Enzyme-linked immune-sorbent assay (ELISA) of anisakid larvae in seafood

Anisakis, a detection method technology, applied in the direction of measuring device, color/spectral characteristic measurement, instrument, etc., can solve the problems of long detection time, inaccurate characterization, cumbersome operation, etc., achieve high sensitivity and accuracy, and easy operation The effect of fast and shortened analysis time

Inactive Publication Date: 2009-12-16
OCEAN UNIV OF CHINA
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AI Technical Summary

Problems solved by technology

[0003] At present, the detection of Anisakis in seafood mainly includes lamp detection method and enzyme digestion method. The former has a high rate of missed detection and cannot be accurately identified, while the latter has the problems of long detection time and cumbersome operation.

Method used

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  • Enzyme-linked immune-sorbent assay (ELISA) of anisakid larvae in seafood

Examples

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Effect test

Embodiment 1

[0018] Example 1: Rapid detection of Anisakis in marine fish fillets

[0019] 1. Preparation of Anisakis worm antigen

[0020] Anisakis was isolated and collected from fish fillets by enzymatic digestion, and species identification was carried out by morphological observation. The isolated Anisakis body was washed with normal saline, placed in a glass homogenizer, and 0.01 Homogenize at 4°C after mol / L PBS, centrifuge at 9700g for 15min, and take the supernatant. Then add 1 / 2 volume of n-hexane to the supernatant to degrease. The extract was dialyzed against redistilled water overnight at 4°C, freeze-dried, and stored at -20°C.

[0021] 2. Preparation and purification of specific antibodies

[0022] Select 20 healthy female BALB / C mice aged 6-8 weeks, and carry out the immunization test by intraperitoneal injection, 0.3mg / mouse (calculated as protein), immunize once every 10 days, 5 consecutive immunizations, and the last immunization After 7 days, the eyeballs were remove...

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Abstract

The invention discloses enzyme-linked immune-sorbent assay (ELISA) of anisakid larvae in seafood, comprising the following steps: separating and purifying somatic antigens of the anisakid larvae and preparing specific polyclonal antibodies by biological immune technology; taking a 96 well polystyrene board as a solid phase carrier, coating the 96 well polystyrene board by a corresponding complete antigen in advance and sealing the 96 well polystyrene board by a carrier protein; during detection, using extract without substances to be detected as negative control at the same time and using standard solution of the antigen of the anisakid larvae for establishing a standard curve; detecting absorption value of specimen by adopting conventional indirect competitive ELISA and computing corresponding inhibition ratio, and further determining the concentration of the substances to be detected (by the somatic antigens) in the specimen according to the standard curve. The ELISA features high sensitivity, good accuracy and simple and quick operation, effectively shortens analysis time and can be applied to quick screening and detecting of the anisakid larvae in the seafood such as fish.

Description

technical field [0001] The invention relates to a detection method for detecting pathogenic parasites in seafood, in particular to an ELISA detection method for detecting anisakis. The invention is funded by the National 863 Project (No. 2007AA091806) and the special fund for the construction of modern agricultural industrial technology system (nycytx-50-G09). Background technique [0002] Anisakid larvae (anisakid larvae) is anisakid larvae, which is the most harmful type of parasite in seafood. Its pathogenic third-stage larvae mainly parasitize marine fish. The strongest and most widely distributed ones are Anisakis nematodes and Pseudo-Neogroundascaris nematodes. With the gradual rise of seafood raw food, the parasite is very likely to become one of the important hazard factors affecting the safety of seafood consumption in my country in the future. [0003] At present, the detection of Anisakis in seafood mainly includes lamp detection method and enzyme digestion meth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/531G01N21/31
Inventor 曹立民许旭林洪隋建新
Owner OCEAN UNIV OF CHINA
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