Saxitoxin artificial antigen, anti-saxitoxin antibody prepared by the saxitoxin artificial antigen, and their preparation methods and application

A technology of saxitoxin and artificial antigen, which is applied in the field of immune analysis, can solve the problems of lack of development, and achieve the effect of simple and effective steps, high titer and strong specificity

Active Publication Date: 2011-10-05
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, ELISA studies on STX

Method used

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  • Saxitoxin artificial antigen, anti-saxitoxin antibody prepared by the saxitoxin artificial antigen, and their preparation methods and application
  • Saxitoxin artificial antigen, anti-saxitoxin antibody prepared by the saxitoxin artificial antigen, and their preparation methods and application
  • Saxitoxin artificial antigen, anti-saxitoxin antibody prepared by the saxitoxin artificial antigen, and their preparation methods and application

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Embodiment 1

[0036] The preparation of embodiment 1 immune antigen

[0037] Dissolve 100 μg saxitoxin (STX) in 1 mL dimethyl sulfoxide (DMSO) to make a 0.1 μg / μL mother solution; water-soluble carbodiimide (EDC), N-hydroxysuccinimide ( NHS), ovalbumin (OVA) with 0.01M phosphate buffer (PBS, pH7.5) to make 1mg / mL working solution; take 100μL STX mother solution, add 20μL EDC and 12μL NHS working solution, shake and react at room temperature for 3h ;Add the reactants in the above steps to 100μL OVA solution, and shake at 4°C for 12h; transfer the above reactants to a dialysis card, and dialyze with 300mL pH7.5 PBS buffer solution and DMSO mixture for 72h, every 12h The medium was changed once, and gradient dialysis was used for dialysis. The volume ratio of PBS buffer and DMSO in the solution for the first dialysis was 100:4-5, the volume ratio for the second dialysis was 100:3-4, and the volume ratio for the third dialysis was 100:3-4. The volume ratio of the dialysis is 100:2-3, the volum...

Embodiment 2

[0039] The preparation of embodiment 2 coating antigen

[0040] Dissolve 100 μg saxitoxin (STX) in 1 mL dimethyl sulfoxide (DMSO) to make a 0.1 μg / μL mother solution; water-soluble carbodiimide (EDC), N-hydroxysuccinimide ( NHS), bovine serum albumin (BSA) and 0.01M phosphate buffered saline (PBS, pH7.5) were used to make 1mg / mL working solution; take 100μL STX mother solution, add 20μL EDC and 12μL NHS working solution, shake at room temperature 3h; add the reactants in the above steps to 100μL BSA solution, and shake at 4°C for 12h; The medium was changed once every 12 hours, and gradient dialysis was used for dialysis. The volume ratio of PBS buffer solution and DMSO in the solution of the first dialysis was 100:4-5, the volume ratio of the second dialysis was 100:3-4, and the volume ratio of the third dialysis was 100:3-4. The volume ratio of the second dialysis is 100:2-3, the volume ratio of the fourth dialysis is 100:1-2, the volume ratio of the fifth dialysis is 100:0...

Embodiment 3

[0041] The preparation of embodiment 3 saxitoxin polyclonal antibody

[0042] 6-week-old female BALB / c mice were selected as immunized animals, and the immunized antigen was thawed and then diluted with normal saline. For the first immunization, 200 μL of STX-OVA (containing 5 μg of STX) and an equal volume of Freund’s complete adjuvant were used, fully emulsified to make an emulsion, and intraperitoneally injected into mice, and then the same amount of STX-OVA and an equal volume of Freund’s adjuvant were taken every two weeks. Fully emulsified with Shi's incomplete adjuvant and injected intraperitoneally. After three immunizations, the tail blood was taken to measure the titer of the mouse serum. If the titer was not high, the immunization was continued. After the titer was greater than the required value, blood was taken, placed at room temperature for 30 minutes, centrifuged at 8000r / min for 20 minutes, and the supernatant serum was collected, purified with a protein A an...

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Abstract

The invention relates to a saxitoxin artificial antigen, an anti-saxitoxin antibody prepared by the saxitoxin artificial antigen, and their preparation methods and application. Through carbodiimide method, amino groups in saxitoxin (STX) are connected with carboxyl groups in ovalbumin (OVA) as a carrier protein. A high purity saxitoxin immunization antigen STX-OVA is obtained through dialysis utilizing a gradient method. The prepared high purity saxitoxin artificial immunization antigen STX-OVA is utilized for preparation of a polyclonal antibody and the polyclonal antibody is utilized for preparation of an enzyme-linked immunosorbent assay kit utilized for detecting saxitoxin. In the invention, saxitoxin artificial antigens of STX-OVA and STX-BSA are synthesized successfully to immunize BALB/c mice and then a polyclonal antibody with high titer and strong singularity is obtained. An enzyme-linked immunosorbent assay kit prepared by an established enzyme-linked immunoassay can be utilized for the rapid detection of saxitoxin.

Description

technical field [0001] The invention belongs to the technical field of immune analysis, and in particular relates to a saxitoxin artificial antigen and antibody, a preparation method thereof, and the preparation and application of an ELISA detection kit. Background technique [0002] At present, paralytic shellfish poison (PSP) has become the most widely distributed, most frequent and most harmful type of marine biotoxin in the world. It inhibits nerve conduction by affecting sodium ion channels. Saxitoxin (STX) is one of the main components of paralytic shellfish toxin, and is a derivative of tetrahydropurine. Its external form is a white, highly hygroscopic solid, soluble in water, Slightly soluble in methanol and ethanol. PSP composed of STX and natural derivatives has a high lethality rate. The mild poisoning dose of STX to adults is 110 μg, and the lethal dose is 540-1000 μg. LD 50 9 μg / kg (mouse, ip). [0003] The analysis methods of shellfish toxins mainly include ...

Claims

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Application Information

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IPC IPC(8): C07K14/77C07K16/18C07K16/06G01N33/543
Inventor 周德庆刘帅帅赵峰朱兰兰柳淑芳
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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