CN106800598B - Antibody preservation solution and preparation method thereof - Google Patents

Abstract

The invention discloses an antibody preservation solution, which comprises: buffer solution, 4-6% trehalose, 0.005-0.01% sodium azide, 30%-50% glycerol, 0.002-0.0025% first polypeptide, 0.002% -0.0025% of the second polypeptide, wherein the buffer is pH7.0-7.5, 40-100mmol/L Tris-HCl buffer, the concentrations of trehalose, sodium azide, glycerol, the first polypeptide, and the second polypeptide All 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 invention also discloses a preparation method of the antibody preservation solution. The antibody preservation solution of the present invention can store the antibody at room temperature for a long time, and can keep the antibody with high activity.

Description

Antibody preservation solution and preparation method thereof

technical field

The invention relates to the field of biotechnology, in particular to an antibody preservation solution and a preparation method thereof.

Background technique

Antibodies refer to immunoglobulins produced by plasma cells differentiated from B lymphocytes under the stimulation of antigens by the body's immune system, which can specifically bind to corresponding antigens. Antibodies are widely used because they can specifically and stably bind to specific antigens. In the process of antibody utilization, whether it is properly stored or not directly affects the activity and use effect of the antibody. For most antibodies, it is usually stored at low temperature. If this method is used to store the antibody, due to repeated freezing and thawing of the antibody, the antibody will be denatured and more Aggregates are produced, thereby reducing antibody activity.

How to provide an antibody preservation solution that can be stored for a long time at room temperature is one of the problems to be solved urgently in the field of antibody application.

SUMMARY OF THE INVENTION

The main technical problem to be solved by the present invention is to provide an antibody preservation solution and a preparation method thereof, which can preserve the antibody at room temperature for a long time, and can keep the antibody relatively active.

In order to solve the above technical problems, the present invention provides an antibody preservation solution, the antibody preservation solution includes: buffer, 4-6% trehalose, 0.005-0.01% sodium azide, 30%-50% glycerol, 0.002-0.0025% One polypeptide, 0.002-0.0025% of the second polypeptide, wherein the buffer is pH7.0-7.5, 40-100mmol/L Tris-HCl buffer, trehalose, sodium azide, glycerol, the first polypeptide, the third The concentrations of the two polypeptides are all 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 antibody preservation solution includes: buffer, 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, The buffer is pH7.0-7.5, 45-90mmol/L Tris-HCl buffer. The concentrations of trehalose, sodium azide, glycerol, the first polypeptide and the second polypeptide are all mass ratios. The amino acid sequence is Val-Pro-Thr-Gly-Ala-Pro, and the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.

Among them, the antibody preservation solution includes: buffer, 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% first polypeptide, 0.0022% second polypeptide, and the buffer solution is pH7.2, 50mmol/L Tris -HCl buffer, the concentrations of trehalose, sodium azide, glycerol, the first polypeptide, and the second polypeptide are all mass ratios, and 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.

Among them, the mass ratio is g/L.

In order to solve the above-mentioned technical problems, the present invention provides a preparation method of an antibody preservation solution, comprising the following steps: adding 4-6% trehalose, 0.005- 0.01% sodium azide, 30%-50% glycerol, 0.002-0.0025% first polypeptide, 0.002-0.0025% second polypeptide, mix well, and prepare the antibody preservation solution; among them, trehalose, sodium azide, glycerol , the concentrations of 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 antibody preservation solution includes: buffer, 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; The buffer is pH7.0-7.5, 45-90mmol/L Tris-HCl buffer.

Among them, the antibody preservation solution includes: buffer, 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% first polypeptide, 0.0022% second polypeptide; the buffer solution is pH7.2, 50mmol/L Tris -HCl buffer.

Among them, the mass ratio is g/L.

The beneficial effects of the present invention are: different from the situation in the prior art, the antibody preservation solution of the present invention includes: buffer, 4-6% trehalose, 0.005-0.01% sodium azide, 30%-50% glycerol, 0.002- 0.0025% of the first polypeptide, 0.002-0.0025% of the second polypeptide, wherein the buffer is pH7.0-7.5, 40-100mmol/L Tris-HCl buffer, and 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. The preservation solution of the above components has the following characteristics: the antibody can be stored for a long time at room temperature, and the antibody maintains a relatively high activity. The addition of the peptide can prevent the hydrolysis and enzymolysis of the substances in the preservation solution, and keep the substances in the preservation solution stable. Specifically, the first polypeptide is used to prevent hydrolysis, and the second polypeptide is used to prevent proteolysis.

Detailed ways

Example 1

The preparation method of the antibody preservation solution is as follows: add 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% first polypeptide, 0.0022% second polyamide to pH7.2, 50mmol/L Tris-HCl buffer Peptide, mix well, and prepare antibody preservation solution. Wherein, the concentrations of trehalose, sodium azide, glycerol, the first polypeptide and the second polypeptide are all mass ratios, specifically g/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.

