN202211085928.5A CN202211085928A CN117705549A CN 117705549 A CN117705549 A CN 117705549A CN 202211085928 A CN202211085928 A CN 202211085928A CN 117705549 A CN117705549 A CN 117705549A
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- 239000003085 diluting agent Substances 0.000 title claims abstract description 88
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 52
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 52
- 239000003223 protective agent Substances 0.000 title claims abstract description 26
- KDXKERNSBIXSRK-UHFFFAOYSA-N lysine Chemical compound NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 53
- 235000018102 proteins Nutrition 0.000 claims abstract description 51
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- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 45
- 239000004472 Lysine Substances 0.000 claims abstract description 37
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 36
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 17
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 15
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- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a protein protecting agent, diluent and application, and relates to the field of in-vitro diagnostic reagents. A protein protecting agent which is an amphiphilic amino acid and/or a zwitterionic polypeptide; the amphiphilic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; zwitterionic polypeptides are polymers of lysine and glutamic acid. The protein protectant can be used for preparing a diluent of a chemiluminescent reagent; the diluent for chemiluminescence prepared from the protein protecting agent can effectively protect magnetic particles or acridinium ester antigen-antibody conjugate, avoid aggregation and precipitation of antibodies due to allosteric factors, and enable the magnetic particles or the acridinium ester antigen-antibody conjugate in the diluent to be preserved for a long time.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to a protein protecting agent, diluent and application.
Background
The chemiluminescent immunoassay (chemiluminescence immunoassay, CLIA) is an emerging immunoassay technology after an enzyme-linked immunoassay (EIA), a Radioimmunoassay (RIA), a Fluorescent Immunoassay (FIA) and a time-resolved fluoroimmunoassay (TRFIA), has the advantages of high sensitivity, high specificity, low reagent price, stable reagent, high detection speed, simple operation, high automation degree and the like, and is the most rapid immunoassay method developed, popularized and applied at present. In practical application of in vitro diagnostic reagent products, magnetic particles or acridinium ester are used as a solid phase end and a labeling end of a reagent to participate in a detection process of a target antibody in a sample to be detected after being coupled with the antibody, and stable preservation of the magnetic particles or the acridinium ester antigen-antibody conjugate is a very important problem, and instability of the magnetic particles or the acridinium ester antigen-antibody conjugate can greatly influence the performance (specificity and sensitivity) of the reagent, and a plurality of factors influencing the stability of the reagent, including external conditions (such as temperature and illumination) and a buffer system of a preservation solution, pH, type and concentration of protein, ionic strength, type of saccharide and surfactant and the like. Under extreme conditions (37 ℃) or during long-term storage, antibodies in the working solution are easy to denature, degrade and aggregate due to hydrophobic effect, so that the spatial stability is reduced, dimers or multimers are generated, even aggregation and precipitation are even caused, the stability of the reagent is reduced, and the requirement of long-term stable storage of the reagent in the market cannot be met.
Currently, BSA (bovine serum albumin) and casein are the main components of protein stabilizers in common dilutions of chemiluminescent reagents, but due to their sources and manufacturing processes, the BSA and casein often contain impurities and are prone to batch-to-batch differences, proteases contained in the BSA may promote degradation of antibodies in the dilutions, and animal-derived IgG contained in the BSA and casein may cause non-specific adsorption of the reagents, resulting in high background or false positive signals. Therefore, it is important to develop a diluent which has a better protecting effect on magnetic particles or acridinium ester antigen-antibody conjugate, can be stored for a long period of time, does not affect other properties such as reagent sensitivity, specificity and repeatability, and has wide applicability.
Disclosure of Invention
In order to solve the problems that the diluent for chemiluminescence in the prior art causes non-specific adsorption of reagents, generates high background or false positive signals, has poor protection effect on magnetic particles or acridinium ester antigen-antibody conjugate, cannot be stored for a long time and the like, the invention provides a protein protecting agent which can be used for preparing the diluent for the chemiluminescence reagent; the invention also provides a diluent for chemiluminescence prepared from the protein protective agent, which can effectively protect magnetic particles or acridinium ester antigen-antibody conjugate, avoid aggregation and precipitation of antibodies due to allosteric factors, and enable the magnetic particles or the acridinium ester antigen-antibody conjugate in the diluent to be stored for a long time.
The invention solves the technical problems by adopting the following technical scheme.
In a first aspect, embodiments of the present invention provide a protein protectant that is an amphiphilic amino acid and/or zwitterionic polypeptide; preferably, the amphiphilic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
In a second aspect, embodiments of the present invention provide for the use of a protein protectant in the preparation of a diluent for a chemiluminescent reagent; preferably, the mass percentage concentration of the protein protectant in the diluent is 0.5% -2%; most preferably, the diluent further comprises: buffer system, polyol, surfactant and saccharide.
