CN114324882A - Protein stabilizer and application thereof - Google Patents
Protein stabilizer and application thereof Download PDFInfo
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- CN114324882A CN114324882A CN202011087597.XA CN202011087597A CN114324882A CN 114324882 A CN114324882 A CN 114324882A CN 202011087597 A CN202011087597 A CN 202011087597A CN 114324882 A CN114324882 A CN 114324882A
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Abstract
The invention relates to the field of in-vitro diagnosis products, and particularly provides a protein stabilizer and application thereof. The protein stabilizer can realize the stabilization of protein (especially low-concentration protein) by combining buffer solution, cosolvent and a small amount of inert protein, prolongs the stability of effective period and thermal stability, is particularly suitable for preparing a calibrator of an in vitro diagnosis product, and can obviously improve the stability of a low-value calibrator.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis products, and particularly relates to a protein stabilizer and application thereof.
Background
In vitro diagnostic products, a complete set of reagents often includes calibrators from the lower to the upper limit of the detection range, which meet certain shelf-life stability, thermal stability, as required by industry standards. Some manufacturers have stringent requirements for the thermal stability of the calibrator (e.g., about 50 ℃ for 3 days). While some antigens are diluted to very low concentration levels (e.g., below 10 ng/ml) to ensure sensitivity, low-value calibrators are used, and higher concentrations of low-concentration proteins are more susceptible to factors such as adsorption to the vessel wall, conformational changes, and temperature, which can lead to denaturation and inactivation and destabilization. At present, a single antigen diluent is difficult to maintain the stability of a low-concentration level protein under a high-temperature condition, although a common stabilizer on the market enhances the stabilizing effect by adding high-concentration animal serum, an antioxidant or a reducing agent, and metal ions or coenzyme, the methods have the defects of complex components, difficult guarantee of batch-to-batch consistency and thermal stability, and easy denaturation and inactivation of the protein. Therefore, there is a need for a diluent that solves the problem of high temperature stability of low-concentration proteins, has no biological safety risk, and has good batch-to-batch consistency.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide a protein stabilizer.
The second purpose of the invention is to provide the application of the protein stabilizer.
The third purpose of the invention is to provide an in vitro diagnostic reagent calibrator.
The fourth object of the present invention is to provide an in vitro diagnostic reagent.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a protein stabilizer is composed of the following components:
a buffer solution;
saccharides: sucrose and trehalose;
inert protein: BSA;
a stabilizer: glycerol and mannitol;
surfactant (b): TritonX-100 and Tween-20.
Further, the protein stabilizer is characterized in that the pH value of the buffer solution is 6.39-6.62;
optionally, the buffer comprises Tris, HEPES, MOPS, PB, or Citrate;
optionally, the buffer is at a final concentration of 20mM to 30mM in the protein stabilizing agent;
optionally, the inert protein comprises BSA or casein;
optionally, the inert protein is present in the protein stabilizing agent at a final concentration of 0.5 w/v% to 2 w/v%;
optionally, the final concentration of the surfactant in the protein stabilizing agent is 0.01 w/v% -0.2 w/v%;
optionally, the final concentration of sucrose in the protein stabilizing agent is 3 w/v% -5 w/v%;
optionally, the trehalose is at a final concentration of 3 w/v% to 5 w/v% in the protein stabilizing agent.
Further, the protein stabilizer consists of the following components: 20-30mM Tris-HCl, 3-5 w/v% sucrose, 3-5 w/v% trehalose, 0.5-2 w/v% BSA, 1.5-3.5 v/v% glycerol, 1.5-3.5 w/v% mannitol, 0.01-0.1 v/v% TritonX-100 and 0.01-0.2 v/v% Tween-20, pH 6.39-6.62.
Further, the protein stabilizer also contains a preservative, and the preservative can be 0.01-0.2 v/v% of Procling300 or sodium azide.
Further, the protein is a carbohydrate protein tumor associated antigen.
Further, the protein comprises squamous cell carcinoma antigen, CA125, CA153, CA19-9, or CA 724.
The protein stabilizer is applied to the preparation of in vitro diagnostic reagent calibrator or quality control product.
An in vitro diagnostic reagent calibrator or quality control product adopts the protein stabilizer as a diluent.
An in vitro diagnostic reagent comprises the in vitro diagnostic product calibrator or quality control product.
