CN106800598B - Antibody preserving fluid and preparation method thereof - Google Patents
Antibody preserving fluid and preparation method thereof Download PDFInfo
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- CN106800598B CN106800598B CN201710071642.4A CN201710071642A CN106800598B CN 106800598 B CN106800598 B CN 106800598B CN 201710071642 A CN201710071642 A CN 201710071642A CN 106800598 B CN106800598 B CN 106800598B
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Abstract
The invention discloses an antibody preservation solution which comprises a buffer solution, 4-6% of trehalose, 0.005-0.01% of sodium azide, 30-50% of glycerol, 0.002-0.0025% of first polypeptide and 0.002-0.0025% of second polypeptide, wherein the buffer solution is a Tris-HCl buffer solution with the pH value of 7.0-7.5 and the concentration of 40-100 mmol/L, the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antibody preservation solution and a preparation method thereof.
Background
The antibody refers to immunoglobulin which is produced by plasma cells differentiated from B lymphocytes under the stimulation of antigen by immune system of body and can be specifically combined with corresponding antigen. Antibodies are widely used because they can specifically and stably bind to specific antigens. In the process of using the antibody, the activity and the using effect of the antibody are directly influenced by the proper storage, and for most antibodies, the antibody is generally stored at low temperature, and due to repeated freezing and thawing of the antibody, the antibody is denatured, multimers are generated, and the activity of the antibody is reduced.
How to provide an antibody preservation solution which can be preserved at room temperature for a long time is one of the problems to be solved in the field of antibody application.
Disclosure of Invention
The invention mainly solves the technical problem of providing an antibody preservation solution and a preparation method thereof, which can preserve an antibody at room temperature for a long time and can keep the antibody with high activity.
In order to solve the technical problems, the invention provides an antibody preservation solution which comprises a buffer solution, 4-6% of trehalose, 0.005-0.01% of sodium azide, 30-50% of glycerol, 0.002-0.0025% of a first polypeptide and 0.002-0.0025% of a second polypeptide, wherein the buffer solution is a Tris-HCl buffer solution with the pH value of 7.0-7.5 and the concentration of 40-100 mmol/L, the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratio, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
The antibody preservation solution comprises a buffer solution, 4.5-5.5% of trehalose, 0.006-0.009% of sodium azide, 35-45% of glycerol, 0.0021-0.0024% of first polypeptide and 0.0021-0.0024% of second polypeptide, wherein the buffer solution is a Tris-HCl buffer solution with the pH value of 7.0-7.5 and the concentration of 45-90 mmol/L, the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
The antibody preservation solution comprises buffer solution, 5% of trehalose, 0.007% of sodium azide, 40% of glycerol, 0.0022% of first polypeptide and 0.0022% of second polypeptide, the buffer solution is a Tris-HCl buffer solution with the pH value of 7.2 and the concentration of 50 mmol/L, the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
Wherein the mass ratio is g/L.
In order to solve the technical problems, the invention provides a preparation method of an antibody preservation solution, which comprises the following steps of adding 4-6% of trehalose, 0.005-0.01% of sodium azide, 30-50% of glycerol, 0.002-0.0025% of first polypeptide and 0.002-0.0025% of second polypeptide into a Tris-HCl buffer solution with the pH value of 7.0-7.5 and the concentration of 40-100 mmol/L, and uniformly mixing to prepare the antibody preservation solution, wherein the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
The antibody preservation solution comprises buffer solution, 4.5-5.5% of trehalose, 0.006-0.009% of sodium azide, 35-45% of glycerol, 0.0021-0.0024% of first polypeptide and 0.0021-0.0024% of second polypeptide, wherein the buffer solution is Tris-HCl buffer solution with the pH value of 7.0-7.5 and the concentration of 45-90 mmol/L.
The antibody preservation solution comprises buffer solution, 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% first polypeptide and 0.0022% second polypeptide, wherein the buffer solution is Tris-HCl buffer solution with the pH value of 7.2 and the concentration of 50 mmol/L.
Wherein the mass ratio is g/L.
