CN111103432B - Freeze-drying auxiliary preparation for thyroid stimulating hormone receptor and freeze-drying method - Google Patents
Freeze-drying auxiliary preparation for thyroid stimulating hormone receptor and freeze-drying method Download PDFInfo
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- CN111103432B CN111103432B CN202010013742.3A CN202010013742A CN111103432B CN 111103432 B CN111103432 B CN 111103432B CN 202010013742 A CN202010013742 A CN 202010013742A CN 111103432 B CN111103432 B CN 111103432B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
The invention relates to the technical field of immunoassay, in particular to a freeze-drying auxiliary preparation of a thyroid stimulating hormone receptor and a freeze-drying method. The freeze-drying auxiliary preparation consists of non-reducing disaccharide, glycine, serum albumin, polyethylene glycol and buffer solution; the contents of all components are as follows: 2-20 wt% of non-reducing disaccharide; 5-30mmol/L of glycine; 2-10 wt% of serum albumin; 0.5 wt% -2 wt% of polyethylene glycol; the balance of the buffer solution. The freeze-drying auxiliary preparation for the thyroid stimulating hormone receptor can effectively assist freeze-drying of the thyroid stimulating hormone receptor, so that the freeze-dried thyroid stimulating hormone receptor has stable shape, good freeze-drying and redissolving effects, no precipitate and no influence on biological activity.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a freeze-drying auxiliary preparation of a thyroid stimulating hormone receptor and a freeze-drying method.
Background
Autoimmune thyroid disease (AITD) is a typical organ-specific Autoimmune disease. Thyroid stimulating hormone receptor (thyrotropin receptor) is a protein-coupled macromolecular glycoprotein expressed on the surface of thyroid follicular cells and is the main antigen for inducing AITD. The antigen-antibody reaction model and the detection method established by the purified thyroid stimulating hormone receptor antigen have important significance for the differential diagnosis of AITD diseases.
In clinical immunoassays, the antigen is usually obtained by a relatively complicated process such as cell culture, expression, purification, and the like. Because the antigen obtained by the method is extremely unstable in a liquid state, at present, most thyroid stimulating hormone receptors are stored and transported by freeze drying. However, the freeze-drying process is a complex phase transition process in which the resulting low temperature stress, freezing stress, drying stress cause structural changes in the thyroid stimulating hormone receptor, decreased solubility, poor re-solubility, decreased biological activity, and the like.
In conclusion, a freeze-drying auxiliary preparation capable of solving the problems of structural change, low solubility, poor re-solubility, reduced biological activity and the like of a thyroid stimulating hormone receptor after freeze-drying is a technical problem to be solved by the technical personnel in the field. No studies have been made on lyophilized auxiliary preparations for thyroid stimulating hormone receptors. The freeze-drying auxiliary preparation of other bioactive substances is not suitable for freeze-drying of the thyroid stimulating hormone receptor, and still causes the problems of reduced solubility, poor re-solubility, reduced bioactivity and the like of the thyroid stimulating hormone receptor.
Disclosure of Invention
In view of the above, the present invention provides a freeze-drying auxiliary preparation for thyroid stimulating hormone receptor and a freeze-drying method. The freeze-drying auxiliary preparation can effectively assist the freeze-drying of the thyroid stimulating hormone receptor, so that the thyroid stimulating hormone receptor is stable in form after freeze-drying, good in freeze-drying and redissolving effects and free of precipitate.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a freeze-drying auxiliary preparation of a thyroid stimulating hormone receptor, which consists of non-reducing disaccharide, glycine, serum albumin, polyethylene glycol and buffer solution.
Preferably, the freeze-drying auxiliary preparation comprises the following components in percentage by weight:
preferably, the freeze-drying auxiliary preparation comprises the following components in percentage by weight:
more preferably, the freeze-drying auxiliary preparation comprises the following components in percentage by weight:
preferably, the non-reducing disaccharide is sucrose and/or trehalose.
Preferably, the serum albumin is bovine serum albumin and/or human serum albumin.
Preferably, the polyethylene glycol is one or more of polyethylene glycol 6000, polyethylene glycol 10000 and polyethylene glycol 20000.
Preferably, the buffer is one of Bis-Tris propane buffer, Bis-Tris buffer and phosphate buffer.
Preferably, the pH value of the buffer solution is 6.5-8.0.