Further, the in vitro diagnostic product detects squamous cell carcinoma antigen, CA125, CA153, CA19-9, or CA 724.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a protein stabilizer, which consists of the following components: Tris-HCl, sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20. The stabilizer can overcome the defect that a large amount of animal serum, antioxidant or reducing agent, metal ions and the like are required to be added to a common stabilizer to improve the stability of protein, can realize the stability of protein (particularly low-concentration protein) through the combination of buffer solution, cosolvent and a small amount of inert protein, prolongs the stability of effective period and thermal stability, is particularly suitable for preparing a calibrator of an in vitro diagnosis product, can obviously improve the stability of a low-value calibrator, and generates certain economic benefit for a clinical diagnosis reagent manufacturer. For example, for 1.5ng/ml squamous cell carcinoma antigen calibrator, under the condition of 37 ℃ hot pressing acceleration test for 7d, the loss of the biological activity is not more than 10% compared with that of 4 ℃, the stability of low-concentration protein is greatly improved, and the overall period of validity of the product is effectively prolonged. Meanwhile, the stabilizer has the advantages of fewer component types, simpler preparation, better batch consistency, low cost and good broad spectrum property, and is suitable for various common proteins. In addition, because no animal serum is used, biological source pollution is avoided.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a general linear model of Bias42/4 versus pH at a concentration of 1.5ng/ml in thermal stress acceleration test 3d in example 2;
FIG. 2 is a graph of normalized effect Pareto in a mixed linear model of Bias42/4 with variables at a concentration of 3d 1.5ng/ml for hot stress accelerated test in example 2;
FIG. 3 is a general linear model of Bias52/4 versus pH at 1.5ng/ml concentration for hot stress accelerated test 3d in example 2;
FIG. 4 is a graph of normalized effect Pareto in a mixed linear model of Bias52/4 with variables at a concentration of 3d 1.5ng/ml for hot stress accelerated test in example 2;
FIG. 5 is a general linear model of Bias37/4 versus pH at a concentration of 5d 1.5ng/ml for hot stress accelerated test in example 2;
FIG. 6 is a graph of normalized effect Pareto in a mixed linear model of Bias37/4 with variables at a concentration of 5d 1.5ng/ml for hot stress accelerated test in example 2;
FIG. 7 is a general linear model of Bias42/4 versus pH at a concentration of 5d 1.5ng/ml for hot stress accelerated test in example 2;
FIG. 8 is a graph of normalized effect Pareto in a mixed linear model of Bias42/4 with variables at a concentration of 5d 1.5ng/ml for hot stress accelerated test in example 2;
FIG. 9 is a general linear model of Bias52/4 versus pH at a concentration of 5d 1.5ng/ml for hot stress accelerated test in example 2;
FIG. 10 is a graph of normalized effect Pareto in a mixed linear model of Bias52/4 with various variables at a concentration of 5d 1.5ng/ml for hot stress accelerated test in example 2.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
The protein stabilizer provided by the invention comprises the following components: Tris-HCl, sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20.
The protein stabilizer is added with the following auxiliary materials: firstly, buffering pair: such as Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), Phosphate (PBS), 2- (N-morpholine) ethanesulfonic acid Monohydrate (MES), etc.; II, co-solvent: sugars and polyols, amino acids and their derivatives, salts, macromolecular polymers, surfactants, and the like; thirdly, protein: animal serum albumin; fourthly, antioxidant protective agent and reducing agent: antioxidants such as citric acid, metal chelating agents such as ethylenediaminetetraacetic acid (EDTA), and the like; penta, substrates, coenzymes, or activating ions: such as Ca2+、Zn2+、Mg2+Etc.; sixthly, phospholipid and fatty acid.
In the invention, the inventor finally obtains the protein stabilizer provided by the invention by screening hundreds of formulas on the basis of a large amount of experimental verification data, the stabilizer can overcome the defect that the stability of the protein is improved by adding a large amount of animal serum, antioxidant or reducing agent, metal ions and the like to a common stabilizer, and the stability of the protein (especially low-concentration protein) can be realized and the stability and the heat stability of the effective period are prolonged by the combination of buffer solution (Tris-HCl), cosolvent (sucrose, trehalose, glycerol, mannitol, TritonX-100 and Tween-20) and a small amount of inert protein (BSA), so that the protein stabilizer is particularly suitable for preparing a calibrator of an in vitro diagnosis product, the stability of the low-value calibrator can be obviously improved, and certain economic benefits are generated for clinical diagnosis reagent manufacturers. Meanwhile, the stabilizer has fewer components, simpler preparation, better batch consistency (avoiding the use of animal serum), low cost and good broad spectrum, and is suitable for various common proteins. In addition, because no animal serum is used, biological source pollution is avoided.
