Cationized diarrhetic shellfish toxin monoclonal antibody as well as preparation method and application thereof

Abstract

The invention provides a cationized diarrhetic shellfish toxin monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of food detection. The preparation method of the cationized diarrhetic shellfish toxin monoclonal antibody comprises the following steps: adding betaine into N, N-dimethylformamide, sequentially adding a coupling agent and an antibody, reacting, and dialyzing to obtain the cationized diarrhetic shellfish toxin monoclonal antibody. The amino group on the surface of the antibody is subjected to directional chemical modification and is converted into a permanent positive charge quaternary ammonium group (-N + (CH3) 3), so that the problem of charge loss caused by deprotonation in an organic solvent or a high-pH environment is thoroughly solved. The method can be widely applied to the fields of aquaculture, food processing and market supervision, DSP poisoning events can be effectively prevented, public health risks are reduced, and meanwhile detection cost and environmental pollution are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a cationic monoclonal antibody against diarrhetic shellfish toxin, and a preparation method and application thereof. Background Art

[0002] Diarrheal shellfish toxins (DSPs) are a class of fat-soluble polyether compounds with high thermal stability and are easily enriched in the digestive glands of shellfish, posing a serious threat to food safety and human health. Existing detection technologies such as enzyme-linked immunosorbent assay (ELISA) require the use of 30-50% methanol to extract toxins, but high concentrations of organic solvents will destroy the three-dimensional structure of antibodies, resulting in a 30% to 50% decrease in antigen binding activity, significantly reducing detection sensitivity and increasing the risk of false negatives. Traditional antibody modification methods (such as amino blocking or hydrophilic coating) have difficulty maintaining charge stability in extreme solvent environments, resulting in antibody deprotonation and inactivation, forcing the detection process to rely on complex solvent replacement steps, which takes more than 2 hours, seriously restricting the practicality and reliability of on-site rapid detection.

[0003] Therefore, there is an urgent need to provide a substance with a short detection time and higher tolerance to organic solvents to achieve the purpose of rapid on-site detection of diarrheal shellfish toxins in food. Summary of the Invention

[0004] The purpose of the present invention is to provide a cationized monoclonal antibody to diarrhetic shellfish toxin and its preparation method and application. The cationized monoclonal antibody to diarrhetic shellfish toxin has greatly improved tolerance to organic solvents compared to unmodified antibodies.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a cationic monoclonal antibody to diarrhetic shellfish toxin, the structural formula of the cationic monoclonal antibody to diarrhetic shellfish toxin is shown in Formula I:

[0007]

[0008] The present invention also provides a method for preparing the cationic diarrhetic shellfish toxin monoclonal antibody, comprising the following steps:

[0009] Betaine is added to N,N-dimethylformamide, and a coupling agent and an antibody are added in sequence, followed by reaction and dialysis to obtain the cationic diarrhetic shellfish toxin monoclonal antibody.

[0010] Preferably, the mass volume ratio of the betaine to N,N-dimethylformamide is 2.34-4.69 mg:1 ml; the N,N-dimethylformamide further contains 1-hydroxybenzotriazole, and the mass volume concentration of the 1-hydroxybenzotriazole is 13.51-40.54 g / L.

[0011] Preferably, the mass ratio of the betaine to the coupling agent is 2.34-4.69:3.83-9.59; and the coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0012] Preferably, the antibody is a monoclonal antibody against diarrhetic shellfish toxin; the mass ratio of the antibody to betaine is 50-100:2.34-4.69.

[0013] Preferably, the reaction temperature is 20-30° C., and the reaction time is 5-7 h.

[0014] Preferably, after 6 to 8 hours of reaction, 1.92 to 7.67 mg of a coupling agent is added again, wherein the coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and the dialysis solvent is phosphate buffer.

[0015] The present invention also provides the use of the cationic diarrhetic shellfish toxin monoclonal antibody in detecting diarrhetic shellfish toxin.

[0016] The present invention also provides the use of the cationic diarrhetic shellfish toxin monoclonal antibody in the preparation of a product for detecting diarrhetic shellfish toxin.