In a third aspect, embodiments of the present invention provide a diluent for a chemiluminescent reagent, the diluent comprising: a protein protecting agent which is an amphiphilic amino acid and/or a zwitterionic polypeptide; preferably, the mass percentage concentration of the protein protecting agent is 0.5% -2%; most preferably, the amphiphilic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
Compared with the prior art, the embodiment of the application has at least the following advantages or beneficial effects:
the protein protecting agent is amphiphilic amino acid and/or zwitterionic polypeptide, can be used for preparing a diluent of a chemiluminescent reagent, and can be used for effectively protecting magnetic particles or acridinium ester antigen-antibody conjugate, avoiding aggregation and precipitation of antibodies due to allosteric, and enabling the magnetic particles or the acridinium ester antigen-antibody conjugate in the diluent to be preserved for a long time. In addition, the main components of the diluent are convenient to obtain, the amphiphilic compound can avoid the defects of traditional protein protectants such as BSA and casein, reduce high background or false positive signals caused by nonspecific adsorption due to the introduction of animal-derived IgG, and simultaneously have no influence on the specificity, repeatability and other performances of the reagent, thus being suitable for popularization and use.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other, and the present invention will be described in detail with reference to specific embodiments.
A protein protecting agent which is an amphiphilic amino acid and/or a zwitterionic polypeptide; the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
A protein protecting agent comprises one or more of tryptophan, methionine, leucine and L-proline.
The application of the protein protectant in preparing the diluent of the chemiluminescent reagent comprises the following steps of 0.5% -2% of the protein protectant in mass percent.
Use of a protein protectant in the preparation of a diluent for a chemiluminescent reagent, said diluent further comprising: buffer system, polyol, surfactant and saccharide.
A diluent for a chemiluminescent reagent, the diluent comprising: a protein protecting agent, wherein the protein protecting agent is amphiphilic amino acid and/or zwitterionic polypeptide; preferably, the mass percentage concentration of the protein protecting agent is 0.5% -2%; most preferably, the amphiphilic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
A diluent for a chemiluminescent reagent comprising the following raw materials:
a protein protecting agent with the mass percentage concentration of 0.1-2%, wherein the protein protecting agent comprises amphiphilic amino acid and/or zwitterionic polypeptide; preferably a protein protectant with a mass percent concentration of 0.5% -2%;
a buffer system with a concentration of 0.01-0.1 mol/L; preferably a buffer system with a concentration of 0.01-0.05 mol/L;
polyol with volume percentage concentration of 1-20%; preferably a polyol having a concentration of 5 to 10% by volume;
surfactant with mass percentage concentration of 0.01-0.1%;
saccharide with mass percentage concentration of 1-10%;
the amphipathic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; and/or, the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
Applicants have unexpectedly found that amphiphilic amino acids having nonpolar side chains (such as tryptophan, methionine, leucine and L-proline) or zwitterionic polypeptides having alternating charges (such as zwitterionic polypeptides resulting from multimerization of positively charged lysine and negatively charged glutamic acid), and the like, are very effective in preventing antibody production of dimers or multimers. Presumably, the amphiphilic compound can actively intervene between the hydrophobic site of the antibody molecule and the water molecule, and aggregation of the amphiphilic compound can be effectively prevented by interaction of the hydrophobic residues of the antibody, so that the solubility and stability of the antibody in an aqueous solution are improved.
In some embodiments of the invention, the protein protectant is a mixture of L-proline and methionine.
Here, the mass fraction of L-proline may be, but is not limited to, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%; methionine may be, but is not limited to, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2% by mass.
In some embodiments of the invention, the protein protectant is leucine. The mass fraction of leucine may be, but is not limited to, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%.
In some embodiments of the invention, the protein protectant is a polymer of lysine and glutamic acid, and the molar ratio of lysine to glutamic acid is (0.5-1.5): 1. The polymer is a zwitterionic polypeptide having alternating charges, resulting from the multimerization of positively charged lysines and negatively charged glutamates.
In some embodiments of the invention, the molar ratio of lysine to glutamic acid is 1:1.
In some embodiments of the invention, the molecular weight of the zwitterionic polypeptide described above is 70000-150000Da.
In some embodiments of the invention, the polyol is selected from one or more of sorbitol, ethylene glycol, glycerol, mannitol.
The volume fraction of the polyol may be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%.
In some embodiments of the invention, the saccharide is selected from sucrose or trehalose at a concentration of 5% to 10% by mass.
The addition of saccharides and polyols to the diluent formulation can protect the antibody from degradation, and the addition of a small amount of sorbitol to the diluent formulation containing sucrose or trehalose can better preserve the natural structure of the antibody and improve the antibody stability.
In some embodiments of the invention, the surfactant is poloxamer 188 at a concentration of 0.05-0.1% by mass or poloxamer 407 at a concentration of 0.05-0.1% by mass.
It should be noted that the mass fraction of the saccharide may be, but is not limited to, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%; the mass fraction of surfactant may be, but is not limited to, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%.
The surfactant helps to stably disperse the protein in the buffer system, and the present invention is not particularly limited, and any surfactant commonly used in the field of diagnostic reagents may be used. Exemplary surfactants may be selected from one or more of the Tween, TRITON, PLURONIC series, for example, from one or more of Tween20, tween-80, span-60, triton X-45, triton X-100, poloxamer 188 and poloxamer 407.
Poloxamer 188 or poloxamer 407 is preferable, and the poloxamer is a novel polymer nonionic surfactant, is easy to dissolve in water, has stronger surface activity and gel effect, has the characteristics of mild physical property and stable chemical property, and can increase the solubility of protein and prevent protein aggregation when being matched with other reagents of the invention, thereby improving the stability of the protein.
