Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation and use methods of microcystin-LR monoclonal antibody immunoaffinity column

A microcystin and anti-immune technology, applied in measuring devices, instruments, scientific instruments, etc., can solve problems that affect the accuracy of results, low MC content, complex components of cyanobacteria samples, etc.

Inactive Publication Date: 2009-06-03
江苏省苏微微生物研究有限公司
View PDF0 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the complex composition of the cyanobacteria sample, the MC content in it is low (mass percentage is about 1 / 10,000), if there are many impurities remaining in the pretreatment process, the accuracy of the results can be seriously affected in the subsequent quantitative analysis

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation and use methods of microcystin-LR monoclonal antibody immunoaffinity column
  • Preparation and use methods of microcystin-LR monoclonal antibody immunoaffinity column
  • Preparation and use methods of microcystin-LR monoclonal antibody immunoaffinity column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Preparation of MC-LR monoclonal antibody

[0041] 1. Synthesis of complete antigen

[0042] (1) Synthesis of MC-LR-BSA immunogen

[0043] The synthesis of the complete antigen MC-LR-BSA adopts the water-soluble carbodiimide method, using 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) as a "bridging agent" to make MC The carboxyl group (-COOH) of -LR reacts with EDC to generate an intermediate product, and then reacts with the amino group on the BSA protein molecule to make a complete antigen MC-LR-BSA conjugate as an immunogen. The synthesis method is as follows:

[0044] 0.5mg of MC-LR was dissolved in 0.5mL of methanol, divided into two equal parts of 0.25mL, blown with nitrogen, and evaporated to dryness at 35°C. One portion containing 0.25 mg of MC-LR was dissolved in 1 mL of PBS (pH 7.4) buffer, 5 mg of EDC was added, shaken until completely dissolved, adjusted to pH 5 with 0.1 M hydrochloric acid solution, and stirred slowly at room temperat...

Embodiment 2

[0049] Example 2: Activation of Sepharose 4B

[0050] Sepharose 4B was activated by the cyanogen bromide method. The cyanogen bromide method has good activation effect, high coupling rate, and little effect on antibodies. The activation process is as follows:

[0051] (1) Take 10mL Sepharose 4B and put it in a Buchner funnel to drain it, wash it twice with 30mL water and drain it, add a small amount of 0.1mol / L NaHCO with pH8.3 3 After washing, immediately transfer to a 100mL beaker and stir slowly under ice bath.

[0052] (2) Weigh 1g of cyanogen bromide in a fume hood, add 10mL of water to dissolve, then pour into Sepharose4B in batches, stir while adding, measure the pH value at the same time, and keep the pH at about 10.5 by adding 2mol / L NaOH dropwise . After the cyanogen bromide has completely reacted and the pH remains basically unchanged, the stirring can be stopped.

[0053] (3) Add the activated Sepharose 4B into small ice cubes, quickly pour it into the Buchner ...

Embodiment 3

[0054] Embodiment 3, the preparation of MC-LR monoclonal antibody IAC

[0055] 1. Preparation of MC-LR monoclonal antibody and Sepharose 4B affinity adsorbent

[0056] The operation steps for preparing MC-LR monoclonal antibody and Sepharose 4B affinity adsorbent are as follows:

[0057] (1) Coupling

[0058] The MC-LR monoclonal antibody prepared and purified above was placed in 0.1M NaHCO containing 0.5M NaCl at pH 8.3 3 The buffer was dialyzed against the coupling buffer for 12 h. Put the above-mentioned activated Sepharose 4B gel in a sand core funnel and quickly wash it with the coupling buffer, then quickly pour it into the MC-LR monoclonal antibody solution for coupling, and monitor the coupling process by UV scanning. Wash away the uncoupled anti-MC-LR monoclonal antibody with more than 5 times the volume of coupling buffer to obtain the agarose-antibody coupling complex. Collect all the eluate, and calculate the amount of uncoupled MC-LR monoclonal antibody by mea...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Preparation and use methods of a microcystin-LR (MC-LR) monoclonal antibody immunoaffinity column (IAC) belong to the technical field of immunoaffinity chromatography and MC detection. The IAC is prepared by fixing MC-LR monoclonal antibody in the column, and can acquire the accurate contents of MC-LR and MC-RR through solvent extraction of cyanobacteria sample, enrichment of a solid-phase extraction column, IAC purification and quantitative determination by liquid chromatography-mass spectrometry. The determination can be interfered seriously by impurities to influence the result accuracy by only using SPE purification without IAC purification. The methods can be used for MC detection in water sample, other algae and similar biological samples.

Description

technical field [0001] A method for preparing and using a microcystin-LR monoclonal antibody immunoaffinity column belongs to the technical field of immunoaffinity chromatography and microcystin detection. Background technique [0002] Cyanobacteria are one of the oldest organisms on the earth, and the eutrophication of water body is conducive to the reproduction and growth of cyanobacteria. The ecological abnormal phenomenon that the cyanobacteria in the water body multiply and gather in a large number in a short period of time is called a bloom (also known as lake indigo). Recent surveys show that 54% of lakes in the Asia-Pacific region are eutrophic, the proportions in Europe, Africa, North America and South America are 53%, 28%, 48% and 41% respectively, and 60% in my country. In recent years, several large freshwater lakes in my country have a large number of cyanobacteria blooms. When cyanobacteria blooms appear, the water surface is covered by thick blue-green lake ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N30/50G01N30/06
Inventor 赵晓联肖付刚汤坚赵春城蔡建荣
Owner 江苏省苏微微生物研究有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com