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Method for producing algae toxin immune affinity chromatographic column and use method thereof

A technology of algae toxin and chromatographic column, which is applied in the direction of chemical instruments and methods, analytical materials, algae/bryopeptide, etc., can solve the problems that the measured value is far from the actual value, interfere with the detection result, etc., and achieve a significant purification effect, The preparation method is simple and the effect is obvious

Inactive Publication Date: 2009-07-29
无锡市疾病预防控制中心 +2
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0009] At present, the detection of algae toxins is mainly performed by instruments such as HPLC, LC-MS or GC-MS. The pretreatment should be enriched by solid-phase extraction column, but the solid-phase extraction column is not specific, and many impurities will be introduced, which will interfere with the detection. As a result, sometimes the measured value is far from the actual value

Method used

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  • Method for producing algae toxin immune affinity chromatographic column and use method thereof
  • Method for producing algae toxin immune affinity chromatographic column and use method thereof
  • Method for producing algae toxin immune affinity chromatographic column and use method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1, the preparation of algal toxin immunoaffinity chromatography column

[0042] 1. Dissolve 0.1mg of microcystin-LR polyclonal antibody in 5mL of 0.1M NaHCO containing 0.5M NaCl at pH 8.3 3 in the buffer.

[0043] 2. Weigh 0.2 g of cyanogen bromide-activated agarose freeze-dried powder, swell it with an appropriate amount of 1 mM dilute hydrochloric acid, and then wash it on a sintered glass filter to remove impurities.

[0044] 3. Dilute the swollen agarose gel in 0.1M NaHCO containing 0.5M NaCl at pH 8.3 3 Mix evenly with the antibody solution prepared in (1) in the buffer, and fully react with shaking at room temperature for 3 hours.

[0045] 4. Use 0.1M NaHCO containing 0.5M NaCl at pH 8.3 3 The buffer washes away the free antibody that is not bound to the agarose gel to obtain the agarose-antibody conjugated complex.

[0046] 5. Transfer the agarose-antibody coupling complex into a 1M ethanolamine solution containing 0.5M NaCl at pH 8.0 and keep for ...

Embodiment 2

[0051] Example 2. Application of algal toxin immunoaffinity chromatography column in the detection of microcystin-LR in Taihu Lake water samples polluted by cyanobacteria

[0052] 1. The method of using algal toxin immunoaffinity chromatography column

[0053] (1) The prepared cyanotoxin immunoaffinity chromatography column was pretreated with 5 mL methanol and 5 mL distilled water.

[0054] (2) 1L of water sample is passed through a glass GF / C microfiber filter, and the filtrate is passed through a treated algal toxin immunoaffinity chromatography column.

[0055] (3) Wash the column with 5mL PBS and 5mL distilled water to remove impurities;

[0056] (4) The microcystin-LR adsorbed in the column was eluted with 10 mL of pure methanol, the eluent was evaporated to dryness under reduced pressure, and the residue was dissolved in 0.5 mL of 30% methanol solution before HPLC analysis;

[0057] (5) The algae toxin purification sample is introduced into HPLC analysis, and the chro...

Embodiment 3

[0063] Example 3, the application of cyanotoxin immunoaffinity chromatography column in the detection of microcystin-LR in cyanobacteria powder

[0064] 1. Pretreatment of cyanobacteria powder

[0065] Add 1 g of crushed blue-green algae powder to 30 mL of 5% acetic acid, stir and extract for 60 minutes, centrifuge, collect the supernatant, precipitate and repeat the extraction twice, combine the supernatant and filter, and the filtrate is set aside.

[0066] 2. The method of using the algal toxin immunoaffinity chromatography column

[0067] The immunoaffinity column purification method of the filtrate is the same as that of the water sample in Example 2.

[0068] 3. Test results

[0069] The content of microcystin-LR in cyanobacteria powder is much higher than that in water. According to the approximate content of the filtrate purified by immunoaffinity chromatography column in HPLC for the first time, dilute it to within the linear range of 0.01-0.5 μg / mL of Microcystin-...

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Abstract

The invention discloses a method for preparing and using a cyanotoxin immunoaffinity chromatography column, which belongs to the field of biotechnology. The invention relates to the preparation of a algae toxin immunoaffinity chromatography packing material, an immunoaffinity chromatography column and a use method thereof by using an antibody capable of specifically recognizing algae toxin. Its purpose is to facilitate the "one-step" enrichment and purification of algal toxins from the extracts of water, aquatic products, algae and their products, and the purified samples are analyzed qualitatively and quantitatively by HPLC or LC-MS. The algae toxin immunoaffinity chromatography column prepared in the present invention has strong specificity for one algae toxin, remarkable purification effect, and is obviously superior to the existing solid phase extraction method. The algae toxin immunoaffinity chromatography column improves the efficiency of enrichment and purification, thereby improving the sensitivity and accuracy of the detection method, and the preparation method is simple and reusable.

Description

technical field [0001] The invention discloses a method for preparing and using a cyanotoxin immunoaffinity chromatography column, which belongs to the field of biotechnology. Background technique [0002] Recent surveys show that due to pollution, 54% of lakes in the Asia-Pacific region are eutrophic, and the proportions in Europe, Africa, North America and South America are 53%, 28%, 48% and 41% respectively, and 60% in my country. . In the eutrophic freshwater water body, when there are suitable conditions, 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 water bloom (also known as lake indigo). [0003] When cyanobacteria blooms appear, the water surface is covered by thick blue-green lake indigo, and even accumulates in large quantities on the shore. In the process of the death and decomposition of a large number of algae, not only emits a foul smell, but also destroys ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/405G01N33/559B01J20/291
Inventor 肖付刚张敬平赵晓联钮伟民汤坚赵春城蔡建荣孙蔚榕
Owner 无锡市疾病预防控制中心
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