The activity of the antibodies preserved in different ways is tested below to illustrate the utility of the antibody preservation solution of this embodiment. Antibody preservation methods are as follows: the antibody preservation solution prepared in Example 1 is used to preserve the antibody; the antibody is preserved in the existing room temperature antibody preservation solution; the antibody is preserved in the existing low temperature (-20° C.) antibody preservation solution. Among them, the antibody is a growth hormone monoclonal antibody, and the antibody is preserved for 1 year in each storage method. For the detection of antibody activity, enzyme-linked immunosorbent assay (ELISA) was used for detection.

The detection method is specifically: dilute the growth hormone antigen with 0.05mol/L, pH9.6 sodium carbonate-sodium bicarbonate buffer containing 5ppm Proclin300 to obtain a coating solution, wherein the growth hormone antigen in the coating solution is diluted. The concentration is 10 μg/ml; the blocking solution is 0.05 mol/L sodium carbonate-sodium bicarbonate buffer containing 5.0 g/L trehalose, 0.5% BSA, pH 9.6; 90 μl/L of coating solution is added to the blank reaction plate Well, incubate at 37°C for 2 h, discard the residual liquid, wash three times with washing solution PBST on a plate washer, and pat dry; add 200 μl/well of blocking solution, incubate at 25 °C overnight, discard the residual liquid, and wash the plate. Wash 3 times with washing solution PBST on the machine, and pat dry; add 90 μl/well of the above-preserved antibodies in different ways, incubate in a 37 °C incubator for 1 h, discard the residual liquid, wash 3 times with washing solution PBST on the plate washer, and pat dry ; Add 90 μl/well of enzyme-labeled antibody, incubate in a 37°C incubator for 1 h, discard the residual liquid, wash 3 times with washing solution PBST on a plate washer, and pat dry; add 90 μl/well of freshly prepared chromogenic solution, protect from light Incubate at 37°C for 20min; add sulfuric acid to stop the reaction; measure the absorbance of each well at 450nm wavelength on a microplate reader. Among them, the concentrations of the antibodies stored in different ways were the same, and the dilution of the antibodies was 1:4000.

Test results: the absorbance value of the antibody preserved in the antibody preservation solution prepared in Example 1 was 1.9, the absorbance value of the antibody preserved in the existing room temperature antibody preservation solution was 0.5, and the absorbance value of the antibody preserved in the existing low temperature (-20°C) antibody preservation solution was 0.5. The absorbance value was 1.1.

According to the test results, under the same conditions, the activity of the antibody preserved in the antibody preservation solution prepared in Example 1 is higher than that of the antibody preserved in other ways.

Example 2

The preparation method of the antibody preservation solution is as follows: add 5.3% trehalose, 0.008% sodium azide, 43% glycerol, 0.0023% first polypeptide, 0.0023% second most to pH7.2, 50mmol/L Tris-HCl buffer Peptide, mix well, and prepare antibody preservation solution. Wherein, the concentrations of trehalose, sodium azide, glycerol, the first polypeptide and the second polypeptide are all mass ratios, specifically g/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.

The activity of the antibodies preserved in different ways is tested below to illustrate the utility of the antibody preservation solution of this embodiment. Antibody preservation methods are as follows: the antibody preservation solution prepared in Example 2 is used to preserve the antibody; the existing room temperature antibody preservation solution is used to preserve the antibody; the existing low temperature (-20° C.) antibody preservation solution is used to preserve the antibody. Among them, the antibody is a growth hormone monoclonal antibody, and the antibody is preserved for 1 year in each storage method. For the detection of antibody activity, enzyme-linked immunosorbent assay was used for detection.

The detection method is specifically: dilute the growth hormone antigen with 0.05mol/L, pH9.6 sodium carbonate-sodium bicarbonate buffer containing 5ppm Proclin300 to obtain a coating solution, wherein the growth hormone antigen in the coating solution is diluted. The concentration is 10 μg/ml; the blocking solution is 0.05 mol/L sodium carbonate-sodium bicarbonate buffer containing 5.0 g/L trehalose, 0.5% BSA, pH 9.6; 90 μl/L of coating solution is added to the blank reaction plate Well, incubate at 37°C for 2 h, discard the residual liquid, wash three times with washing solution PBST on a plate washer, and pat dry; add 200 μl/well of blocking solution, incubate at 25 °C overnight, discard the residual liquid, and wash the plate. Wash 3 times with washing solution PBST on the machine, and pat dry; add 90 μl/well of the above-preserved antibodies in different ways, incubate at 37 °C for 1 h, discard the residual liquid, wash 3 times with washing solution PBST on the plate washer, and pat dry ; Add 90 μl/well of enzyme-labeled antibody, incubate in a 37°C incubator for 1 h, discard the residual liquid, wash 3 times with washing solution PBST on a plate washer, and pat dry; add 90 μl/well of freshly prepared chromogenic solution, protect from light Incubate at 37°C for 20min; add sulfuric acid to stop the reaction; measure the absorbance of each well at 450nm wavelength on a microplate reader. Among them, the concentrations of the antibodies stored in different ways were the same, and the dilution of the antibodies was 1:4000.