[0017] The present invention also provides a kit for detecting diarrhetic shellfish toxin, comprising the cationic diarrhetic shellfish toxin monoclonal antibody.

[0018] The beneficial effects of the present invention compared with the prior art are:

[0019] The present invention converts the amino groups on the surface of the antibody into permanent positively charged quaternary ammonium groups (-N + (CH3)3), which completely solves the problem of charge loss caused by deprotonation in organic solvents or high pH environments. Experiments show that the activity retention rate of the modified antibody in a 50% methanol system exceeds 90%, which is more than 2 times higher than that of the unmodified antibody (activity <40%). The present invention has realized the integrated process of organic solvent extraction-direct detection for the first time, shortening the sample pretreatment time from 2 hours to 15 minutes, while reducing the amount of methanol used by 50%, greatly reducing the harm of toxic reagents to operators and the environment.

[0020] The breakthrough of this invention lies in overcoming the core technical bottleneck of poor antibody solvent tolerance in rapid detection of fat-soluble toxins, filling a gap in the field of antibody modification with high organic solvent compatibility. By stabilizing the antibody conformation through charge engineering, not only does it significantly improve detection accuracy and repeatability (coefficient of variation <5%), but it also promotes the development of on-site rapid detection products such as immunochromatographic test strips, achieving efficient "one-step" screening.

[0021] The present invention can be widely used in aquaculture, food processing and market supervision. It can effectively prevent DSP poisoning incidents, reduce public health risks, and at the same time reduce detection costs and environmental pollution. It has both technological innovation, industrial practicality and social benefits, and is of milestone significance for food safety assurance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0023] Figure 1 The present invention provides a synthetic route for the cationic diarrhetic shellfish toxin monoclonal antibody. DETAILED DESCRIPTION

[0024] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The present invention provides a cationic monoclonal antibody to diarrhetic shellfish toxin, the structural formula of the cationic monoclonal antibody to diarrhetic shellfish toxin is shown in Formula I:

[0030]

[0031] The present invention also provides a method for preparing the cationic diarrhetic shellfish toxin monoclonal antibody, comprising the following steps:

[0032] Betaine is added to N,N-dimethylformamide, and a coupling agent and an antibody are added in sequence, followed by reaction and dialysis to obtain the cationic diarrhetic shellfish toxin monoclonal antibody.

[0033] In the present invention, the mass volume ratio of betaine to N,N-dimethylformamide is preferably 2.34-4.69 mg:1 ml, more preferably 2.50-4.00 mg:1 ml, more preferably 3.00-3.50 mg:1 ml, and further preferably 3.25 mg:1 ml; the N,N-dimethylformamide preferably further contains 1-hydroxybenzotriazole, and the mass volume concentration of the 1-hydroxybenzotriazole is preferably 13.51-40.54 g / L, more preferably 15.00-35.00 g / L, and further preferably 20.00-30.00 g / L. 0g / L, and further preferably 25.00g / L; the mass ratio of betaine to coupling agent is preferably 2.34-4.69:3.83-9.59, further preferably 2.50-4.00:4.00-9.00, further preferably 3.00-3.50:5.00-8.00, further preferably 3.25:6.00-7.00, and further preferably 3.25:6.50; the coupling agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the antibody is preferably a monoclonal antibody against diarrhoeal shellfish toxin; the antibody and betaine The mass ratio is preferably 50-100:2.34-4.69, more preferably 60-90:2.50-4.00, more preferably 70-80:3.00-3.50, and further preferably 75:3.25; the reaction temperature is preferably 20-30°C, more preferably 22-28°C, more preferably 24-26°C, and further preferably 25°C; the reaction time is preferably 5-7h, more preferably 6h; after the reaction for 6-8h, preferably 1.92-7.67mg of coupling agent is added again, and more preferably after the reaction for 6.5-7.5h 2.00 to 7.00 mg of the coupling agent is added again, more preferably 4.00 to 6.00 mg of the coupling agent is added again after the reaction for 7 hours, and further preferably 5.00 mg of the coupling agent is added again after the reaction for 7 hours; the coupling agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; after the addition of the coupling agent, the reaction is preferably continued for 5 to 7 hours, more preferably 6 hours; the dialysis solvent imine hydrochloride is phosphate buffer, and the pH of the phosphate buffer is preferably 6.8 to 7.2, more preferably 7.0; the dialysis can remove unreacted amines and by-products.