In a preferred embodiment, the stabilizing agent comprises 20mM-30mM Tris-HCl, (3-5) w/v% (w/v) sucrose, (3-5) w/v% (w/v) trehalose, (0.5-2) w/v% BSA, (1.5-3.5) v/v% glycerol, (1.5-3.5) w/v% mannitol, (0.01-0.1) v/v% TritonX-100 and (0.01-0.2) v/v% Tween-20, pH 6.39-6.62.
"w/v%" means the number of grams of a substance contained in 100ml of a solution; "v/v%" refers to the number of milliliters of a substance contained in 100ml of solution. Wherein, the concentration of Tris-HCl can be but is not limited to 20mM, 22mM, 24mM, 26mM, 28mM or 30 mM; the concentration of sucrose may be, but is not limited to, 3 w/v%, 3.5 w/v%, 4 w/v%, 4.5 w/v%, or 5 w/v%; the concentration of trehalose may be, but is not limited to, 3 w/v%, 3.5 w/v%, 4 w/v%, 4.5 w/v%, or 5 w/v%; the concentration of BSA may be, but is not limited to, 0.5 w/v%, 0.7 w/v%, 1 w/v%, 1.3 w/v%, 1.5 w/v%, 1.7 w/v%, or 2 w/v%; the concentration of glycerol may be, but is not limited to, 1.5 v/v%, 1.8 v/v%, 2 v/v%, 2.3 v/v%, 2.5 v/v%, 2.7 v/v%, 3 v/v%, 3.2 v/v%, or 3.5 v/v%; the concentration of mannitol may be, but is not limited to, 1.5 w/v%, 1.8 w/v%, 2 w/v%, 2.3 w/v%, 2.5 w/v%, 2.7 w/v%, 3 w/v%, 3.2 w/v%, or 3.5 w/v%; the concentration of TritonX-100 may be, but is not limited to, 0.01 v/v%, 0.03 v/v%, 0.05 v/v%, 0.07 v/v%, or 0.1 v/v%; the concentration of Tween-20 may be, but is not limited to, 0.01 v/v%, 0.05 v/v%, 0.1 v/v%, 0.15 v/v%, or 0.2 v/v%; the pH may be, but is not limited to, 6.39, 6.44, 6.49, 6.54, 6.59, or 6.62.
In a preferred embodiment, the protein stabilizing agent further comprises a preservative, and the preservative can be 0.01-0.2 v/v% of Procling300 or sodium azide. The concentration of the preservative may be, but is not limited to, 0.01 v/v%, 0.05 v/v%, 0.1 v/v%, 0.15 v/v%, or 0.2 v/v%.
In a more preferred embodiment, the protein stabilizing agent consists of: 25mM Tris-HCl, 4 w/v% sucrose, 4 w/v% trehalose, 1 w/v% BSA, 2.5 v/v% glycerol, 2.5 w/v% mannitol, 0.05 v/v% TritonX-100, 0.1 v/v% Tween-20 and 0.1 v/v% Proclin-300, pH 6.44.
In some embodiments, the protein stabilizing agent provided by the present invention has a good effect on the stability of carbohydrate tumor associated antigens, such as squamous cell carcinoma antigen, CA125 (ovarian cancer antigen), CA153, CA19-9, or CA724 (gastric cancer antigen), and the like. Particularly, the stability effect of the protein in a low concentration range of 1ng/ml-60ng/ml is more remarkable.