In some embodiments of the invention, the buffer system described above includes one or more of Hepes buffer, PBS buffer, and 2-morpholinoethanesulfonic acid buffer.
The buffer system of the invention is a mixture composed of weak acid and salt thereof, weak base and salt thereof, and can offset and lighten the influence of external strong acid or strong base on the pH value of the solution to a certain extent, thereby keeping the pH value of the solution relatively stable. The buffer system of the present invention includes one or more of Hepes buffer system, PBS buffer system (i.e., phosphate buffer system) and 2-morpholinoethanesulfonic acid buffer system (MES).
Here, the concentration of the buffer may be, but not limited to, 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L, 0.06mol/L, 0.07mol/L, 0.08mol/L, 0.09mol/L, 0.1mol/L.
In some embodiments of the present invention, the diluent further comprises the following raw materials in percentage by mass: 0.3-1% of preservative and/or 0.5-2% of inorganic salt.
In some embodiments of the invention, the preservative is PC-300 or PC-950.
Here, the mass percentage concentration of the preservative may be, but is not limited to, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.
A preservative for extending the shelf life of the agent. The type of the preservative is not particularly limited, and any preservative commonly used in the field of diagnostic reagents can be used. For example, selected from the kroevin series of preservatives, the ProClin series of preservatives, and sodium azide. The KroVin series preservative is selected from KroVin 100, kroVin 400, kroVin 500 and KroVin 750; the ProClin series preservative may be selected from ProClin50, proClin150, proClin200, proClin300, proClin950 and ProClin5000.
In some embodiments of the invention, the inorganic salt is a sodium salt and/or a potassium salt.
In some embodiments of the invention, the inorganic salt is sodium chloride with a mass percentage concentration of 0.5-1%. The inorganic salts provide the electrolyte for the buffer system of the present invention.
The mass fraction of the inorganic salt may be, but is not limited to, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%.
In some embodiments of the invention, the above-described diluent comprises the following raw materials:
a protein protectant with a mass percent concentration of 0.1-2%; preferably, the protein protectant comprises amphiphilic amino acids and/or zwitterionic polypeptides;
a buffer system with a concentration of 0.01-0.1 mol/L;
polyol with volume percentage concentration of 1-20%;
surfactant with mass percentage concentration of 0.01-0.1%;
saccharide with mass percentage concentration of 1-10%;
preservative with mass percentage concentration of 0.3-1%;
the mass percentage concentration of the inorganic salt is 0.5-2%.
In some embodiments of the invention, the above-described diluent comprises the following raw materials:
a protein protective agent with the mass fraction of 0.1-2%, wherein the protein protective agent comprises amphiphilic amino acid and/or zwitterionic polypeptide;
a buffer system with a concentration of 0.01-0.1 mol/L;
polyol with volume percentage concentration of 1-20%;
surfactant with mass percentage concentration of 0.01-0.1%;
saccharide with mass percentage concentration of 1-10%;
preservative with mass percentage concentration of 0.3-1%.
Example 1
The diluent for the chemiluminescent reagent provided in the embodiment comprises glycerol with the volume percentage of 1%, and Hepes buffer with the volume percentage of 0.02 mol/L; the material also comprises the following raw materials in percentage by mass: 5% sucrose, 0.05% poloxamer 188, 1% L-proline, 1% methionine and 0.3% PC-300.
The preparation method comprises the following steps:
1) Preparing 0.02mol/L Hepes buffer solution, and regulating the pH value to 7.2 by NaOH;
2) According to the formula amount, adding sucrose, poloxamer 188, L-proline, methionine, glycerol and PC-300 into Hepes buffer solution with the concentration of 0.02mol/L, and stirring until the mixture is completely dissolved to obtain diluent.
Example 2
The diluent for the chemiluminescent reagent provided in the embodiment comprises 1% by volume of sorbitol and 0.02mol/L PBS buffer; the material also comprises the following raw materials in percentage by mass: 5% trehalose, 0.05% poloxamer 188, 1% leucine, 0.9% sodium chloride and 0.6% pc-950.
The preparation method comprises the following steps:
1) Preparing 0.02mol/L PBS buffer solution, and regulating the pH value to 7.4;
2) According to the formula amount, trehalose, poloxamer 188, leucine, sorbitol and PC-950 are added into PBS buffer solution with the concentration of 0.02mol/L, and the mixture is stirred until the mixture is completely dissolved, thus obtaining the diluent.
Example 3
The diluent for the chemiluminescent reagent provided in the embodiment comprises glycerol with the volume percentage of 1%, and Hepes buffer with the volume percentage of 0.02 mol/L; the material also comprises the following raw materials in percentage by mass: 5% sucrose, 0.05% poloxamer 188, 0.5% zwitterionic polypeptide (a polymer of lysine and glutamic acid) and 0.3% pc-300.
The preparation method comprises the following steps:
1) Preparing 0.02mol/L Hepes buffer solution, and regulating the pH value to 7.6 by NaOH;
2) According to the formula amount, sucrose, poloxamer 188, zwitterionic polypeptide (polymer of lysine and glutamic acid), glycerol and PC-300 are added into Hepes buffer solution with the concentration of 0.02mol/L, and the mixture is stirred until the mixture is completely dissolved, so that diluent is obtained.
