Modification method for improving acid resistance of paralytic shellfish toxin monoclonal antibody

Abstract

The invention belongs to the technical field of biology, and particularly relates to a modification method for improving acid resistance of a paralytic saxitoxin monoclonal antibody, which comprises the following steps: adding the paralytic saxitoxin monoclonal antibody into a DMF (Dimethyl Formamide) solution containing 1-hydroxybenzotriazole; adding a coupling reagent and a beta-aminoethanesulfonic acid aqueous solution, and stirring for reaction; adding a coupling reagent, and continuously reacting; and after the reaction is completed, carrying out centrifugal dialysis in a phosphate buffer solution to obtain the anionized paralytic shellfish toxin antibody. The stability of the paralytic saxitoxin monoclonal antibody in a low-pH environment is effectively improved by a method of modifying carboxyl with sulfo groups. The adverse effect of acid on the antibody stability in the process of extracting paralytic saxitoxin with acid is avoided.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a modification method for improving the acid resistance of a monoclonal antibody to paralytic shellfish toxins. Background Art

[0002] Paralytic shellfish toxins (PSPs) are a class of highly toxic compounds produced by specific shellfish, and their detection is critical to ensuring food safety. In the process of developing immunoassay kits for paralytic shellfish toxins, a key technical challenge lies in the extraction process of the toxins. Since paralytic shellfish toxins usually require acidic conditions for extraction, the acidic components in the extract often have an adverse effect on the monoclonal antibodies used in subsequent tests. Specifically, at low pH values ​​(i.e., peracidic environments), the aspartic acid and glutamic acid carboxyl groups in the structure of the paralytic shellfish toxin monoclonal antibody will be protonated, causing the protein to carry a large amount of positive charge. This charge state will induce electrostatic repulsion, which in turn reduces the stability of the protein, making it more susceptible to denaturation, ultimately affecting the ability of the antibody to bind to the antigen, and even causing the antibody to be completely inactivated.

[0003] Traditionally, researchers have tried a variety of methods to overcome this problem, but the results are often limited and cannot meet the high requirements for antibody stability and activity in practical applications. Therefore, finding a method that can effectively improve the acid resistance of monoclonal antibodies against paralytic shellfish toxins has become the key to improving the performance and reliability of immunoassay kits.

[0004] In recent years, antibody modification technology has made significant progress, providing a new way to improve the physical and chemical properties and biological activity of antibodies. Among them, changing the charge state of the antibody surface by specific chemical modification means, thereby improving its stability under different environmental conditions, has become a research hotspot. However, there is no record in the prior art of using antibody modification technology to improve the acid resistance of monoclonal antibodies to paralytic shellfish toxins. Summary of the invention

[0005] The object of the present invention is to provide a modification method for improving the acid resistance of a monoclonal antibody to paralytic shellfish toxins, which effectively improves the stability of the monoclonal antibody to paralytic shellfish toxins in a low pH environment.

[0006] The objective of the present invention is achieved through the following technical solutions: The present invention provides a modification method for improving the acid resistance of a monoclonal antibody against paralytic shellfish toxins, comprising the following steps: (1) Adding the monoclonal antibody against paralytic shellfish toxins into a DMF solution containing 1-hydroxybenzotriazole; (2) Add coupling reagent and β-aminoethanesulfonic acid aqueous solution and stir to react; (3) Add coupling reagent and continue the reaction; (4) After the reaction is completed, the anionized paralytic shellfish toxin antibody is obtained by centrifugation and dialysis in a phosphate buffer solution.

[0007] Furthermore, in step (1), the concentration of 1-hydroxybenzotriazole is 50-500 mmol.

[0008] Furthermore, in step (2), the coupling reagent is 10-30 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the concentration of the β-aminoethanesulfonic acid aqueous solution is 10-30 mmol, and the pH value is 4-7.

[0009] Furthermore, in step (3), the coupling reagent is 28 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0010] Furthermore, in step (4), the pH of the phosphate buffer solution is 7.0.

[0011] The present invention also provides an anionized paralytic shellfish toxin antibody prepared by the modification method.

[0012] The present invention also provides a use of the anionized paralytic shellfish toxin antibody in the preparation of an immunoassay kit, wherein the anionized paralytic shellfish toxin antibody can improve the stability of the immunoassay kit. Beneficial Effects

[0013] The present invention effectively improves the stability of the monoclonal antibody of paralytic shellfish toxin in a low pH environment by modifying the carboxyl group with a sulfonic acid group. This solves the adverse effects of acid on the stability of the antibody during the acid extraction of paralytic shellfish toxins. The modified antibody maintains good activity in the range of pH 6-7. Although the activity of the antibody is lost below pH 5, the activity of the modified antibody is better than that of the unmodified antibody in the entire experimental pH range. In particular, at pH 6.0, the activity of the modified antibody decreases less than that at pH 7.0, showing strong acid resistance. Since paralytic shellfish toxins require acid extraction, and the modified antibody has better acid resistance, it can be used to develop a more stable and reliable immunoassay kit to improve the accuracy and reliability of the test results. This modification method is not only applicable to monoclonal antibodies of paralytic shellfish toxins, but also can provide useful reference and reference for other antibodies that need to be used in an acidic environment, thereby broadening the application range of antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 This is a graph showing the activity test results of unmodified paralytic shellfish toxin monoclonal antibodies; Figure 2 This is a graph showing the activity test results of the modified paralytic shellfish toxin monoclonal antibody (i.e., anionized paralytic shellfish toxin antibody). DETAILED DESCRIPTION

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0018] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0019] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field; Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0022] The paralytic shellfish toxin monoclonal antibody used in the present invention, HOBt was purchased from Shanghai Sangon, EDC was purchased from Shanghai Sangon, β-aminoethanesulfonic acid was purchased from Shanghai Sangon, and phosphate buffer solution was purchased from Shanghai Sangon.