Squamous Cell Carcinoma Antigen (SCCA) is a TA-4 subcomponent Squamous epithelial carcinoma Antigen extracted from metastatic lesions of cervical cancer. It is a tumor marker that is very specific and the earliest used for diagnosis. Originally isolated from cervical cancer tissues, belong to the family of serine protease inhibitors with respect to biological activity. It includes two genes, SCCA1 and SCCA 2. SCCA is found in a wide range of normal tissues (in microscopic quantities) of different organs and in malignant diseased epithelial cells. There are at least 4 forms of SCCA in serum, free SCCA1, free SCCA2 and the corresponding serine protease conjugates. SCCA inhibits apoptosis in normal squamous epithelial cells and is involved in differentiation of the squamous epithelial layer, in tumor cell growth, which aids in the diagnosis and monitoring of cancers of all squamous epithelial cell origin, for example: cervical cancer, lung cancer (non-small cell lung cancer), head and neck cancer, esophageal cancer, and vulvar cell cancer. Squamous cell carcinoma antigens are present in very low amounts in normal tissues and therefore require to be formulated with antigens to correspondingly low concentrations for calibration during the development of the in vitro diagnostic reagents. The protein stabilizer provided by the invention has good effect on the stability of low-concentration antigen of squamous cell carcinoma, for example, the loss of biological activity of 1.5ng/ml squamous cell carcinoma antigen calibrator is not more than 10% compared with 4 ℃ under the condition of 37 ℃ hot pressing acceleration test for 7 d.
The invention also provides a preparation method of the low-concentration protein stabilizer, which is obtained by mixing Tris-HCl, sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100, Tween-20 and Proclin-300. Further, Tris-HCl is dissolved in ultrapure water to prepare buffer solution, and then other substances are added and mixed evenly.
The low-concentration protein stabilizer provided by the invention can be used as a diluent of a calibrator or a quality control product in an in-vitro diagnostic reagent, the stability of the prepared calibrator or quality control product and the reagent containing the calibrator or quality control product is greatly improved, and the expiration date is obviously prolonged.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Materials, reagents, and the like used in the embodiments are commercially available unless otherwise specified; the experimental methods are not specially explained, and are all conventional methods. The information of each material and reagent is as follows:
| reagent and apparatus | Manufacturer and product catalog number | Purity or accuracy of |
| Tris | Amresco，0497-5kg | VWR Ultra Pure |
| Sucrose | Raw worker, A502792-0500 | >99％ |
| Trehalose | Aladdin，T100010-500g | ≥99％ |
| Glycerol | Beijing chemical engineering, 20181220 | Analytical purity |
| Mannitol | Aladdin，M108828-500g | AR,98.0％ |
| Proclin-300 | Sigma-Aldrich，1002772587 | Reagent Grade |
| BSA | Proliant Biologicals，BB81430101 | Reagent Grade |
| Triton X-100 | Raw Process, A110694-0500 | >98％ |
| Tween-20 | Sigma-Aldrich，P1379-500ml | Reagent Grade |
| PH meter | Mettler Toledo S210 | 0.01 |
Example 1 stability assessment embodiment:
the assessment standard is as follows: the formula can ensure that the loss of the biological activity of the calibrator does not exceed +/-10 percent compared with the loss of the biological activity at 4 ℃ under the condition of 37 ℃ hot pressing acceleration test for 7 d.
The stability assessment process is set as follows: under a streptavidin two-step detection mode, the high-concentration and low-concentration calibrators are subjected to thermal stress acceleration tests at 37 ℃, 45 ℃ and 52 ℃ for 3d, 5d and 7 d.
The specific embodiment is as follows:
1. preparing recombinant antigen calibrators with different concentrations: the recombinant squamous cell carcinoma antigen stock solution is diluted into two concentrations of high concentration and low concentration by antigen diluent, wherein the concentrations are respectively 1.5ng/ml, 60ng/ml or 1.5ng/ml and 15 ng/ml.
2. Subpackaging antigen calibrators with different concentrations: the antigen calibrator prepared from different antigen dilutions was dispensed into 300. mu.l each tube through a 1.5ml centrifuge tube.
3. Labeling: labeling the split charging tube of the high-concentration and low-concentration calibrator formed by diluting different antigen diluents, wherein the label comprises diluent types, a standing temperature, a concentration value and thermal disruption starting time.
4. Placing a calibrator, and setting detection time: the calibrator with different concentrations diluted by different antigen diluents with the same temperature is placed at the same temperature, and the temperature gradient is set to be 4 ℃, 37 ℃, 45 ℃ and 52 ℃. The detection time is set to be 3d, 5d, 7d, and is not equal.