The antibody preservation solution has the beneficial effects that the antibody preservation solution is different from the prior art, and comprises a buffer solution, 4-6% of trehalose, 0.005-0.01% of sodium azide, 30-50% of glycerol, 0.002-0.0025% of a first polypeptide and 0.002-0.0025% of a second polypeptide, wherein the buffer solution is a Tris-HCl buffer solution with the pH value of 7.0-7.5 and the pH value of 40-100 mmol/L, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
Detailed Description
Example 1
The preparation method of the antibody preservation solution comprises the following steps of adding 5% of trehalose, 0.007% of sodium azide, 40% of glycerol, 0.0022% of first polypeptide and 0.0022% of second polypeptide into a Tris-HCl buffer solution with the pH value of 7.2 and the concentration of 50 mmol/L, uniformly mixing, and preparing the antibody preservation solution, wherein the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, specifically g/L, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
The antibody preservation method comprises the steps of preserving the antibody by using the antibody preservation solution prepared in the example 1, preserving the antibody by using the existing room-temperature antibody preservation solution, preserving the antibody by using the existing low-temperature (-20 ℃) antibody preservation solution, preserving the antibody by using the growth hormone monoclonal antibody in each preservation mode for 1 year, and detecting the activity of the antibody by using enzyme linked immunosorbent assay (E L ISA).
The detection method specifically comprises the steps of diluting growth hormone antigen with 0.05 mol/L pH9.6 sodium carbonate-sodium bicarbonate buffer solution containing 5ppm Proclin300 to obtain coating solution, wherein the concentration of the growth hormone antigen in the coating solution is 10 mu g/ml, the sealing solution is 0.05 mol/L pH9.6 sodium carbonate-sodium bicarbonate buffer solution containing 5.0 g/L trehalose and 0.5% BSA, the coating solution is 90 mu l/hole, incubation is carried out for 2h in a 37 ℃ incubator, residual liquid is removed, washing is carried out for 3 times by using washing solution PBST on a plate washing machine and patted dry, 200 mu l/hole of the sealing solution is added, incubation is carried out at 25 ℃ overnight, residual liquid is removed, washing is carried out for 3 times by using PBST on the plate washing machine and dried, antibodies stored in different manners are added, the residual liquid is 90 mu l/hole, incubation is carried out for 1h in the 37 ℃ incubator for 1h, incubation is carried out by using the plate washing machine for 3 times, the washing solution is carried out for 3 times by using fresh PBST, the incubation is carried out at 37 ℃ for 3 times, the same incubation, the antibodies are added in the incubator and the incubation is added in the incubation at a different manners, the same wavelength, the pH value is added, the antibody is added in the absorbance of the staining machine, and the absorbance of the antibody is added, the antibody is measured, and the antibody is measured.
And (3) detection results: the absorbance of the antibody preserved in the antibody preservation solution prepared in example 1 was 1.9, the absorbance of the antibody preserved in the conventional room-temperature antibody preservation solution was 0.5, and the absorbance of the antibody preserved in the conventional low-temperature (-20 ℃) antibody preservation solution was 1.1.
From the results of the detection, it was demonstrated that the activity of the antibody preserved in the antibody preservation solution prepared in example 1 was higher than that of the antibody preserved in the other manner under the same conditions.
Example 2
The preparation method of the antibody preservation solution comprises the following steps of adding 5.3% of trehalose, 0.008% of sodium azide, 43% of glycerol, 0.0023% of first polypeptide and 0.0023% of second polypeptide into 50 mmol/L Tris-HCl buffer solution with the pH value of 7.2, uniformly mixing, and preparing the antibody preservation solution, wherein the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, specifically g/L, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
The activities of the antibodies preserved in different ways were examined below to illustrate the utility of the antibody preservation solution of this example. Antibody preservation methods include: preserving the antibody by using the antibody preservation solution prepared in example 2; preserving the antibody by using the existing room-temperature antibody preserving fluid; the antibody is preserved in the existing low-temperature (-20 ℃) antibody preservation solution. Wherein the antibody is a growth hormone monoclonal antibody, and the antibody is preserved for 1 year in each preservation mode. For the detection of the antibody activity, enzyme-linked immunosorbent assay is adopted for detection.
The detection method specifically comprises the steps of diluting growth hormone antigen with 0.05 mol/L pH9.6 sodium carbonate-sodium bicarbonate buffer solution containing 5ppm Proclin300 to obtain coating solution, wherein the concentration of the growth hormone antigen in the coating solution is 10 mu g/ml, the sealing solution is 0.05 mol/L pH9.6 sodium carbonate-sodium bicarbonate buffer solution containing 5.0 g/L trehalose and 0.5% BSA, the coating solution is 90 mu l/hole, incubation is carried out for 2h in a 37 ℃ incubator, residual liquid is removed, washing is carried out for 3 times by using washing solution PBST on a plate washing machine and patted dry, 200 mu l/hole of the sealing solution is added, incubation is carried out at 25 ℃ overnight, residual liquid is removed, washing is carried out for 3 times by using PBST on the plate washing machine and dried, antibodies stored in different manners are added, the residual liquid is 90 mu l/hole, incubation is carried out for 1h in the 37 ℃ incubator for 1h, incubation is carried out by using the plate washing machine for 3 times, the washing solution is carried out for 3 times by using fresh PBST, the incubation is carried out at 37 ℃ for 3 times, the same incubation, the antibodies are added in the incubator and the incubation is added in the incubation at a different manners, the same wavelength, the pH value is added, the antibody is added in the absorbance of the staining machine, and the absorbance of the antibody is added, the antibody is measured, and the antibody is measured.
