Method for detecting ciguatoxin on basis of capillary electrophoresis/electrochemistry and enzyme-linked immunoassay

An enzyme-linked immunoassay and capillary electrophoresis technology, which is applied in the direction of material analysis, analysis of materials, and measurement devices by electromagnetic means, can solve problems such as unreported, and achieve improved detection sensitivity, increased quantity, high selectivity and specialization. Oneness effect

Inactive Publication Date: 2011-02-02
QINGDAO UNIV OF SCI & TECH
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Problems solved by technology

But so far, the detection method of ciguatoxin based on capillary electroph

Method used

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  • Method for detecting ciguatoxin on basis of capillary electrophoresis/electrochemistry and enzyme-linked immunoassay
  • Method for detecting ciguatoxin on basis of capillary electrophoresis/electrochemistry and enzyme-linked immunoassay
  • Method for detecting ciguatoxin on basis of capillary electrophoresis/electrochemistry and enzyme-linked immunoassay

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Embodiment 1

[0031] Example 1 Capillary Electrophoresis Electrochemical Enzyme-linked Immunoassay for Detection of Ciguatoxin in Standard Samples

[0032] The instruments and reagents used are as described above.

[0033] Using colloidal gold as a solid phase carrier, the enzyme and antibody are immobilized on the surface at the same time, increasing the amount of enzyme immobilized and improving the detection sensitivity. In order to load more enzyme molecules on each colloidal gold, when the colloidal gold reacts with the enzyme, HRP remains in excess. image 3 It is the capillary electrophoresis spectrum of the colloidal gold enzyme-labeled antibody probe, peak 1 is the electrophoretic peak formed by the excess HRP catalyzed substrate in the solution, and peak 2 is the electrophoretic peak formed by the colloidal gold enzyme-labeled antibody probe catalyzed substrate, indicating that the enzyme Successfully marked on colloidal gold.

[0034] Using non-competitive mode, a series of dif...

Embodiment 2

[0037] Example 2 Using capillary electrophoresis electrochemical enzyme-linked immunoassay method to detect ciguatoxin in simulated fish samples

[0038] Add ciguatera toxin standard substance to fish samples to simulate actual sample detection: incubate fish meat and viscera samples in a 70°C water bath for 15 minutes, after homogenization by homogenizer, add a certain amount of ciguatera toxin standard substance, and wash with acetone (3L / kg sample) and 80% acetone (0.5L / Kg sample) extraction, filtered, acetone extract was evaporated to dryness with a rotary evaporator, and the residue was washed with 90% methanol (0.5L / Kg sample) and n-hexane (1:1 v :v 2) extraction, the methanol phase was distilled to dryness again with a rotary evaporator, the residue was extracted with 25% ethanol (0.5L / Kg sample) and ether (1:1 v:v), the ether fraction was collected, and the rotary evaporator distilled After drying, the residue was dissolved in chloroform-methanol (97:3 v:v), and dried...

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Abstract

The invention discloses a novel method for determining amnesic shellfish poisoning (ASP) in shellfish samples on the basis of capillary electrophoresis/electrochemistry (CE/EC) and enzyme-linked immunoassay. The method comprises the following steps: preparing colloidal-gold-supporting HRP enzyme-labeled (horse radish peroxidase) ciguatoxin (CTX) antibody probes; conducting the reaction on sample solution and colloidal-gold-supporting HRP enzyme-labeled CTX antibody with an excessive amount by a non-competitive manner; dividing colloidal-gold-supporting HRP enzyme-labeled antibody-antigen conjugate and residual colloidal-gold-supporting HRP enzyme-labeled antibody probes into different zones in separation capillary tubes by the migration rate; sequentially feeding the colloidal-gold-supporting HRP enzyme-labeled antibody-antigen conjugate and the residual colloidal-gold-supporting HRP enzyme-labeled antibody probes into reacted capillary tubes; respectively catalyzing aminophenol (AP) as a H2O2-oxidized (hydrogen peroxide) substrate in the buffer solution in the reaction capillary tubes after CE separation to generate 3-aminophenoxazine with the electrochemical activity; and feeding and detecting the 3-aminophenoxazine in an electrochemical detection cell. The linear range and limit of detection (LOD) for detecting CTX standard solution are 1.0pg/mL-50.0pg/mL and 0.3pg/mL respectively.

Description

technical field [0001] The invention relates to a capillary electrophoresis electrochemical enzyme-linked immunoassay technique, in particular to a capillary electrophoresis enzyme-linked immunoassay method for detecting ciguatoxin. Background technique [0002] Ciguatoxin (CTX), also known as ciguatoxin, is a more harmful marine algae toxin, mainly derived from a dinoflagellate 2 Gambier. The food-borne disease caused by humans ingesting coral fish containing ciguatera toxin is called ciguatera poisoning, which is currently the most reported marine biotoxin disease in the world. Since the content of ciguatera toxin in fish is very low, and CTX-contaminated fish has no abnormality in sense, smell and taste, it is not easy to be detected by simple conventional methods. Therefore, a simple, fast and reliable detection method has not been established so far. At present, there are many detection methods at home and abroad, but each method has its advantages and disadvantages. ...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/535G01N27/447
Inventor 李雪梅葛安青李晓琳张召香梅振华张书圣
Owner QINGDAO UNIV OF SCI & TECH
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