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Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column

A technology of microcystin and immunophile, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of high price, low detection limit, interference detection results, etc.

Active Publication Date: 2009-06-03
启东市三江建筑机械有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, HPLC and LC-MS methods are commonly used for the detection of MC-LR in water. Due to the low content, solid phase extraction (SPE) is generally used for enrichment before sample injection, but SPE is not specific, and there are many impurities in water, which will Introduce many impurities and interfere with the detection results, especially in HPLC method, sometimes the measured value is far from the actual value
Although the LC-MS method has a low detection limit and relatively accurate measurement results, it is expensive and generally not available in laboratories.

Method used

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  • Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column
  • Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column
  • Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Preparation of MC-LR polyclonal antibody

[0040] 1. Synthesis of complete antigen

[0041] (1) Synthesis of MC-LR-BSA immunogen

[0042]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:

[0043] Dissolve 0.5mg of MC-LR in 0.5mL of methanol, divide it into two equal portions of 0.25mL, pass nitrogen gas, and evaporate 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 temperature for...

Embodiment 2

[0053] Example 2: Activation of Sepharose 4B

[0054] 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:

[0055] (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.

[0056] (2) Weigh 1g of cyanogen bromide in a fume hood, add 10mL of water to dissolve, then pour into agarose in batches, stir while adding, and measure the pH value at the same time, and keep the pH at 10.5 by adding 2mol / L NaOH dropwise about. After the cyanogen bromide has completely reacted and the pH remains basically unchanged, the stirring can be stopped.

[0057] (3) Add the activated Sepharose 4B into small ice cubes, quickly pour it into the Buchner f...

Embodiment 3

[0058] Example 3: Preparation of MC-LR polyclonal antibody IAC

[0059] The MC-LR polyclonal antibody is coupled with Sepharose 4B to obtain an affinity adsorbent, which is filled into the column to prepare the MC-LR polyclonal antibody immunoaffinity column. The operation steps are as follows:

[0060] (1) Coupling

[0061] Place the MC-LR polyclonal antibody prepared and purified above 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 polyclonal antibody solution for coupling, and monitor the coupling process by UV scanning. Wash away the unconjugated anti-MC-LR polyclonal antibody with more than 5 times the volume of coupling buffer to obtain the agarose-antibody coupling complex. All the eluates were collected, and the amount of uncoupled MC-LR polyclonal anti...

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Abstract

Preparation and use methods of a microcystin-LR (MC-LR) polyclonal antibody immunoaffinity column (IAC) belong to the technical field of immunoaffinity chromatography and MC detection. The IAC is prepared by fixing MC-LR polyclonal antibody in the column, so that MC-LR in the sample solution can be adsorbed by the IAC and can be enriched and purified after elution, then the quantitative and qualitative analysis can be carried out by HPLC. As the pre-processing process, the IAC enrichment is superior to that of the prior solid-phase extraction (SPE) method. HPLC test shows that MC-LR peak of the MC-LR polyclonal antibody can be separated from peaks of other impurities by IAC so as not to influence the detection result. However, the SPE method is difficult to distinguish the MC-LR peak due to the presence of so many impurities. The MC-LR polyclonal antibody can be specifically combined with the MC-LR, so that IAC has excellent specificity to remove the majority of interferents in one step. The SPE has no specificity and cannot remove the interferents so as to influence the final detection result.

Description

technical field [0001] A method for preparing and using a microcystin-LR polyantibody immunoaffinity column belongs to the technical field of immunoaffinity chromatography and microcystin detection. Background technique [0002] With the development of industry and agriculture and the acceleration of urbanization, industrial and domestic sewage enters the water environment, resulting in increased eutrophication of water bodies. The increase in the content of nitrogen and phosphorus in the water has caused the frequent occurrence of toxic cyanobacteria blooms. In recent years, my country's drinking water sources such as Taihu Lake, Chaohu Lake, Dianchi Lake and other lakes, some reservoirs and rivers have been attacked by cyanobacteria blooms. According to the "Report on the Protection and Development of the Yangtze River" released in April 2007, after the Three Gorges reservoir area was impounded to 135 meters in 2003, 12 first-class tributaries of the Yangtze River had "wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/50G01N30/06
Inventor 张敬平肖付刚钮伟民汤坚周磊戴维杰毛云中
Owner 启东市三江建筑机械有限公司
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