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Magnetic lateral flow immunoassay for rapid detection of TTX and preparation of detection test strip

A technology of magnetic immunochromatography and test strips, which is applied in the field of food inspection, can solve the problems of not being able to detect TTX quickly and easily, and achieve the effect of meeting the needs of qualitative detection, rapid quantitative detection, and short time consumption

Active Publication Date: 2015-04-08
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Purpose of the present invention: aiming at the shortcomings and defects that the existing detection technology cannot detect TTX quickly and easily, provide a kind of magnetic immunochromatography method and magnetic bead-labeled chromatography test paper that use immunomagnetic beads as markers for rapid detection of TTX Strip preparation

Method used

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  • Magnetic lateral flow immunoassay for rapid detection of TTX and preparation of detection test strip
  • Magnetic lateral flow immunoassay for rapid detection of TTX and preparation of detection test strip
  • Magnetic lateral flow immunoassay for rapid detection of TTX and preparation of detection test strip

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1: Magnetic immunochromatography detects TTX in the muscle extract

[0061] (1) Preparation of detection antigen (TTX-BSA conjugate): mix TTX and bovine serum albumin (BSA) at a ratio of 1:5 (w / w), add 4% formaldehyde solution as a coupling agent, and incubate at 30°C Down spin reaction 3d. After the reaction was completed, the mixture was taken out, and the TTX-BSA conjugate was desalted with a commercial PD-10 desalting column according to the attached product instruction manual, and the desalting medium was 10 mMPBS (pH 7.4).

[0062] (2) Antibody preparation and purification: BALB / C mice were immunized with TTX-KLH as an immunogen, and monoclonal antibody cell lines were prepared and screened according to conventional hybridoma technology and limiting dilution method. Then the anti-TTX specific antibody cell line was proliferated and cultured, and injected into BALB / C mice to prepare ascites. After the prepared ascitic fluid was concentrated with 50% (w...

Embodiment 2

[0068] Embodiment 2: Magnetic immunochromatography detects TTX in puffer fish visceral tissue

[0069] Except for the sample detection step, the detection sample is to use the confirmed non-toxic liver extract to serially dilute the TTX standard. Other steps are with example 1.

Embodiment 3

[0070] Embodiment 3: Magnetic immunochromatography detects TTX in wild puffer fish tissue

[0071] (1) Sample preparation of puffer fish tissue

[0072] Take puffer fish muscle tissue as an example. Take 5g of puffer fish muscle tissue, cut it into pieces, add 25ml of 0.1% acetic acid, boil and stir for 10min. After cooling to room temperature, centrifuge at 4000rpm for 10min, and transfer the supernatant into a new 50ml centrifuge tube. Add 20ml of 0.1% acetic acid to the precipitate, and boil for 3min. After cooling to room temperature, all liquid and boiled samples were added to centrifuge tubes and centrifuged at 4000rpm for 10min. Take the supernatant, measure the volume, put it in a separatory funnel, add an equal volume of ether, and defat twice. Put the aqueous layer into a 50ml volumetric flask. Use 1M sodium hydroxide to adjust the pH value of the extract to 6.5-7.4, then add PBS to 50ml, and store the extract at 4°C for later use.

[0073] (2) Sample testing ...

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Abstract

The invention relates to a magnetic lateral flow immunoassay (LFIA) for a rapid detection of Tetrodotoxin (TTX) and a preparation method of a detection test strip, belonging to the technical field of TTX detection. The magnetic LFIA for the rapid detection of TTX is characterized in that: a sample pad, a combination pad labeled by magnetic beads and modified by anti-TTX specific rat monoclonal antibody, a chromatography membrane pre-coated with a detection line T of TTX-BSA and a control line C of IgG, a water absorbent pad are successively pasted on a base plate staggeredly by the distance between adjacent part being 1 mm, then a transparent plastic sealing membrane is coated on the top layer to form a magnetic LFIA test strip for the rapid detection of TTX; immunomagnetic beads are captured by chromatography, the rapid qualitative detection of TTX is realized according to the formed 1-2 macroscopic color developing strips after sample chromatography; the prepared magnetic LFIA test strip through a magnetism analyzer is detected, and the quantitative determination of TTX can be realized according to the detection values of the formed magnetic signal of the detection line and control line after sample chromatography.

Description

technical field [0001] The invention belongs to the field of food inspection, in particular to a magnetic immunochromatographic method for rapid detection of TTX and the preparation of detection test strips, belonging to the detection technology category. Background technique [0002] Puffer fish meat is delicious and nutritious, and Asian residents such as China and Japan have the habit of eating it. However, the ovaries, livers, intestines and other internal organs of puffer fish are rich in Tetrodotoxin (TTX for short). Due to the stable nature of this toxin, Coupled with the lack of effective rescue measures after poisoning, there are reports of poisoning caused by eating poisonous puffer fish every year, which seriously threatens the lives of consumers. [0003] TTX is one of the most toxic natural neurotoxins, widely distributed in nature. In addition to being present in pufferfish species, the toxin has also been detected in certain salamanders, toads, octopuses, con...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/12G01N33/577G01N33/531
Inventor 卢瑛郑诚刘源王锡昌
Owner SHANGHAI OCEAN UNIV
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