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A kit of enzyme-linked immunity detection for toxin of microcapsule alga

A technology of microcystin enzyme and microcystin, which is applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems of low sensitivity, high detection cost, cumbersome detection process, etc., and achieves convenient use, structural Simple, Sensitive Effects

Active Publication Date: 2007-12-26
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Conventional physical and chemical detection methods, especially liquid chromatography and mass spectrometry instruments, are bulky, expensive, require indoor environment with high environmental conditions, and require specialized technicians to operate, complicated pre-treatment, and expensive detection; and due to micro There are many isomers of cystic toxins with similar properties, and the lack of corresponding standards has become a limitation for large-scale screening of microcystins
Although the phosphatase inhibition test and cell test can detect toxins well, there are shortcomings such as low sensitivity or cumbersome detection process, and small sample throughput.

Method used

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  • A kit of enzyme-linked immunity detection for toxin of microcapsule alga
  • A kit of enzyme-linked immunity detection for toxin of microcapsule alga
  • A kit of enzyme-linked immunity detection for toxin of microcapsule alga

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0044] Example 1. Obtaining the Hybridoma Cell Line MC8C10 CGMCC No.2101 and Identification of the Anti-Microcystin-LR Monoclonal Antibody MC8C10 Produced

[0045] 1. Obtaining the hybridoma cell line MC8C10 CGMCC No.2101

[0046] 1. Synthesis of Microcystin-LR Complete Antigen MC-LR-BSA

[0047] Microcystin-LR was chemically modified with 2-mercaptoethylamine, and an active group—amino group was introduced into its seventh amino acid (Mdha), and then the modified microcystin was synthesized by glutaraldehyde method. -LR was coupled with bovine serum albumin, and the complete antigen was obtained after filtration chromatography, and the coupling ratio of the complete antigen was determined by MALDI-TOF / MS. The specific method is as follows:

[0048] (1) Amino modification of Microcystin-LR

[0049] ① Fully mix 2-mercaptoethylamine and MC-LR in an alkaline carbonate buffer (pH=8.0) according to a molar ratio of 3000:1; shake the mixture well and react at 50°C for 1.5 hours; ...

Embodiment 2

[0096] Example 2, Preparation and Identification of Anti-Microcystin-LR Polyclonal Antibody

[0097] 1. Preparation of polyclonal antibody against microcystin-LR

[0098] Clinically healthy male New Zealand white rabbits with a body weight of 1.5-2 kg were injected subcutaneously at 6 points on the back with the microcystin-LR complete antigen MC-LR-BSA prepared in Example 1 at a dose of 1.2 mg immunogen / rat. Freund's complete adjuvant was used for the first immunization, followed by booster immunization with immunogen containing Freund's incomplete adjuvant every four weeks thereafter, a total of four times before and after, and 10 days after the last immunization, slaughtered, blood was collected, and serum was separated and refrigerated for later use.

Embodiment 3

[0099] Embodiment 3, microcystin-LR competition ELISA kit

[0100] 1. Composition of Microcystin-LR Competition ELISA Kit

[0101] The kit includes the following reagents and microtiter plate installed in the kit box:

[0102] a) Preparation of anti-microcystin-LR standard solution: MC-LR purchased from Alexis Company (Lausen, Switzerland) was used as a standard product (product number ALX-350-012), and high-purity water was used to prepare MC-LR standard solution respectively , with concentrations of 0.5 μg / L, 2 μg / L and 8 μg / L, respectively, filled into MC-LR standard reagent bottles.

[0103] b) Antibody solution preparation: Dilute the monoclonal antibody MC8C10 (1 mg solid) of Example 1 with phosphate buffer to a working concentration of 1:6000, and then add 1% (mass percentage) bovine serum albumin (BSA) and 0.1% thimerosal (mass percentage), filled in the reagent bottle.

[0104] c) Preparation of enzyme-labeled secondary antibody solution: horseradish peroxidase-goa...

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Abstract

An enzyme-linked immunoassay kit of microcapsule phycotoxin consists of coated antigen of complete antigen A and multiclone antibody of microcapsule phycotoxin-LR as well as enzyme labeled two-antis. The said complete antigen A is prepared for leading in an amino group on N-methy dehydroalanine at the seventh amino acid residual base of microcapsule phycotoxin to obtain microcapsule phycotoxin being modified by amino group, coupling modified microcapsule phycotoxin with carrier protein to obtain coupled object being used as said complete antigen A.

Description

technical field [0001] The invention relates to a microcystin ELISA detection kit, in particular to a microcystin-LR ELISA detection kit. Background technique [0002] Eutrophication of water body causes algae to proliferate abnormally and release Microcystins (MCs), among which Microcystin-LR (Microcystin-LR, MC-LR) is currently known to be the most acutely toxic and harmful Freshwater cyanotoxins. Microcystin-LR is a group of cyclic heptapeptides whose chemical structural formula is shown in formula I: [0003] (Formula I) [0004] Frequent outbreaks of microcystin blooms seriously threaten ecological security and human drinking water safety, and timely and accurate monitoring of microcystins in water is very important. At present, the research and development of algal toxin detection technology in water is very active, mainly including high performance liquid chromatography (High Performance Liquid Chromatography, HPLC), enzyme-linked immunosorbent assay (Enzyme-Link...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531G01N33/53C12N5/12
Inventor 何苗盛建武施汉昌
Owner TSINGHUA UNIV
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