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Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof

A tetrodotoxin and artificial antigen technology, applied in the field of antibody preparation and artificial antigen, can solve the problems of cumbersome operation and low detection sensitivity of tetrodotoxin

Inactive Publication Date: 2010-07-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The object of the present invention is to provide an artificial antigen and its corresponding specific antibody for fast and easy detection of tetrodotoxin according to the problems of low tetrodotoxin detection sensitivity and complicated operation in the prior art

Method used

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  • Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof
  • Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof
  • Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0029] The preparation of embodiment 1 tetrodotoxin artificial antigen

[0030] (1) Preparation of immunogen

[0031] Preparation of TTX-HCHO-KLH: Dissolve 2.2mg tetrodotoxin in 3mL PB (0.04M pH7.4), take 1mL KLH solution (concentration: 10mg / mL) with a syringe, slowly add KLH solution under stirring, add 150uL drop by drop 37wt% formaldehyde, stirred at room temperature for 72 hours in the dark. Dialyze each time with 1L of PBS (0.05M pH7.0) at 4°C for 48h. Antigens were identified by UV scanning.

[0032] (2) Preparation of coated antigen

[0033] Preparation of TTX-HCHO-BSA: 1.1mg tetrodotoxin was dissolved in 1.5mL PB (0.04M pH7.4), 7.5mgBSA was added, 60uL of 37wt% formaldehyde was added dropwise, and the reaction was stirred at room temperature for 72h in the dark. The reaction solution was collected and dialyzed with 1 L of PBS (0.05M pH7.0) at 4°C for 48 hours each time. Ultraviolet scanning confirmed that the dialysis was complete and the reaction solution was co...

Embodiment 2

[0035] The preparation of the specific antibody of embodiment 2 tetrodotoxin artificial antigen

[0036] (1) Immunized animals

[0037] For the first immunization, a certain amount of protein concentration of immune antigens (TTX-HCHO-BSA, TTX-HCHO-KLH) was added to an equal volume of Freund's complete adjuvant, emulsified completely, and mice were injected subcutaneously and intraperitoneally on the back. Each mouse was injected with 100 μL each time, containing 100 μg of immunogen (calculated according to protein content). Afterwards, immunize once every two weeks and emulsify with an equal volume of Freund's incomplete adjuvant. Tail blood was taken to test the serum titer.

[0038] One week after the second immunization, use TTX-HCHO-OVA as the coating antigen, detect the antiserum antibody titer of TTX-HCHO-BSA immunized mice by indirect ELISA, and use TTX-HCHO-OVA to TTX-HCHO-KLH immunized mice Or TTX-HCHO-BSA for coating antigen detection. Then check every 2 weeks. ...

Embodiment 3

[0074] The rapid detection of embodiment 3 tetrodotoxin

[0075] (1) Indirect competitive time-resolved fluorescent immunoassay

[0076] Eu 3+ Preparation and purification of labeled goat anti-mouse antibody: 50 μL (20 mg / mL) of goat anti-mouse secondary antibody was added with 250 μL (1 mg / mL) of Eu 3+ -N 2 -[β-isocyanic acid-benzyl]-diethylenetriaminetetraacetic acid (Eu 3+ -DTTA) into a vial, add 200 μL of solution A, adjust the pH to 9.5, shake gently, and react in the dark at room temperature for 18 hours. The reaction solution was chromatographed on a Sephardex G25 column (1×40cm) equilibrated with 50mmol / L Tris-HCl pH7.6 buffer solution, A 280 Monitor collected protein peaks.

[0077] Record and collect the first peak as the europium-labeled goat anti-mouse secondary antibody, and the second peak as the chelate of the europium reagent. Collect 1 mL of the product in each tube, put 5 μL of each tube into an empty 96-well microtiter plate, add 200 μL of fluorescence...

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Abstract

The invention discloses an artificial antigen of tetraodotoxin and a corresponding specific antibody and a preparation method and an application thereof. In the artificial antigen of the tetraodotoxin, the mass ratio of the tetraodotoxin to carrier protein is 1:10. In the invention, the artificial antigen is prepared from the tetraodotoxin which is coupled with carrier protein by a Mannich method, and the specific antibody is prepared from the artificial antigen immunization animals. The invention can be used for the extraction and purification of the artificial antigen aiming at the specific antibody of the artificial antigen of the tetraodotoxin, and can also be used for setting time-resolved immunofluorometric assay or an enzyme linked immunosorbent assay to detect the tetraodotoxin quickly and sensitively.

Description

technical field [0001] The invention relates to the field of preparation of tetrodotoxin artificial antigen and antibody, in particular to an artificial tetrodotoxin antigen, corresponding specific antibody, preparation method and application thereof. Background technique [0002] The chemical research of tetrodotoxin (TTX) began in 1909, and the structure of TTX was determined by Woodward after 1964. In 1972, Kishi et al. successfully synthesized tetrodotoxin by chemical methods. The molecular formula of tetrodotoxin is C 11 h 17 N 3 o 8 , with a molecular weight of 319.27. The molecule is mainly composed of 3 nitrogen atoms, which form a special structure with hydrogen and oxygen atoms: it contains 1 carbon ring, 1 guanidine group, 6 hydroxyl groups, and a semialdonolactone at the C-5 and C-10 positions Connect the separate rings. Some experts call it "one of nature's strangest molecules" and one of the deadliest poisons in the world. 1g of tetrodotoxin is 10,000 ti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K16/44G01N33/577G01N33/533C07D491/22
Inventor 雷红涛钟青萍刘燕婷张挺孙远明余海虎
Owner SOUTH CHINA AGRI UNIV
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