Test results: the absorbance value of the antibody preserved in the antibody preservation solution prepared in Example 2 was 1.95, the absorbance value of the antibody preserved in the existing room temperature antibody preservation solution was 0.5, and the absorbance value of the antibody preserved in the existing low temperature (-20°C) antibody preservation solution was 0.5. The absorbance value was 1.1.

According to the test results, under the same conditions, the activity of the antibody preserved in the antibody preservation solution prepared in Example 2 is higher than that of the antibody preserved in other ways.

Comparative Example 1

The preparation method of the antibody preservation solution is as follows: add 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% first polypeptide to pH7.2, 50mmol/L Tris-HCl buffer, mix well, and prepare Antibody preservation solution. Wherein, the concentrations of trehalose, sodium azide, glycerol, and the first polypeptide are all mass ratios, specifically g/L; 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 is as follows: add 5% trehalose, 0.007% sodium azide, 40% glycerol, 0.0022% second polypeptide to pH7.2, 50mmol/L Tris-HCl buffer, mix well, and prepare Antibody preservation solution. Wherein, the concentrations of trehalose, sodium azide, glycerol, and the second polypeptide are all mass ratios, specifically g/L; the amino acid sequence of the second polypeptide is Ile-Pro-Gly-Pro-Val-Gly.

The activity of the antibodies preserved in different antibody preservation solutions is tested below to illustrate the utility of the antibody preservation solution of the present invention. The antibody preservation solution includes: 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; and the antibody preservation solution prepared in Comparative Example 2. Among them, the preserved antibody is growth hormone monoclonal antibody, and each antibody preservation solution preserves the antibody for 1 year. For the detection of antibody activity, enzyme-linked immunosorbent assay was used for detection.

The detection method is specifically: dilute the growth hormone antigen with 0.05mol/L, pH9.6 sodium carbonate-sodium bicarbonate buffer containing 5ppm Proclin300 to obtain a coating solution, wherein the growth hormone antigen in the coating solution is diluted. The concentration is 10 μg/ml; the blocking solution is 0.05 mol/L sodium carbonate-sodium bicarbonate buffer containing 5.0 g/L trehalose, 0.5% BSA, pH 9.6; 90 μl/L of coating solution is added to the blank reaction plate Well, incubate at 37°C for 2 h, discard the residual liquid, wash three times with washing solution PBST on a plate washer, and pat dry; add 200 μl/well of blocking solution, incubate at 25 °C overnight, discard the residual liquid, and wash the plate. Wash 3 times with washing solution PBST on the machine, and pat dry; add 90 μl/well of the above-mentioned antibodies preserved in different preservation solutions, incubate at 37 °C for 1 h, discard the residual liquid, wash 3 times with washing solution PBST on the plate washer, and pat Dry; add 90 μl/well of enzyme-labeled antibody, incubate in a 37°C incubator for 1 h, discard the residual liquid, wash 3 times with washing solution PBST on a plate washer, and pat dry; add 90 μl/well of freshly prepared chromogenic solution to avoid The light was placed in a 37°C incubator for 20 min; sulfuric acid was added to stop the reaction; the absorbance of each well at a wavelength of 450 nm was measured on a microplate reader. Among them, the concentration of antibodies stored in different preservation solutions is the same, and the dilution of antibodies is 1:4000.

Test results: the absorbance value of the antibody preserved in the antibody preservation solution prepared in Example 1 was 1.9, the absorbance value of the antibody preserved in the antibody preservation solution prepared in Example 2 was 1.95, and the absorbance value of the antibody preserved in the antibody preservation solution prepared in Comparative Example 1 was 1.95. The value was 1.2, and the absorbance value of the antibody preserved in the antibody preservation solution prepared in Comparative Example 2 was 1.26.

According to the detection results, under the same conditions, the activity of the antibody preserved in the antibody preservation solution prepared by the present invention is higher than that of the antibody preserved in other antibody preservation solutions.

To sum up, the antibody preservation solution of the present invention has the following characteristics: the antibody can be stored for a long time at room temperature, and the antibody maintains a relatively high activity, wherein the room temperature is 26-29°C, and the storage period is more than 7 years; the first polypeptide And the addition of the second polypeptide can prevent the hydrolysis and enzymolysis of the substances in the preservation solution, and keep the substances in the preservation solution stable. Specifically, the first polypeptide is used to prevent hydrolysis, and the second polypeptide is used to prevent proteolysis. .

The above descriptions are only the embodiments of the present invention, and do not limit the scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the description of the present invention, or directly or indirectly applied in other related technical fields, will not limit the scope of the invention. Similarly, it is included in the scope of patent protection of the present invention.

SEQUENCE LISTING

<110> Guangzhou Guiyu Biotechnology Co., Ltd.

<120> A kind of antibody preservation solution and preparation method thereof

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PRT

<213> Artificial sequences

<400> 1

Val Pro Thr Gly Ala Pro

1 5

<210> 2

<211> 6

<212> PRT

<213> Artificial sequences

<400> 2

Ile Pro Gly Pro Val 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.