Example 4
The diluent for the chemiluminescent reagent provided in the embodiment comprises 1% of glycerol and 1% of sorbitol by volume, and 0.05mol/L MES buffer solution; the material also comprises the following raw materials in percentage by mass: 2% sucrose, 0.01% poloxamer 188, 0.5% zwitterionic polypeptide (a polymer of lysine and glutamic acid), 0.9% sodium chloride and 0.6% pc-950.
The preparation method comprises the following steps:
1) Preparing 0.05mol/L MES buffer solution, and regulating the pH value to 6.0;
2) According to the formula, sucrose, poloxamer 118, zwitterionic polypeptide (polymer of lysine and glutamic acid), sorbitol, glycerol, sodium chloride and PC-950 are added into 0.05mol/L MES buffer solution, and stirred until the mixture is completely dissolved, thus obtaining the diluent.
Comparative example 1
This comparative example is substantially the same as example 1, except that: 1.0% by mass of L-proline and 1.0% by mass of methionine were replaced with 1.0% by mass of casein.
Comparative example 2
This comparative example is substantially the same as example 1, except that: 1.0% by mass of L-proline and 1.0% by mass of methionine were replaced with 1.0% by mass of glycine.
Comparative example 3
This comparative example is substantially the same as example 1, except that: 1.0% by mass of L-proline and 1.0% by mass of methionine were replaced with 1.0% by mass of lysine (monomer).
Comparative example 4
This comparative example is substantially identical to example 2, except that: leucine at 1.0% by mass was replaced with casein at 1.0% by mass.
Comparative example 5
This comparative example is substantially identical to example 2, except that: leucine at 1.0% by mass was replaced with serine at 1.0% by mass.
Comparative example 6
This comparative example is substantially identical to example 3, except that: the zwitterionic polypeptide (lysine+glutamic acid) was replaced with 0.5% casein by mass.
Comparative example 7
This comparative example is substantially identical to example 3, except that: the zwitterionic polypeptide (lysine+glutamic acid) with the mass percentage of 0.5% is replaced by glutamic acid with the mass percentage of 0.5%.
Comparative example 8
This comparative example is substantially identical to example 3, except that: the zwitterionic polypeptide (lysine+glutamic acid) with the mass percent of 0.5 percent is replaced by polylysine with the mass percent of 0.5 percent.
Comparative example 9
This comparative example is substantially identical to example 4, except that: the zwitterionic polypeptide (lysine+glutamic acid) was replaced with 0.5% bsa by mass at 0.5% by mass.
Comparative example 10
This comparative example is substantially identical to example 4, except that: the zwitterionic polypeptide (lysine+glutamic acid) was replaced with 0.5% casein by mass.
Test example 1
1. L-proline and methionine mass percent selection
Combining L-proline and methionine with different mass fractions (shown in Table 1), preparing a diluent according to the preparation method of example 1 (other conditions are unchanged, only the mass fractions of L-proline and methionine are changed), diluting the acridinium ester-HIV antigen antibody conjugate to a working concentration (0.58 mug/mL) by using the prepared diluent, dividing the diluted conjugate into 3 bottles, placing 3mL of each bottle, placing in a 37 ℃ oven for acceleration experiments, and detecting HIV project calibrators on days 1, 3 and 7 respectively, wherein the detection results are shown in Table 1.
The luminescence value was detected on an i3000 fully automated chemiluminescence immunoassay using a human immunodeficiency virus antigen-antibody detection kit (direct chemiluminescence method). The calibrator for the test example comprises human immunodeficiency virus type 1 antibody, human immunodeficiency virus type 2 antibody and recombinant human immunodeficiency virus type 1p24 antigen.
The detection principle is as follows:
the first step, HIV-1/HIV-2 antibody and HIV-1p24 antigen in the calibrator are combined to HIV-1/HIV-2 antigen and HIV-1p24 monoclonal antibody coated magnetic particles to form antigen-antibody complex; secondly, adding acridinium ester marked HIV-1/HIV-2 antigen and HIV-1p24 monoclonal antibody after washing to form double antigen and double antibody sandwich complex; and (3) washing again, adding a substrate solution, performing chemiluminescence reaction, and measuring a luminescence signal value (RLU), wherein the luminescence signal value is in direct proportion to the content of the antibody and the antigen in the calibrator.
TABLE 1 measurement results 1 of thermal acceleration stability at 37℃
As can be seen from Table 1, the optimal addition amounts were found when the mass percentages of L-proline and methionine were 1.0% and 1.0%, respectively.
2. Measurement of the thermal acceleration stability of the Diluent at 37℃provided in example 1
The acridinium ester-HIV antigen-antibody conjugate was diluted to working concentration (0.58. Mu.g/mL) with the dilutions prepared in examples 1 and comparative examples 1-3, respectively, and the diluted conjugate was aliquoted into 16 bottles (3 bottles for determination of thermal acceleration stability at 37 ℃,7 bottles for determination of stability at 2-8 ℃,6 bottles for determination of long term stability), 3mL per bottle. 3 bottles were taken and placed in a 37 ℃ oven for acceleration experiments, and HIV project calibrators were detected on days 1, 3 and 7, respectively, and the detection results are shown in Table 2.