[0023] The modification method for improving the acid resistance of the monoclonal antibody to paralytic shellfish toxins in the present invention comprises the following steps: (1) Add the paralytic shellfish toxin monoclonal antibody to a DMF solution containing 50-500 mmol 1-hydroxybenzotriazole (HOBt) to activate the carboxyl group of the antibody.

[0024] (2) Add 10-30 mmol of coupling reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0025] (3) Add 10-30 mmol of an aqueous solution of β-aminoethanesulfonic acid with a pH value of 4-7.

[0026] (4) The reaction is carried out under stirring for 6-24 hours.

[0027] (5) Add another 28 mmol of EDC and continue the reaction.

[0028] (6) After the reaction is completed, the reaction solution is centrifuged and dialyzed in a phosphate buffer solution at pH 7.0 to remove unreacted amines and by-products to obtain anionized paralytic shellfish toxin antibodies (Ab-SO3 - ).

[0029] In the present invention, the reaction between the sulfonic acid group and the basic group can effectively reduce the reaction between the antibody and the acid, thereby improving the acid resistance of the paralytic shellfish toxin monoclonal antibody.

[0030] Example 1 The modification method used in this example to improve the acid resistance of the monoclonal antibody to paralytic shellfish toxins is as follows: (1) A monoclonal antibody against paralytic shellfish toxin was added to a DMF solution containing 250 mmol 1-hydroxybenzotriazole to activate the carboxyl group of the antibody.

[0031] (2) Add 20 mmol of coupling reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0032] (3) Add 20 mmol of β-aminoethanesulfonic acid aqueous solution with a pH value of 6.

[0033] (4) The reaction is carried out under stirring for 6-24 hours.

[0034] (5) Add another 28 mmol of EDC and continue the reaction.

[0035] (6) After the reaction is completed, the reaction solution is centrifuged and dialyzed in a phosphate buffer solution at pH 7.0 to remove unreacted amines and by-products, thereby obtaining anionized paralytic shellfish toxin antibodies (Ab-SO3 - ).

[0036] Test Example 1 Antibody Activity Test The anionized paralytic shellfish toxin antibody Ab-SO3 prepared in Example 1 - The unmodified PSP toxin monoclonal antibody was placed in buffer solutions of different pH values ​​(pH 5.0, 5.5, 6.0, 6.6, 7.0, 7.5) and the activity of the antibody at each pH value was measured under the same conditions. The activity of the antibody was evaluated by comparing the binding ability of the antibody to the antigen. The data were recorded and analyzed to evaluate the stability of the modified antibody at different pH values.

[0037] According to the antibody activity test results, the activity changes of the modified antibody at different pH values ​​were analyzed, and the activity of the antibody before and after modification at the same pH value was compared to evaluate the effect of the modification method on improving the acid resistance of the antibody.

[0038] The final result is Figure 1-2 As shown by Figure 1-2 It can be seen that the anionized paralytic shellfish toxin antibody Ab-SO3 - It exhibits good activity in the pH range of 6-7, especially at pH 6.0, where its activity decreases less than that of the unmodified antibody at pH 7.0. When the pH is lower than 6, the activity of the antibody before and after modification decreases, and the activity loss is about 50%.

[0039] However, in the entire experimental pH range, the anionized paralytic shellfish toxin antibody Ab-SO3 - The activity retention was better than that of the unmodified paralytic shellfish toxin monoclonal antibody, indicating that the modification method effectively improved the acid resistance of the antibody.

[0040] In summary, the present invention successfully improves the acid resistance of the monoclonal antibody against paralytic shellfish toxins by modifying the sulfonic acid group with the carboxyl group. The modified antibody exhibits better stability under acidic conditions, which helps to improve the performance and reliability of the immunoassay kit. The present invention verifies the effectiveness and practicality of the proposed modification method, and provides strong technical support for the detection of paralytic shellfish toxins.

[0041] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A modification method for improving the acid resistance of a monoclonal antibody against paralytic shellfish toxins, characterized in that: The following steps are involved: (1) Adding the paralytic shellfish toxin monoclonal antibody to a DMF solution containing 1-hydroxybenzotriazole; (2) Add coupling reagent and β-aminoethanesulfonic acid aqueous solution and stir to react; (3) Add coupling reagent and continue the reaction; (4) After the reaction is completed, the anionized paralytic shellfish toxin antibody is obtained by centrifugation and dialysis in a phosphate buffer solution.

2. The method for modifying a monoclonal antibody against paralytic shellfish toxins to improve acid resistance according to claim 1, wherein: In step (1), the concentration of 1-hydroxybenzotriazole is 50-500 mmol.

3. The method for modifying a monoclonal antibody against paralytic shellfish toxins to improve acid resistance according to claim 1, wherein: In step (2), the coupling reagent is 10-30 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the concentration of the β-aminoethanesulfonic acid aqueous solution is 10-30 mmol, and the pH value is 4-7.

4. The method for modifying a monoclonal antibody against paralytic shellfish toxins to improve acid resistance according to claim 1, wherein: In step (3), the coupling reagent is 28 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

5. The method for modifying a monoclonal antibody against paralytic shellfish toxins to improve acid resistance according to claim 1, wherein: In step (4), the pH of the phosphate buffer solution is 7.

0.

6. An anionized paralytic shellfish toxin antibody prepared by the modification method according to any one of claims 1 to 5.

7. Use of the anionized paralytic shellfish toxin antibody according to claim 6 in the preparation of an immunoassay kit, characterized in that: The anionized paralytic shellfish toxin antibody can improve the stability of the immunoassay kit.

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