And (3) detection results: the absorbance of the antibody preserved in the antibody preservation solution prepared in example 2 was 1.95, the absorbance of the antibody preserved in the conventional room-temperature antibody preservation solution was 0.5, and the absorbance of the antibody preserved in the conventional low-temperature (-20 ℃) antibody preservation solution was 1.1.
From the results of the detection, it was demonstrated that the activity of the antibody preserved in the antibody preservation solution prepared in example 2 was higher than that of the antibody preserved in the other manner under the same conditions.
Comparative example 1
The preparation method of the antibody preservation solution comprises the following steps of adding 5% of trehalose, 0.007% of sodium azide, 40% of glycerol and 0.0022% of first polypeptide into 50 mmol/L Tris-HCl buffer solution with the pH value of 7.2, uniformly mixing to prepare the antibody preservation solution, wherein the concentrations of the trehalose, the sodium azide, the glycerol and the first polypeptide are mass ratios, specifically g/L, and the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro.
Comparative example 2
The preparation method of the antibody preservation solution comprises the following steps of adding 5% of trehalose, 0.007% of sodium azide, 40% of glycerol and 0.0022% of second polypeptide into a Tris-HCl buffer solution with the pH value of 7.2 and the concentration of 50 mmol/L, uniformly mixing, and preparing the antibody preservation solution, wherein the concentrations of the trehalose, the sodium azide, the glycerol and the second polypeptide are mass ratios, specifically g/L, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
The activities of the antibodies preserved in the different antibody preservation solutions were examined below to illustrate the utility of the antibody preservation solution of the present invention. The antibody preservation solution comprises: the antibody preservation solution prepared in example 1; the antibody preservation solution prepared in example 2; the antibody preservation solution prepared in comparative example 1; antibody preservation solution prepared in comparative example 2. Wherein the preserved antibody is a growth hormone monoclonal antibody, and each antibody preservation solution preserves the antibody for 1 year. For the detection of the antibody activity, enzyme-linked immunosorbent assay is adopted for detection.
The detection method specifically comprises the steps of diluting growth hormone antigen with 0.05 mol/L pH9.6 sodium carbonate-sodium bicarbonate buffer solution containing 5ppm Proclin300 to obtain coating solution, wherein the concentration of the growth hormone antigen in the coating solution is 10 mu g/ml, the sealing solution is 0.05 mol/L pH9.6 sodium carbonate-sodium bicarbonate buffer solution containing 5.0 g/L trehalose and 0.5% BSA, the coating solution is 90 mu l/hole, incubation is carried out for 2h in a 37 ℃ incubator, residual liquid is removed, washing is carried out for 3 times by using washing solution PBST on a plate washing machine and dried, 200 mu l/hole of the sealing solution is added, incubation is carried out at 25 ℃ overnight, residual liquid is poured off, washing is carried out for 3 times by using PBST on the plate washing machine and dried, antibodies stored by using different kinds of the above-mentioned solutions are added, incubation is carried out for 1h in the 37 ℃ incubator for 1h, the residual liquid is poured off, washing is carried out for 3 times by using the washing solution on the plate washing machine, the incubation is carried out for 3 times by using the incubator, antibodies are added into the incubator, the incubation liquid is added, the incubation liquid with the same temperature of the incubation liquid, the incubation is added, the incubation liquid is added, the antibodies are added to the antibodies, the antibodies are added to the antibodies.
And (3) detection results: the absorbance of the antibody preserved in the antibody preservation solution prepared in example 1 was 1.9, the absorbance of the antibody preserved in the antibody preservation solution prepared in example 2 was 1.95, the absorbance of the antibody preserved in the antibody preservation solution prepared in comparative example 1 was 1.2, and the absorbance of the antibody preserved in the antibody preservation solution prepared in comparative example 2 was 1.26.
According to the detection results, the activity of the antibody preserved in the antibody preservation solution prepared by the invention is higher than that of the antibody preserved in other antibody preservation solutions under the same conditions.