[0034] The present invention also provides the use of the cationic diarrhetic shellfish toxin monoclonal antibody in detecting diarrhetic shellfish toxin.

[0035] The present invention also provides the use of the cationic diarrhetic shellfish toxin monoclonal antibody in the preparation of a product for detecting diarrhetic shellfish toxin.

[0036] The present invention also provides a kit for detecting diarrhetic shellfish toxin, comprising the cationic diarrhetic shellfish toxin monoclonal antibody.

[0037] Example 1

[0038] A method for preparing a cationic monoclonal antibody to diarrhetic shellfish toxin comprises the following steps:

[0039] 1 mL of 2.34 g / L betaine aqueous solution was added to 1 mL of N,N-dimethylformamide solution containing 13.51 g / L 1-hydroxybenzotriazole, 1 mL of 3.83 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 50 mg of diarrheal shellfish toxin monoclonal antibody (purchased from Wuhan Yingke Biotechnology Co., Ltd.) were added, and the reaction was stirred at 25°C for 6 hours. 1 mL of 1.92 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and the reaction was continued for 7 hours; the solution was dialyzed using a phosphate buffer solution at pH 7.0 to obtain the cationized diarrheal shellfish toxin monoclonal antibody.

[0040] Example 2

[0041] A method for preparing a cationic monoclonal antibody to diarrhetic shellfish toxin comprises the following steps:

[0042] 1 mL of 4.69 g / L betaine aqueous solution was added to 1 mL of N,N-dimethylformamide solution containing 40.54 g / L 1-hydroxybenzotriazole, 1 mL of 9.59 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 mg of diarrheal shellfish toxin monoclonal antibody were added, and the mixture was stirred at 20°C for 7 hours. 1 mL of 7.67 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and the reaction was continued for 6 hours. The mixture was dialyzed using a pH 6.8 phosphate buffer to obtain the cationized diarrheal shellfish toxin monoclonal antibody.

[0043] Example 3

[0044] A method for preparing a cationic monoclonal antibody to diarrhetic shellfish toxin comprises the following steps:

[0045] 1 mL of 3.00 g / L betaine aqueous solution was added to 1 mL of N,N-dimethylformamide solution containing 25.00 g / L 1-hydroxybenzotriazole, 1 mL of 5.00 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 70 mg of diarrheal shellfish toxin monoclonal antibody were added, and the mixture was stirred at 30°C for 5 hours. 1 mL of 5.00 g / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and the reaction was continued for 5 hours. The mixture was dialyzed using a pH 7.2 phosphate buffer to obtain the cationized diarrheal shellfish toxin monoclonal antibody.

[0046] Test Example 1

[0047] At 25°C, 150 μL of 10 μg / L diarrhetic shellfish toxin monoclonal antibody solutions before and after amino modification (prepared according to the method in Example 1) was mixed with 150 μL of 0.01 mol / L pH 7.4 PBS solution and added to a BSA-OA-coated ELISA plate. The plate was incubated for 25 minutes, washed three times, and a secondary antibody aqueous solution containing horseradish peroxidase (HRP) was added at a volume ratio of 1:1000 and incubated for 40 minutes. The plate was then transferred to 150 μL of 0.01 mol / L pH 7.4 PBS solution containing 0.6 μL / ml guaiacol, and 15 μL of 1.0 mol / L hydrogen peroxide was added. The plate was mixed and the absorbance change within 0 to 1 minute was recorded at 470 nm, and a linear equation was plotted.

[0048] After testing the DSP monoclonal antibody before amino modification, the linear equation was plotted as y = 0.0037x + 0.05, where y represents the absorbance and x represents time (s). The initial rate was determined as a slope of 0.0037. After testing the DSP monoclonal antibody after amino modification, the linear equation was plotted as y = 0.0033x + 0.13, where y represents the absorbance and x represents time (s). The initial rate was determined as a slope of 0.0033. This indicates that amino modification does not significantly alter the activity of the DSP monoclonal antibody in aqueous solution.