The invention also provides a freeze-drying method of the thyroid stimulating hormone receptor, which comprises the step of mixing the thyroid stimulating hormone receptor with the freeze-drying auxiliary preparation and then carrying out freeze-drying.
Preferably, the volume ratio of the thyroid stimulating hormone receptor to the freeze-drying auxiliary preparation is 1: (20-30).
The invention provides a freeze-drying auxiliary preparation of a thyroid stimulating hormone receptor and a freeze-drying method. The freeze-drying auxiliary preparation consists of non-reducing disaccharide, glycine, serum albumin, polyethylene glycol and buffer solution; the contents of all components are as follows: 2-20 wt% of non-reducing disaccharide; 5-30mmol/L of glycine; 2-10 wt% of serum albumin; 0.5 wt% -2 wt% of polyethylene glycol; the balance of the buffer solution. The invention has the technical effects that:
the freeze-drying auxiliary preparation for the thyroid stimulating hormone receptor can effectively assist freeze-drying of the thyroid stimulating hormone receptor, so that the freeze-dried thyroid stimulating hormone receptor has stable shape, good freeze-drying and redissolving effects, no precipitate and no influence on biological activity.
Detailed Description
The invention discloses a freeze-drying auxiliary preparation of a thyroid stimulating hormone receptor and a freeze-drying method, and the freeze-drying auxiliary preparation can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a thyroid stimulating hormone receptor freeze-drying auxiliary preparation, which comprises a buffer medium with the pH range of 6.5-8.0, 5-10% of thyroid stimulating hormone receptor by volume, non-reducing disaccharide with the amount of about 2-20% by weight, bovine serum albumin with the amount of about 2-10% by weight, polyethylene glycol with the amount of about 0.5-2% by weight and glycine with the concentration of about 5-30 mmol/L.
The freeze drying process is a multi-step process and can generate various effects such as low temperature, freezing and dehydration; even after the freeze-drying process is successfully completed, it is difficult to ensure the stability of the active ingredient in the lyophilized product during long-term storage. In order to prevent the active ingredients in the lyophilized products from being denatured during the freeze-drying and storage processes, auxiliary agents with protective action are required. The freeze-dried thyroid stimulating hormone receptor product obtained by using the freeze-dried thyroid stimulating hormone receptor auxiliary preparation provided by the invention has the advantages of good cake shape, pure white color, good redissolution effect, no appearance of incompatible particles or flaky precipitates and good stability. The freeze-drying auxiliary preparation greatly improves the storage stability of the thyroid stimulating hormone receptor, so that the freeze-drying auxiliary preparation has wider application in the field of immunoassay.
The freeze-drying auxiliary preparation of the thyroid stimulating hormone receptor and the reagent or the instrument used in the freeze-drying method can be purchased from the market.
The invention is further illustrated by the following examples:
examples 1 to 15
Thyroid stimulating hormone receptor protein is combined with buffer solution, non-reducing disaccharide, bovine serum albumin, polyethylene glycol and glycine according to the volume ratio of 1:24 in the table 1 in different proportions, and then freeze drying is carried out by the same procedure (the freeze drying curves all adopt-80 ℃ for precooling for 2h, sublimation for one time is carried out at-40 ℃ to-15 ℃ for 12h, and drying for two times is carried out at-15 ℃ to 25 ℃ for 8 h). Evaluation indexes of the freeze-dried product include: molding, redissolving (carrying out equal-volume redissolving by using deionized water), clarity, and titer evaluation before and after freeze-drying. Wherein the titer assessment before and after drying is expressed as signal value recovery before and after lyophilization.
Constructing a kit: the solid phase is an antibody of a stimulating thyroid stimulating hormone receptor coated by magnetic beads; the marker is a receptor which is marked by the marker and only binds to the thyroid stimulating hormone receptor antibody; the substance to be detected is a stimulating thyroid stimulating hormone receptor antibody; the method is utilized to respectively construct the detection kit of the stimulated thyroid stimulating hormone receptor antibody. And (3) detecting a calibrator containing a series of stimulating thyroid stimulating hormone receptor antibody concentrations of 0IU/L, 2IU/L, 5IU/L, 10IU/L, 20IU/L and 30IU/L, which are obtained by diluting with a calibrator diluent, in a parallel multi-well manner, measuring on an ampere chart biological full-automatic chemiluminescence apparatus A2000 PLUS system to obtain luminescence values of all embodiments, and meanwhile, calculating a mean value of recovery of concentration signal values of all calibrators. The detailed results are shown in Table 2 below.
