A novel small molecule structure and its application in the detection of blue-green algae hepatotoxins

A blue-green algae liver and small molecule technology, applied in measurement devices, analytical materials, instruments, etc., can solve problems such as differences in antibody characteristics and broad-spectrum limitations, and achieve high sensitivity, broad-spectrum, and easy operation. Effect

Active Publication Date: 2018-09-18
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Most of the antibodies prepared from blue-green algae hepatotoxins (a variety of microcystin analogs and nodular toxins) that have been reported so far use MC-LR and NOD coupling proteins to prepare antigens and antibodies, and their antibody characteristics are also very different , broad-spectrum is also limited by the immunogen and coater

Method used

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  • A novel small molecule structure and its application in the detection of blue-green algae hepatotoxins
  • A novel small molecule structure and its application in the detection of blue-green algae hepatotoxins
  • A novel small molecule structure and its application in the detection of blue-green algae hepatotoxins

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1 Synthesis of novel small molecule LH-1 structure

[0060] 1. Synthesis of new small molecule structure LH-1

[0061] The synthetic route of the novel small molecule structure LH-1 is as follows: figure 2 As shown, the specific reaction process is as follows:

[0062] (a) Aldol condensation of biuret: At room temperature, weigh 26.2g of D-propionylthioazoalkylthione (CAS: 1217320-19-8) and dissolve it in 250mL of anhydrous dichloromethane, then stir in Cool in acetone with dry ice to -78°C, add 12 mL of titanium tetrachloride dropwise to the above solution, stir the resulting mixture for 15 min to form a titanium complex, then add 18.9 mL of distilled N,N-diiso Propylethylamine, the mixture was then stirred for 40 min to form the corresponding titanium enolate, then 20.0 mL of freshly distilled N-methylpyrrolidone was added, and after 10 min, 13.4 mL of freshly distilled phenylacetaldehyde was added, and the above solution was continued at -78 ℃ for another...

Embodiment 2

[0081] Example 2 Synthesis of artificial antigen

[0082] 1. Synthesis of artificial antigen

[0083] (1) Accurately weigh 2 mg of LH-1, 1 mg of NHS and 1 mg of EDC, dissolve in 500 μL of DMF, and stir overnight in the dark at room temperature, no precipitation appears;

[0084] (2) 8 mg of keyhole limpet hemocyanin (KLH) was added to 500 μL of carbonic acid buffer (0.1moL / L, pH 9.6);

[0085] (3) Slowly add the LH-1 solution dropwise into the KLH solution, and stir for 3 hours at room temperature in the dark;

[0086] (4) Dialyze with phosphate buffer saline for two days, 4 times a day, and obtain LH-1 artificial antigen after dialysis.

[0087] (5) The coupling method of MC-LR and ovalbumin (OVA) is the same as above.

[0088] Carbonate solution (0.1M, pH 9.6): weigh 1.59g Na 2 CO 3 and 2.93g NaHCO 3 , Dilute to 1000mL with deionized water.

[0089] Phosphate buffer (0.01M, pH 7.4): Na 2 HPO 4 12H 2 O 2.90g, NaCl 8.50g, KCl 0.20g, KH 2 PO 4 0.20g, add deionized ...

Embodiment 3

[0093] Example 3 Preparation and Purification of LH-1 Polyclonal Antibody with New Small Molecule Structure

[0094] 1. Preparation of novel small molecular structure LH-1 antibody

[0095] New Zealand white rabbits were immunized with LH-1-KLH, 1mg / mL artificial antigen solution was mixed and emulsified with an equal amount of adjuvant, the initial immunization was mixed with Freund's complete adjuvant after emulsification, and the immunogen and Freund's complete adjuvant were mixed and emulsified for the subsequent booster immunization. After emulsification of incomplete adjuvant, the animals were immunized four times, with an interval of 3 weeks between each time.

[0096] 2. Purification of LH-1 polyclonal antibody with new small molecule structure (caprylic acid-ammonium sulfate method):

[0097] (1) Take rabbit serum and centrifuge at 12000r / min for 15min at 4°C to remove the precipitate;

[0098] (2) Mix 1 volume of rabbit serum with 2 volumes of acetate buffer (pH 4....

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PUM

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Abstract

The invention discloses a novel small molecule structure and application thereof in detection of hepatotoxin in blue-green algae. The novel small molecule structure has a structural formula shown as in formula (I) that is shown in the description. The special novel small molecule structure is acquired first herein as an immunogen; the small molecule structure and microalgal toxin MC-LR are conjugated to a carrier protein to obtain an artificial antigen, a polyclonal antibody is prepared with the small molecule and artificial antigen and is used to prepare antibody-labeled nano gold particles, a nitrocellulose membrane is coated with the MC-JR artificial antigen to form a detection strip, a goat anti-rabbit secondary antibody is used as a control strip, and a broad-spectrum immunoassay test strip for quick detection of hepatotoxin in blue-green algae is established according to the competitive immunoassay principle. The immunochromatography test strip has high sensitivity and broad spectrum, is capable of semi-quantitatively detecting hepatotoxin in blue-green algae, is applicable to various microcystin analogues and nodularin, and has the advantages of high speed, good visuality, good operational convenience and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety. More specifically, it relates to a novel small molecule structure and its application in the detection of blue-green algal hepatotoxins. Background technique [0002] With the development of industrialization and urbanization, a large amount of nutrients are injected into the water to cause eutrophication of the water body, which leads to the rapid growth of algae, and the metabolism of algae produces toxic substances including blue-green algae hepatotoxins (microcystins, nodularin) substances that endanger human health. my country is one of the countries with the most serious algae disasters in the world. 80% of the algae blooms in freshwater lakes are poisonous, and microcystins are the most widely distributed and most toxic. [0003] Microcystins (MCs) are mainly produced by freshwater algae Microcystis aeruginosa. MCs are a class of monocyclic heptapeptide hepatotoxins. Due to the diff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/531G01N33/559G01N33/543
CPCG01N33/531G01N33/54346G01N33/559
Inventor 雷红涛张雅琼孙远明沈兴陆宁徐振林沈玉栋杨金易
Owner SOUTH CHINA AGRI UNIV
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