In summary, the antibody preservation solution of the present invention has the following characteristics: the antibody can be stored at room temperature for a long time, and the antibody can keep high activity, wherein the room temperature is 26-29 ℃, and the storage life is more than 7 years; the first polypeptide and the second polypeptide are added to prevent hydrolysis and enzymolysis of substances in the preservation solution and keep the substances in the preservation solution stable, specifically, the first polypeptide is used for preventing hydrolysis, and the second polypeptide is used for preventing proteolysis.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> Guangzhou Guiyu Biotechnology Ltd
<120> an antibody preservation solution and a method for preparing the same
<130>
<160>2
<170>PatentIn version 3.5
<210>1
<211>6
<212>PRT
<213> Artificial sequence
<400>1
Val Pro Thr Gly Ala Pro
1 5
<210>2
<211>6
<212>PRT
<213> Artificial sequence
<400>2
Ile Pro Gly ProVal Gly
1 5
Claims (6)
1. The antibody preservation solution is characterized by comprising a buffer solution, 4-6% of trehalose, 0.005-0.01% of sodium azide, 30-50% of glycerol, 0.002-0.0025% of first polypeptide and 0.002-0.0025% of second polypeptide, wherein the buffer solution is a Tris buffer solution with the pH value of 7.0-7.5 and the concentration of 40-100 mmol/L, the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
2. The antibody preservation solution according to claim 1, wherein the antibody preservation solution comprises a buffer solution, 4.5-5.5% trehalose, 0.006-0.009% sodium azide, 35-45% glycerol, 0.0021-0.0024% first polypeptide and 0.0021-0.0024% second polypeptide, wherein the buffer solution is Tris-HCl buffer solution with pH7.0-7.5 and 45-90 mmol/L, the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
3. The antibody preservation solution according to claim 2, wherein the antibody preservation solution comprises buffer solution, 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% first polypeptide and 0.0022% second polypeptide, wherein the buffer solution is Tris-HCl buffer solution with the pH value of 7.2 and the concentration of 50 mmol/L, the concentrations of the trehalose, the sodium azide, the glycerol, the first polypeptide and the second polypeptide are mass ratios, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
4. A method for preparing an antibody preservation solution is characterized by comprising the following steps:
adding 4-6% trehalose, 0.005-0.01% sodium azide, 30-50% glycerol, 0.002-0.0025% first polypeptide and 0.002-0.0025% second polypeptide into Tris-HCl buffer solution (pH7.0-7.5) and 40-100 mmol/L mmol/HCl, and uniformly mixing to prepare an antibody preservation solution;
the concentration of the trehalose, the sodium azide, the glycerol, the first polypeptide and the concentration of the second polypeptide are mass ratio, the amino acid sequence of the first polypeptide is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.
5. The method for producing an antibody preservation solution according to claim 4, wherein the antibody preservation solution comprises: buffer solution, 4.5-5.5% trehalose, 0.006-0.009% sodium azide, 35% -45% glycerol, 0.0021-0.0024% first polypeptide, 0.0021-0.0024% second polypeptide;
wherein the buffer solution is Tris-HCl buffer solution with the pH value of 7.0-7.5 and the concentration of 45-90 mmol/L.
6. The method for producing an antibody preservation solution according to claim 5, wherein the antibody preservation solution comprises: buffer solution, 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% first polypeptide and 0.0022% second polypeptide;
wherein the buffer solution is Tris-HCl buffer solution with the pH value of 7.2 and the concentration of 50 mmol/L.
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| CN107602695B (en) * | 2017-08-02 | 2020-06-19 | 深圳清华大学研究院 | Fluorescent antibody preservation solution, kit and application thereof, and fluorescent antibody preservation method |
| CN114137202B (en) * | 2022-01-12 | 2023-12-01 | 杭州隆基生物技术有限公司 | Monoclonal antibody preservation solution |
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| PT3721904T (en) * | 2009-11-20 | 2021-11-15 | Biocon Ltd | Formulations of t1h antibody |
| JP2011241206A (en) * | 2010-04-23 | 2011-12-01 | Arkray Inc | Method for stabilizing labeled antibody |
| EP2714735B1 (en) * | 2011-06-03 | 2021-07-21 | XOMA Technology Ltd. | Antibodies specific for tgf-beta |
| EA039663B1 (en) * | 2012-05-03 | 2022-02-24 | Амген Инк. | Use of an anti-pcsk9 antibody for lowering serum cholesterol ldl and treating cholesterol related disorders |
| CN102879589A (en) * | 2012-09-26 | 2013-01-16 | 中国人民解放军总医院 | Human NT-proBNP preparation capable of stable preservation and preparation method thereof |
| CN103224542B (en) * | 2013-04-17 | 2014-09-10 | 江苏澳格姆生物科技有限公司 | Solution for antibody activity preservation under normal temperature and preparation method thereof |
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| TWI797073B (en) * | 2016-01-25 | 2023-04-01 | 德商安美基研究（慕尼黑）公司 | Pharmaceutical composition comprising bispecific antibody constructs |
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