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A broad-spectrum microcystin ELISA detection kit

A technology of microcystin enzyme and microcystin, applied in biological testing, immunoglobulin, measuring devices, etc., can solve the problem that the total amount of microcystin cannot be directly detected

Active Publication Date: 2021-11-09
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing microcystin detection kits can only detect one microcystin isomer, and cannot directly detect the total amount of microcystin

Method used

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  • A broad-spectrum microcystin ELISA detection kit
  • A broad-spectrum microcystin ELISA detection kit
  • A broad-spectrum microcystin ELISA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1, the acquisition and identification of positive hybridoma cell lines and the preparation of broad-spectrum microcystin monoclonal antibodies

[0062] 1. Synthesis of immunogen KLH-MCs

[0063] The above-mentioned 12 microcystin small molecule structural variants introduced an amino active group on the seventh amino acid (Mdha) through chemical modification with 2-mercaptoethylamine, and purified by solid phase extraction with glutaraldehyde two The modified microcystin molecules were coupled with KLH respectively, and then 12 kinds of complete antigens were obtained after gel filtration chromatography, and the immunogen KLH-MCs were obtained after mixing in equal amounts. The specific method is as follows:

[0064] Step 1 Preparation of amino-modified microcystin molecules and KLH conjugates

[0065] 1.1. Preparation of amino-modified MC-LR and KLH conjugates

[0066] Dissolve 2-mercaptoethylamine and microcystin small molecule MC-LR with a molar ratio of 3...

Embodiment 2

[0148] Embodiment 2, the composition of broad-spectrum microcystin ELISA kit

[0149] 1. Composition of broad-spectrum microcystin ELISA kit

[0150] The kit includes the following reagents and microtiter plate installed in the kit box:

[0151] a) Preparation of anti-microcystin standard solution: make a series of 3-fold gradient dilutions of MC-LR standard with sample diluent, and prepare concentrations of 20.000 μg / L, 6.667 μg / L, and 2.222 μg / L respectively , 0.741 μg / L, 0.247 μg / L, 0.082 μg / L, 0.027 μg / L, and 0 μg / L standard solutions were filled into standard reagent bottles respectively.

[0152] b) Antibody solution preparation: the monoclonal antibody MCAB1 of Example 1 was diluted with phosphate buffer, and then bovine serum albumin (BSA) and thimerosal were added to obtain an antibody solution. In the antibody solution, the content of the monoclonal antibody MCAB1 is 0.0625 μg / mL, the content of BSA is 1% (mass percentage), and the content of thimerosal is 0.01% (m...

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Abstract

The invention relates to a broad-spectrum microcystin enzyme-linked immunoassay kit, comprising a coating antigen, a microcystin monoclonal antibody solution and an enzyme-labeled secondary antibody; the microcystin monoclonal antibody solution is the The microcystin monoclonal antibody is diluted with phosphate buffer solution, and the solution obtained by adding bovine serum albumin and thimerosal. The broad-spectrum microcystin enzyme-linked immunoassay kit of the present invention has the advantages of strong broad-spectrum response to MCs isomers, high sensitivity, large analysis throughput, fast detection speed, and low cost, and can be applied in water , Quantitative detection of the total amount of microcystins in freshwater products of animal origin and freshwater products of plant origin.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to an enzyme-linked immunoassay kit for detecting the total amount of microcystin. Background technique [0002] Microcystins (MCs) are secondary metabolites produced by cyanobacteria such as Microcystis and Anabaena during water blooms, and are one of the most widely distributed cyanotoxins in fresh water. MCs are a class of small molecules with a common cyclic heptapeptide structure, consisting of 5 non-proteinogenic amino acids (such as dehydroalanine derivatives and special β-amino acid Adda, etc.) and 2 proteinogenic amino acids (the second and the variable amino acid at the 4th position), and its chemical structural formula is shown in Formula 1. More than 100 microcystin isomers have been reported so far, and the MC isomers widely distributed in nature are MC-LR, MC-RR and MC-YR (L, R, Y represent leucine respectively acid, arginine and tyrosine). [0003] [0004] M...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/545G01N33/53C07K16/44
CPCC07K16/44G01N33/5308G01N33/545G01N33/577
Inventor 周小红
Owner TSINGHUA UNIV
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