A two-step streptavidin detection mode:
uniformly mixing the calibrator and the streptavidin-labeled antibody, incubating at 42 ℃ for 2.5min, adding streptavidin-coupled magnetic beads, incubating at 42 ℃ for 2min, washing with a cleaning solution for 4 times, adding an alkaline phosphatase-labeled antibody, incubating at 42 ℃ for 3.5min, washing with a cleaning solution for 4 times, adding substrate AMPPD, incubating at 37 ℃ for 5min, and detecting.
The preparation method of the antigen diluent comprises the following steps:
1. weighing Tris, adding Pall ultrapure water for dissolving, adjusting the pH to 8.0 by hydrochloric acid, and fixing the volume to be a formula amount of Tris-HCl solution;
2. sequentially weighing sucrose, trehalose, glycerol, mannitol, TritonX-100, Tween-20 and Proclin300, dissolving in a Tris-HCl solution, and fully stirring and dissolving by using a magnetic stirrer;
3. adjusting the pH of the solution prepared in the step 2 to about 7.0 by a pH meter, HCl and NaOH, adding BSA, and stirring by using a magnetic stirrer until the BSA is completely dissolved;
4. the solution was accurately adjusted to the formula value by means of a pH meter, HCl and NaOH, and filtered through Millipore 0.22um microporous membrane after constant volume, and stored at 4 ℃.
EXAMPLE 2 formulation screening of different antigen dilutions
We identified three major factors that play a critical role in recombinant squamous cell carcinoma antigen stability in the laboratory's existing dilutions through exploration: pH, BSA, and polyethylene glycol (PEG 4000). The experiment is carried out through three-factor three-gradient interaction experiment, and the main experiment design is as follows:
the specific formulation of a representative combination of the three-factor three-gradient interaction test design is as follows:
the results of examination with reference to the examination protocol in example 1 (Bias37/4, Bias42/4, Bias52/4 indicate the percentage of loss of biological activity in the squamous cell carcinoma antigen treated at 37 ℃, 42 ℃ and 52 ℃ respectively, as compared with 4 ℃; CV4, CV37, CV42 and CV52 indicate the squamous cell carcinoma antigen treated at 4 ℃, 37 ℃, 42 ℃ and 52 ℃ respectively, and the same applies to the test light emission values CV) were as follows:
through Minitab data analysis, the Bias37/4 under the concentration of 1.5ng/ml in the hot forced acceleration test 3d has no significant correlation with the general linear model and the mixed linear model of each variable, namely the thermal stability at 37 ℃ and the variation trend of each variable cannot be judged at the moment; while the general linear model and the mixed linear model of the Bias42/4 and each variable are significantly related to pH (FIGS. 1 and 2), which shows that pH significantly affects the stability under the condition, and the model predicts that the absolute value of the Bias42/4 is the minimum in the pH range of 7.53-7.74; similarly, Bias52/4 was significantly correlated with pH, both the general linear model and the mixed linear model for each variable (fig. 3 and 4), indicating that pH appears to affect stability under these conditions, and that the model predicts that Bias52/4 is the smallest in absolute value over the pH range of 7.08-7.22.
Similarly, the general linear model and the mixed linear model of the Bias37/4 and each variable at the concentration of 1.5ng/ml are obviously related to the pH (figures 5 and 6) when the heat stress is analyzed for 5d, and the Bias37/4 absolute value is the minimum within the range of 6.39-6.62 by the model prediction; bias42/4 was significantly associated with pH with both the general linear model and the mixed linear model of each variable (fig. 7 and 8), which predicted the smallest absolute value of Bias42/4 in the pH range of 6.50-6.62; bias52/4 was significantly associated with pH with both the general linear model and the mixed linear model of each variable (fig. 9 and 10), which predicted the smallest absolute value of Bias52/4 in the pH range of 6.28-6.51.
In summary, the thermal stability of the 1.5ng/ml low concentration calibrator in the three gradient thermal stress accelerated tests 3d-5d at 37 ℃, 42 ℃ and 52 ℃ is mainly affected by the pH, which is shown by the lower pH requirement at higher temperature and longer time. According to the model, the optimal pH range of 1.5ng/ml calibrator after being thermally broken at 37 ℃ for 5d is predicted to be 6.39-6.62.