TABLE 2 measurement results of thermal acceleration stability at 37 ℃ 2
As can be seen from Table 2, the dilutions provided in example 1 had a 3 day signal retention of 96%, a 7 day signal retention of 91%, and the dilutions provided in comparative examples 1-3 had a 3 day signal retention of 81%, 73%, 78, respectively; the 7-day signal retention rates are 74%, 63% and 71%, respectively, and the experimental results show that the diluent provided in example 1 can significantly improve the thermal acceleration stability of the reagent at 37 ℃.
3. Determination of the stability to opening of the Diluent at 2-8℃provided in example 1
7 bottles of the prepared diluted conjugate are taken, placed in an instrument reagent bin after being opened, and HIV project calibrators are detected on days 1, 7, 14, 21, 28, 35 and 36 respectively under the light-shielding condition of 2-8 ℃, and the detection results are shown in table 3.
Table 3 2-8deg.C stability to open bottle test results
As can be seen from table 3, the 35-day signal retention rate of the diluent provided in example 1 was 98% higher than that of the diluent provided in comparative examples 1 to 3, and in comparative example 1, 1.0% by mass of L-proline and 1.0% by mass of methionine in the diluent were replaced with 1.0% by mass of casein; in comparative example 2, 1.0% by mass of L-proline and 1.0% by mass of methionine in the diluent were replaced with 1.0% by mass of glycine; in comparative example 3, 1.0% by mass of L-proline and 1.0% by mass of methionine in the diluent were replaced with 1.0% by mass of lysine (monomer), glycine and lysine being non-amphiphilic amino acids. The experimental results show that the combination of the amphiphilic amino acids can obviously improve the bottle opening stability of the reagent at 2-8 ℃.
4. Long term stability determination of the dilutions provided in example 1
6 bottles of the prepared diluted conjugate are taken and stored at 2-8 ℃ under a sealed and light-proof condition, and HIV project calibrators are detected in1 st month, 3 rd month, 6 th month, 9 th month, 12 th month and 13 th month respectively, and the detection results are shown in table 4.
TABLE 4 results of Long-term stability measurements
As can be seen from Table 4, the 12 th month signal retention rate of the diluent provided in example 1 was 90%, while the 12 th month signal retention rates of the diluents provided in comparative examples 1 to 3 were 77%, 69% and 74%, respectively, and the above experimental results show that the diluent provided in example 1 can significantly improve the long-term stability of the reagent as compared with the diluents provided in comparative examples 1 to 3.
Test example 2
1. Leucine mass percent selection
Taking leucine with different mass fractions (shown in table 5), preparing a diluent according to the preparation method of example 2 (other conditions are unchanged, only the mass fraction of leucine is changed), diluting the acridinium ester-BNP antibody conjugate to a working concentration by using the prepared diluent, dividing the diluted conjugate into 3 bottles with 3ml each, placing the bottles in a 37 ℃ oven for acceleration experiments, and detecting BNP project calibrators on days 1, 3 and 7 respectively, wherein the detection results are shown in table 5.
The luminescence value was measured on an i3000 full-automatic chemiluminescence immunoassay using a type B natriuretic peptide assay kit (direct chemiluminescence method) in this test example. The calibrator contained two concentrations (0.025. Mu.g/mL and 4. Mu.g/mL) of acridine ester-labeled goat anti-human BNP monoclonal antibodies.
The detection principle is as follows:
and mixing the sample to be detected, the immune magnetic particles coated with the sheep anti-human BNP monoclonal antibody and the acridinium ester marked sheep anti-human BNP monoclonal antibody to form an 'antibody-antigen-antibody' immune complex. After washing, a luminescent substrate is added to generate a chemiluminescent reaction, and the relative luminescence value (RLU) is measured and is in direct proportion to the concentration of BNP in the sample.
Table 5 results of measurement of stability under thermal acceleration at 37 ℃ 1
As can be seen from table 5, when the mass percentages of L-proline and methionine were 1.0% and 1.0%, respectively, the 3-day signal retention and 7-day signal retention in the calibrators of two different concentrations were 95% or more, which is the optimal addition amount.
2. Measurement of the thermal acceleration stability of the Diluent at 37℃provided in example 2
The acridinium ester-BNP antibody conjugate was diluted to the working concentration with the dilutions formulated in example 2 and comparative examples 4 and 5, respectively, and the diluted conjugate was aliquoted into 16 bottles (3 bottles for measuring the thermal acceleration stability at 37 ℃,7 bottles for measuring the open stability at 2-8 ℃,6 bottles for measuring the long-term stability), 3ml per bottle. 3 bottles of each group were randomly taken and placed in a 37 ℃ oven for acceleration experiments, and BNP project calibrators were detected on days 1, 3 and 7 respectively, and the detection results are shown in Table 6.