TABLE 1 Freeze-drying adjuvant formulation
TABLE 2
Note: the average value of the recovery rate of the signal values of each point before and after freeze-drying is the signal value after freeze-drying/the signal value before freeze-drying; and (7) accelerating the stability of the light source by taking the first day as a standard luminous value as a reference, and calculating the average value of the recovery rate of the signal value of each point.
The results show that the freeze-dried products in the embodiments are well formed, and the redissolution time is less than 10 min; the recovery rate of signal values before and after freeze-drying is more than 90%; the stability test is accelerated, and the signal value recovery rate is over 90 percent.
The lyophilized product of example 1 was selected for further real-time stability assessment and accelerated stability assessment, the results of which are shown in table 3.
TABLE 3
Thyroid stimulating hormone receptors are membrane proteins, unstable and difficult to protect. From the table data, under the freeze-drying auxiliary preparation, the luminous count of the calibrator after 12 months still keeps about 90% and tends to a steady state, and the change is small; the accelerated stability results show that the signal value recovery rate is still about 90% after destructive baking at 37 ℃. The results show that the freeze-drying auxiliary preparation has good protection effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A freeze-dried auxiliary preparation of thyroid stimulating hormone receptor is characterized in that the freeze-dried auxiliary preparation consists of non-reducing disaccharide, glycine, serum albumin, polyethylene glycol and buffer; the contents of all components are as follows:
the polyethylene glycol is one or a combination of more of polyethylene glycol 6000, polyethylene glycol 10000 and polyethylene glycol 20000;
the non-reducing disaccharide is sucrose or trehalose;
the buffer solution is Bis-Tris propane buffer solution, Bis-Tris buffer solution or phosphate buffer solution.
2. The lyophilized auxiliary preparation according to claim 1, wherein the lyophilized auxiliary preparation comprises the following components:
3. the lyophilized auxiliary preparation according to claim 1, wherein the lyophilized auxiliary preparation comprises the following components:
4. the lyophilized co-formulation according to claim 1, wherein the serum albumin is bovine serum albumin and/or human serum albumin.
5. The lyophilization aid formulation according to any one of claims 1 to 4, wherein the buffer has a pH of 6.5 to 8.0.
6. A method for lyophilizing a thyroid stimulating hormone receptor, comprising mixing the thyroid stimulating hormone receptor with the lyophilization aid preparation according to any one of claims 1 to 5, and lyophilizing the mixture.
7. The lyophilization process according to claim 6, wherein the volume ratio of the thyroid stimulating hormone receptor to the lyophilization aid formulation is 1: (20-30).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5795789A (en) * | 1997-06-04 | 1998-08-18 | Dade Behring Inc. | Standard solution for the determination of thyroid function |
| CN102050875B (en) * | 2010-12-10 | 2013-05-29 | 天津市协和医药科技有限公司 | Extraction and purification method of TSHR (Thyroid Stimulating Hormone Receptor) |
| BR112013026883A2 (en) * | 2011-04-20 | 2021-12-07 | Sandoz Ag | Pharmaceutical composition, kit comprising a composition and method of producing a pharmaceutical composition |
| CN103472235A (en) * | 2013-08-26 | 2013-12-25 | 河北省科学院生物研究所 | Long-acting protein solution stabilizing agent |
| CN106645689A (en) * | 2016-06-30 | 2017-05-10 | 深圳市亚辉龙生物科技股份有限公司 | Thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit and preparation method thereof |
| CN109187781A (en) * | 2018-09-03 | 2019-01-11 | 美康生物科技股份有限公司 | Serotype freeze-dried powder 25-hydroxyvitamin D3 and D2 quality control substance matter and preparation method thereof |
| CN109298176A (en) * | 2018-10-29 | 2019-02-01 | 深圳天深医疗器械有限公司 | Myocarditis quality-control product and preparation method thereof, myocarditis detection kit and myocarditis detection device |
| CN109593834A (en) * | 2019-01-29 | 2019-04-09 | 百康芯(天津)生物科技有限公司 | Frozen-dried protective system needed for a kind of nucleic acid amplification agents and preparation method thereof |
| CN109738626B (en) * | 2019-02-19 | 2021-11-26 | 复星诊断科技(上海)有限公司 | NGAL latex immunoturbidimetry detection kit and preparation method thereof |
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