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Microcystin monoclonal antibody and its preparation method and application

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

Active Publication Date: 2008-02-20
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
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Embodiment 1

[0034] Example 1. Obtaining the Hybridoma Cell Line MC8C10 CGMCC No.2101 and Identification of the Anti-Microcystin-LR Monoclonal Antibody MC8C10 Produced

[0035] 1. Obtaining the hybridoma cell line MC8C10 CGMCC No.2101

[0036] 1. Synthesis of Microcystin-LR Complete Antigen MC-LR-BSA

[0037] 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:

[0038] (1) Amino modification of Microcystin-LR

[0039]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 ...

Embodiment 2

[0086] Example 2. Using the monoclonal antibody MC8C10 secreted by the hybridoma cell line MC8C10 CGMCC No.2101 to detect microcystin-LR in water samples

[0087] The competition ELISA method is used for detection, and the reagents and microtiter plates used are prepared according to the following methods:

[0088] 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.

[0089] 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 microcystlin monoclonal antibody and the preparation method and the application thereof. The microcystlin monoclonal antibody is a microcystlin-LR monoclonal antibody, and the preparation method thereof is that: 1) an amino group is introduced to a seventh amino acid residue N-methyl dehydroalanine on microcystlin-LR, thus obtaining amino modified microcystlin-LR polypeptide; the amino modified microcystlin-LR polypeptide is coupled with carrier protein to obtain complete A antigen; 2) the immunity mouse of complete A antigen obtained in step 1) to get spleen cell of immunity mouse, which then is fused with myeloma cell of mouse to screen out positive hybrid cancer cell strain; the positive hybrid cancer cell strain is cultured or is injected in sygeneous mouse abdominal cavity to induce ascites to get the microcystlin-LR monoclonal antibody. The monoclonal antibody can be used for examining microcystlin-LR and immunity affinity purification thereof.

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] [0004] (Formula I) [0005] 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 wa...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/577C12N5/18
Inventor 何苗盛建武施汉昌
Owner TSINGHUA UNIV
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