TABLE 6 measurement results of thermal acceleration stability at 37℃2
As can be seen from table 6, the 3-day signal retention rate of the dilutions provided in example 2 in the two calibrators of different concentrations was 99%, the 7-day signal retention rate was 97% and 96%, respectively, which were higher than the 3-day signal retention rate (93%, 94% and 88%, 89%) and the 7-day signal retention rate (83%, 83% and 77%, 78%) of the dilutions provided in comparative examples 4 and 5 in the calibrators of different concentrations, respectively, and the above experimental results indicated that the dilutions provided in example 2 significantly improved the thermal acceleration stability of the reagents at 37 ℃.
3. Determination of the stability to opening of the Diluent at 2-8deg.C provided in example 2
7 bottles of the prepared diluted conjugate are taken, placed in an instrument reagent bin after being opened, and BNP project calibrators are detected on days 1, 7, 14, 21, 28, 35 and 36 respectively under the light-shielding condition of 2-8 ℃, and the detection results are shown in table 7.
Table 7 2-8deg.C stability to open bottle test results
As can be seen from table 7, the 35-day signal retention rate of the dilutions provided in example 2 was 98% in the two calibrators of different concentrations, which is higher than the 35-day signal retention rate (90%, 94% and 89%, 85%) of the dilutions provided in comparative example 4 and comparative example 5 in the calibrators of different concentrations, in comparative example 4, leucine was replaced by 1.0% by mass casein in the dilutions; in comparative example 5, leucine at a mass percent of 1.0% in the diluent was replaced with serine at a mass percent of 1.0%; serine is a non-amphiphilic amino acid. The experimental result further proves that the amphiphilic amino acid can obviously improve the bottle opening stability of the reagent at 2-8 ℃.
4. Long term stability determination of the dilutions provided in example 2
6 bottles of the prepared diluted conjugate are taken and stored at 2-8 ℃ under a sealed and light-proof condition, BNP project calibrators are detected in1 st month, 3 rd month, 6 th month, 9 th month, 12 th month and 13 th month, and the detection results are shown in table 8.
TABLE 8 results of Long-term stability measurements
As can be seen from Table 8, the 12 th month signal retention of the dilutions provided in example 2 was 96% and 98% respectively in the two different concentrations of calibrator, which is higher than the 12 th month signal retention of the dilutions provided in comparative examples 4 and 5 (83%, 84% and 77%, 79%). The above experimental results show that the dilution provided in example 2 significantly improves the long-term stability of the reagent compared to comparative examples 4 and 5.
Test example 3
1. Mass percent selection of zwitterionic polypeptides (lysine+glutamic acid)
The zwitterionic polypeptides with different mass fractions (shown in Table 5) were taken, then a diluent was prepared according to the preparation method of example 3 (other conditions were unchanged, only the mass fraction of the zwitterionic polypeptide was changed), the acridinium ester-TP antigen conjugate was diluted to a working concentration (0.50. Mu.g/mL) with the prepared diluent, the diluted conjugate was divided into 3 bottles, each 3mL was placed in a 37℃oven for acceleration experiments, and Anti-TP item calibrator was tested on days 1, 3, 7, respectively, with the test results shown in Table 9.
Here, the luminescence value was detected on an i3000 full-automatic chemiluminescence immunoassay using a treponema pallidum antibody detection kit (direct chemiluminescence method) in this test example, and the calibrator contained acridinium ester-labeled recombinant treponema pallidum antigen and treponema pallidum antibody.
The detection principle is as follows:
firstly, mixing a sample and magnetic particles coated with treponema pallidum antigen (TP-Ag), wherein treponema pallidum antibodies in the sample are combined with the magnetic particles coated with the treponema pallidum antigen to form an antigen-antibody complex; step two, adding an analysis buffer solution and an acridinium ester marked treponema pallidum antigen after washing to form an antigen-antibody-antigen immune complex; and (3) washing again, adding a substrate solution, performing chemiluminescence reaction, and measuring a luminescence signal value (RLU), wherein the luminescence signal value is in direct proportion to the content of treponema pallidum antibodies in the sample.
Table 9 results of measurement of thermal acceleration stability at 37℃1
As can be seen from table 9, when the mass percentage of the zwitterionic polypeptide (lysine+glutamic acid) was 0.50%, the 3-day signal retention was 93% and the 7-day signal retention was 87% for the optimal addition amount.
2. Measurement of the thermal acceleration stability of the Diluent at 37℃provided in example 3
The acridinium ester-TP antigen conjugate was diluted to a working concentration (0.50. Mu.g/mL) with the dilutions prepared in example 3 and comparative examples 6-8, respectively, and the diluted conjugate was divided into 16 bottles (3 bottles for measuring heat acceleration stability at 37 ℃,7 bottles for measuring open stability at 2-8 ℃,6 bottles for measuring long term stability), 3mL per bottle. 3 bottles of the samples were randomly taken and placed in a 37 ℃ oven for acceleration experiments, and Anti-TP item calibrators were detected on days 1, 3 and 7, respectively, and the detection results are shown in table 10.
Table 10 results of measurement of stability under thermal acceleration at 37 ℃ 2
As can be seen from table 10, the dilution provided in example 3 had a signal retention rate of 93% on day 3 and 88% on day 7, which is higher than the dilutions provided in comparative examples 6-8 (80%, 90%, 89%) and 7-day signal retention rates (65%, 76%, 74%), and the above experimental results indicate that the dilution provided in example 3 significantly improved the thermal acceleration stability of the reagent at 37 ℃.