[0049] Test Example 2

[0050] 150 μL of a 10 μg / L DSP monoclonal antibody solution (prepared according to the method in Example 1) before and after amino modification was mixed with 150 μL of methanol (1:1 mixture) and added to a BSA-OA-coated ELISA plate. The plate was incubated for 25 minutes, washed three times, and then a secondary antibody solution (containing 1:1000 horseradish peroxidase) was added. The plate was then transferred to 150 μL of a 0.01 mol / L PBS solution (pH 7.4) containing 0.6 μL / mL guaiacol. 15 μL of 1.0 mol / L hydrogen peroxide was then added and mixed. The absorbance change at 470 nm was recorded from 0 to 1 minute, and a linear equation was plotted. The effect of a 1:1 methanol-water mixture on the activity of the DSP monoclonal antibody before and after amino modification was tested.

[0051] After testing the DSP monoclonal antibody before amino modification, the linear equation was plotted as y = 0.00042x + 0.14, where y represents the absorbance and x represents time (s). The initial rate was determined as a slope of 0.00042. After testing the DSP monoclonal antibody after amino modification, the linear equation was plotted as y = 0.00281x + 0.12, where y represents the absorbance and x represents time (s). The initial rate was determined as a slope of 0.00281. This indicates that the activity of the DSP monoclonal antibody in aqueous methanol was 6.7 times higher after amino modification than before modification. The modified DSP monoclonal antibody significantly improved its tolerance to organic solvents.

[0052] As can be seen from the above examples, the present invention provides a cationic monoclonal antibody to diarrhetic shellfish toxin, and its preparation method and application. The cationic monoclonal antibody to diarrhetic shellfish toxin is obtained by quaternizing the amino residues, and its tolerance to organic solvents is greatly improved compared with the unmodified antibody.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A cationic monoclonal antibody to diarrhetic shellfish toxin, characterized in that: The structural formula of the cationic monoclonal antibody to diarrhetic shellfish toxin is shown in Formula I:

2. A method for preparing a cationic monoclonal antibody against diarrhetic shellfish toxin according to claim 1, characterized in that: The steps include: Betaine is added to N,N-dimethylformamide, and a coupling agent and an antibody are added in sequence, followed by reaction and dialysis to obtain the cationic diarrhetic shellfish toxin monoclonal antibody.

3. The preparation method according to claim 1, characterized in that The mass volume ratio of the betaine to N,N-dimethylformamide is 2.34-4.69 mg:1 ml; the N,N-dimethylformamide also contains 1-hydroxybenzotriazole, and the mass volume concentration of the 1-hydroxybenzotriazole is 13.51-40.54 g / L.

4. The preparation method according to claim 1, characterized in that The mass ratio of the betaine to the coupling agent is 2.34-4.69:3.83-9.59; the coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

5. The preparation method according to claim 1, characterized in that The antibody is a monoclonal antibody against diarrhoeal shellfish toxin; the mass ratio of the antibody to betaine is 50-100:2.34-4.

69.

6. The preparation method according to claim 1, characterized in that The reaction temperature is 20-30° C., and the reaction time is 5-7 h.

7. The preparation method according to claim 1, characterized in that After the reaction was continued for 6 to 8 hours, 1.92 to 7.67 mg of a coupling agent was added again. The coupling agent was 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The dialysis solvent was phosphate buffer.

8. Use of the cationic monoclonal antibody against diarrhetic shellfish toxin according to claim 1 in detecting diarrhetic shellfish toxin.

9. Use of the cationic monoclonal antibody against diarrhetic shellfish toxin according to claim 1 in the preparation of a product for detecting diarrhetic shellfish toxin.

10. A kit for detecting diarrhetic shellfish toxins, characterized in that: The invention comprises the cationized diarrhetic shellfish toxin monoclonal antibody according to claim 1.

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