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Microcystin monoclonal antibody and its preparation method and application

A monoclonal antibody, microcystin technology, applied in biochemical equipment and methods, microorganisms, anti-animal/human immunoglobulin, etc., to achieve high stability and uniform texture

Active Publication Date: 2009-12-09
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, antibodies are the bottleneck determining the large-scale application of immunoassays for microcystins

Method used

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  • Microcystin monoclonal antibody and its preparation method and application
  • Microcystin monoclonal antibody and its preparation method and application
  • Microcystin monoclonal antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Obtaining of the hybridoma cell line MC8C10CGMCC No.2101 and identification of the anti-microcystin-LR monoclonal antibody MC8C10 produced

[0034] 1. Obtaining the hybridoma cell line MC8C10CGMCC No.2101

[0035] 1. Synthesis of Microcystin-LR Complete Antigen MC-LR-BSA

[0036] 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 (MC-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:

[0037] (1) Amino modification of Microcystin-LR

[0038] 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....

Embodiment 2

[0085] Example 2. Using the monoclonal antibody MC8C10 secreted by the hybridoma cell line MC8C10 CGMCC No.2101 to detect microcystin-LR in water samples

[0086] The competition ELISA method is used for detection, and the reagents and microtiter plates used are prepared according to the following methods:

[0087] a) Preparation of anti-microcystin-LR standard solution: MC-LR purchased from Alexis Company (Lausen, Switzerland) was used as the standard product (product number ALX-350-012), and the following concentrations were prepared with high-purity water (unit: μg / L) Gradient MC-LR standard solution: 1000, 300, 90, 27, 8.1, 2.43, 0.729, 0.219, 0.0656, 0.020, 0.006, 0, as a water sample.

[0088] 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% (mass percentage composition) of thimerosal, filled ...

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Abstract

The invention discloses a microcystin monoclonal antibody as well as its preparation method and application. The microcystin monoclonal antibody of the present invention is a monoclonal antibody of microcystin-LR, which is prepared according to the method comprising the following steps: 1) the amino acid residue N-formaldehyde at the seventh position of microcystin-LR An amino group is introduced on the base dehydroalanine to obtain an amino-modified microcystin-LR polypeptide; the amino-modified microcystin-LR polypeptide is coupled to a carrier protein to obtain a complete antigen A; 2) the The complete antigen A of step 1) is used to immunize mice, the splenocytes of immunized mice are taken, fused with mouse myeloma cells, and a positive hybridoma cell line is screened out; the positive hybridoma cell line is cultivated or the positive hybridoma cell line is The cell line was injected into the peritoneal cavity of syngeneic mice to induce ascites, and the monoclonal antibody to microcystin-LR was obtained. The monoclonal antibody can be used for the detection and immunoaffinity purification of microcystin-LR.

Description

technical field [0001] The invention relates to a microcystin monoclonal antibody and its preparation method and application. 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. Therefore, timely and accurate monitoring of microcystins in water is very important. Immunoassay is a detection method based on antigen-antibody specific reaction. It has the advantages of strong specificity, high sensitivity, large analysis throughput, fast detection speed and low cost. It is used for rapid screening and early warning of l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18G01N33/577C12N5/18
Inventor 何苗盛建武施汉昌
Owner TSINGHUA UNIV
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