3. Determination of the stability to opening of the Diluent at 2-8deg.C provided in example 3
7 bottles of the prepared diluted conjugate are taken, and Anti-TP item calibrators are detected on days 1, 7, 14, 21, 28, 35 and 36 respectively under the light-shielding condition of 2-8 ℃, and the detection results are shown in Table 11.
TABLE 11 results of stability to bottle opening at 2-8deg.C
As can be seen from table 11, the dilution provided in example 3 had a signal retention rate of 98% on day 35, which is higher than that of the dilutions provided in comparative examples 6 to 8 (93%, 94%), and in comparative example 6, the zwitterionic polypeptide (lysine+glutamic acid) was replaced with 0.5% casein by mass, which is 0.5% by mass; in comparative example 7, the zwitterionic polypeptide (lysine+glutamic acid) at a mass percent of 0.5% in the diluent was replaced with 0.5% glutamic acid at a mass percent; in comparative example 8, the zwitterionic polypeptide (lysine+glutamic acid) was replaced with 0.5% polylysine by mass percent in the diluent. The experimental results show that the diluent provided in the embodiment 3 can significantly improve the stability of the reagent at 2-8 ℃ in opening the bottle.
4. Long term stability determination of the dilutions provided in example 3
6 bottles of the prepared diluted conjugate are taken and stored at 2-8 ℃ under a sealed and light-proof condition, and Anti-TP items of calibrator are detected in1 st, 3 rd, 6 th, 9 th, 12 th and 13 th months respectively, and the detection results are shown in table 12.
TABLE 12 results of Long-term stability measurements
As can be seen from Table 12, the dilution provided in example 3 had a 12 th month signal retention of 93% in both calibrations, which is much higher than the 12 th month signal retention (67%, 76%, 75%) of the dilutions provided in comparative examples 6-8. The above experimental results show that the dilution provided in example 3 significantly improves the long-term stability of the reagent as compared to comparative examples 6-8.
Test example 4
The magnetic particle-HIV antigen-antibody conjugate was diluted to a working concentration (concentration of magnetic particles was 0.3 mg/mL) with the dilutions prepared in example 4 and comparative examples 9 and 10, respectively, and the diluted conjugate was divided into 17 bottles of 3mL each. Placing 3 bottles in a 37 ℃ oven for acceleration experiments, and detecting the calibrator on days 1, 3 and 7 respectively; storing 6 bottles at 2-8deg.C under sealed and light-proof conditions, and detecting calibrator at 1 st month, 3 rd month, 6 th month, 9 th month, 12 th month and 13 th month respectively; placing 7 bottles of the calibrator in an instrument reagent bin after opening the bottles, and detecting the calibrator on days 1, 7, 14, 21, 28, 35 and 36 respectively under the condition of avoiding light at the temperature of 2-8 ℃; one bottle was taken for reagent specificity verification. The calibrator comprises recombinant human immunodeficiency virus-1/human immunodeficiency virus-2 antigen and human immunodeficiency virus-1 p24 monoclonal antibody.
1. Determination of thermal acceleration stability at 37℃
The measurement results are shown in Table 13.
Table 13 results of measurement of thermal acceleration stability at 37 ℃
As can be seen from table 13, the diluent provided in example 4 had a 3-day signal retention rate of 97%, a 7-day signal retention rate of 94%, and the diluents provided in comparative examples 9 and 10 had a 3-day signal retention rate of 82%, 72%, and a 7-day signal retention rate of 74% and 62%, respectively, and the above experimental results showed that the diluent provided in example 4 significantly improved the thermal acceleration stability of the reagent at 37 ℃.
2. 2-8deg.C bottle opening stability determination
The measurement results are shown in Table 14.
TABLE 14 results of stability to opening at 2-8deg.C
As can be seen from Table 14, the 35 day signal retention of the dilutions provided in example 4 was 98% higher than the 35 day signal retention of the dilutions provided in comparative examples 9 and 10. In comparative example 9, the mass percent of zwitterionic polypeptide (polymer of lysine+glutamic acid) in the diluent was replaced with 0.5% bsa; in comparative example 10, the zwitterionic polypeptide (polymer of lysine+glutamic acid) was replaced with 0.5% casein by mass in the diluent. The experimental results show that compared with the traditional protein stabilizer BSA and casein, the zwitterionic polypeptide (polymer of lysine and glutamic acid) can remarkably improve the 2-8 ℃ bottle opening stability of the reagent.
3. Long term stability
The results are shown in Table 15.
TABLE 15 results of Long-term stability measurements
As can be seen from table 15, the 12 th month signal retention rate of the diluent provided in example 4 was 91%, and the 12 th month signal retention rates of the diluents provided in comparative examples 9 and 10 were 77% and 67%, respectively, and the above experimental results show that the diluent provided in example 4 can significantly improve the long-term stability of the reagent compared to the diluents provided in comparative examples 9 and 10.
4. Specific detection
The detection results are shown in Table 16.
TABLE 16 specificity measurement results
The clinical samples are all from clinical samples which are confirmed to be negative through a disease control center.