Example 3 protein stabilizer obtention
According to the analysis result of the example 2, a pH gradient is designed to verify that the specific formula is as follows:
the examination was performed according to the examination protocol in example 1, and the results were as follows:
according to the analysis of the results, the stabilizer 32 is only one diluent with the signal value of 3d-7d not more than 10% reduced compared with the signal value of 4 ℃ at the temperature of 37 ℃, namely the formula can pass the thermal stability of 7d at the temperature of 37 ℃.
Example 4 component orthogonal screening of protein stabilizers
The examination was performed according to the examination protocol in example 1, and the results were as follows:
| Bias37/4 1.5ng/ml | Bias37/4 15ng/ml | |
| stabilizer 34 | -17.1％ | -13.2％ |
| Stabilizer 35 | -15.3％ | -12.9％ |
| Stabilizer 36 | -20.4％ | -16.8％ |
| Stabilizer 37 | -18.9％ | -15.4％ |
| Stabilizer 38 | -14.9％ | -11.6％ |
| Stabilizer 39 | -12.4％ | -11.2％ |
| Stabilizer 40 | -8.2％ | -3.1％ |
| Stabilizer 41 | -5.7％ | -2.6％ |
| Stabilizer 42 | -4.6％ | -1.8％ |
As can be seen from the results analysis, the Tris-HCl, sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100, Tween-20 and Proclin-300 components are the simplest combination for protein stabilizers, and the effect of the stabilizers is influenced by the absence of any one of the components.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. A protein stabilizer is characterized by comprising the following components:
a buffer solution;
saccharides: sucrose and trehalose;
an inert protein;
a stabilizer: glycerol and mannitol;
surfactant (b): TritonX-100 and Tween-20.
2. The protein stabilizer according to claim 1, wherein the buffer has a pH of 6.39 to 6.62;
optionally, the buffer comprises Tris, HEPES, MOPS, PB, or Citrate;
optionally, the buffer is at a final concentration of 20mM to 30mM in the protein stabilizing agent;
optionally, the inert protein comprises BSA or casein;
optionally, the inert protein is present in the protein stabilizing agent at a final concentration of 0.5 w/v% to 2 w/v%;
optionally, the final concentration of the surfactant in the protein stabilizing agent is 0.01 w/v% -0.2 w/v%;
optionally, the final concentration of sucrose in the protein stabilizing agent is 3 w/v% -5 w/v%;
optionally, the trehalose is at a final concentration of 3 w/v% to 5 w/v% in the protein stabilizing agent.
3. The protein stabilizer according to claim 1, characterized by consisting of:
20mM-30mM Tris-HCl, 3 w/v% -5 w/v% sucrose, 3 w/v% -5 w/v% trehalose, 0.5 w/v% -2 w/v% BSA, 1.5 w/v% -3.5 v/v% glycerol, 1.5 w/v% -3.5 w/v% mannitol, 0.01 w/v% -0.1 v/v% TritonX-100 and 0.01 w/v% -0.2 v/v% Tween-20, pH6.39-6.62.
4. The protein stabilizer according to claims 1-3, wherein the protein stabilizer further comprises a preservative, and the preservative is selected from 0.01-0.2 v/v% Procling300 or sodium azide.
5. The protein stabilizer according to claim 1 to 3 or 4, wherein the protein is a carbohydrate protein tumor-associated antigen.
6. The protein stabilizer of claim 5, wherein the protein comprises squamous cell carcinoma antigen, CA125, CA153, CA19-9, or CA 724.
7. Use of a protein stabilizing agent as claimed in any one of claims 1 to 6 in the preparation of a calibrator or quality control for in vitro diagnostic reagents.
8. An in vitro diagnostic reagent calibrator or quality control, wherein said calibrator or quality control employs the protein stabilizer of any one of claims 1-6 as a diluent.
9. An in vitro diagnostic reagent comprising the calibrator or quality control according to claim 8.
10. The in vitro diagnostic reagent of claim 9, wherein the in vitro diagnostic reagent detects squamous cell carcinoma antigen, CA125, CA153, CA19-9, or CA 724.
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Inventor after: Xiang Haijun Inventor after: Liu Jiangwu Inventor after: Xia Liangyu Inventor before: Xiang Haijun Inventor before: Liu Jianghong Inventor before: Xia Liangyu |