The instrument automatically calculates a Cutoff value according to the RLU in the calibrator, the result of sample measurement is judged by an S/CO value, and the S/CO value calculation formula is shown in (1):
S/CO＝RLU sample to be measured Cutoff value (1)
The decision criteria are as follows:
negative (-). S/CO <1.000, and HIV-1/2 type antibody and HIV-1p24 antigen are not detected in the sample to be detected.
Positive (+): S/CO is more than or equal to 1.000, and HIV-1/2 type antibody or HIV-1p24 antigen is not detected in the sample to be detected.
As can be seen from Table 16, the test results of the dilutions provided in example 4 of the present invention are consistent with the clinical samples, while the test results of the dilutions provided in comparative examples 9 and 10 show that the dilutions provided in example 4 of the present invention have good specificity, and further demonstrate that the zwitterionic polypeptides can avoid the defects of conventional protein protectants such as casein and BSA, and reduce the high background or false positive signals caused by non-specific adsorption due to the introduction of animal-derived IgG.
In summary, the diluent for the chemiluminescent reagent provided by the embodiment of the invention can effectively protect magnetic particles or acridinium ester antigen-antibody conjugate, avoid antibody allosteric, and thus, generate aggregation and precipitation, so that the magnetic particles or the acridinium ester antigen-antibody conjugate in the diluent can be stored for a long time. The main components of the amphiphilic compound are convenient to obtain, the defects of traditional protein protectants such as BSA and casein are avoided, high background or false positive signals caused by nonspecific adsorption due to the introduction of animal-derived IgG are reduced, and meanwhile, the specificity, repeatability and other performances of the reagent are not influenced, so that the amphiphilic compound is suitable for popularization and use.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Claims (10)
1. A protein protecting agent, which is characterized in that the protein protecting agent is amphiphilic amino acid and/or zwitterionic polypeptide; preferably, the amphiphilic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
2. Use of a protein protectant according to claim 1 for the preparation of a diluent for a chemiluminescent reagent; preferably, the mass percentage concentration of the protein protectant in the diluent is 0.5% -2%; most preferably, the diluent further comprises: buffer system, polyol, surfactant and saccharide.
3. A diluent for a chemiluminescent reagent, the diluent comprising: a protein protecting agent which is an amphiphilic amino acid and/or a zwitterionic polypeptide; preferably, the mass percentage concentration of the protein protecting agent is 0.5% -2%; most preferably, the amphiphilic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
4. A diluent for a chemiluminescent reagent comprising the following materials:
a protein protective agent with the mass percentage concentration of 0.1-2%, wherein the protein protective agent comprises amphiphilic amino acid and/or zwitterionic polypeptide; preferably a protein protectant with a mass percent concentration of 0.5% -2%;
a buffer system with a concentration of 0.01-0.1 mol/L; preferably a buffer system with a concentration of 0.01-0.05 mol/L;
polyol with volume percentage concentration of 1-20%; preferably a polyol having a concentration of 5 to 10% by volume;
surfactant with mass percentage concentration of 0.01-0.1%;
saccharide with mass percentage concentration of 1-10%;
preferably, the amphiphilic amino acid is selected from one or more of tryptophan, methionine, leucine and L-proline; and/or, the zwitterionic polypeptide is a polymer of lysine and glutamic acid.
5. The diluent of claim 4 wherein the protein protectant is a mixture of L-proline and methionine.
6. The diluent of claim 4 wherein the protein protectant is leucine.
7. The diluent of claim 4 wherein the protein protectant is a polymer of lysine and glutamic acid; preferably, the molar ratio of lysine to glutamic acid is (0.5-1.5): 1; most preferably, the molar ratio of lysine to glutamic acid is 1:1.
8. The diluent of claim 4 or 7, wherein the polymer of lysine and glutamic acid has a molecular weight of 70000 to 150000Da.
9. The diluent of any one of claims 4 to 8, wherein the polyol is selected from one or more of sorbitol, ethylene glycol, glycerol, mannitol;
and/or the saccharide is selected from sucrose or trehalose with a mass percentage concentration of 5-10%;
and/or the surfactant is poloxamer 188 with the mass percentage concentration of 0.05-0.1% or poloxamer 407 with the mass percentage concentration of 0.05-0.1%;
and/or the buffer system comprises one or more of Hepes buffer, PBS buffer and 2-morpholinoethanesulfonic acid buffer.
10. The diluent according to any one of claims 4 to 8, further comprising the following raw materials in mass percent: 0.3-1% of preservative and/or 0.5-2% of inorganic salt;
preferably, the preservative is PC-300 or PC-950;
preferably, the inorganic salt is sodium salt and/or potassium salt; more preferably, the inorganic salt is 0.5-1% sodium chloride.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211085928.5A CN117705549A (en) | 2022-09-06 | 2022-09-06 | Protein protecting agent, diluent and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211085928.5A CN117705549A (en) | 2022-09-06 | 2022-09-06 | Protein protecting agent, diluent and application |
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| CN117705549A true CN117705549A (en) | 2024-03-15 |
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| CN202211085928.5A Pending CN117705549A (en) | 2022-09-06 | 2022-09-06 | Protein protecting agent, diluent and application |
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| CN (1) | CN117705549A (en) |
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