Vitamin B12 aptamer affinity column, and preparation method and application thereof

An aptamer and B12 technology, applied in chemical instruments and methods, separation methods, solid adsorbent liquid separation, etc., can solve problems affecting the efficiency of immunoaffinity columns, preservation of antibody inactivation, etc., to reduce cross-reactivity, High purification efficiency and guaranteed correctness

Inactive Publication Date: 2017-05-24
BEIJING MEIZHENG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Improper storage or improper operation can easily cause antibody inactivation and affect the efficiency of the immunoaffinity column

Method used

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  • Vitamin B12 aptamer affinity column, and preparation method and application thereof
  • Vitamin B12 aptamer affinity column, and preparation method and application thereof
  • Vitamin B12 aptamer affinity column, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041]Example 1: Preparation of an affinity column using an amino-modified vitamin B12 aptamer

Embodiment approach

[0042] A preferred embodiment of the present invention to prepare vitamin B12 aptamer affinity column is as follows:

[0043] 1. Vector activation

[0044] Select N-hydroxysuccinimide (NHS) modified agarose carrier sepharose4B for activation

[0045] Take 1g of N-hydroxysuccinimide-modified agarose, add 50ml of 1mM HCl, after swelling for 30min, wash the gel with 100ml of 1mM HCl for 6 times, and then wash with 100ml of ultrapure water

[0046] 2. The activated agarose gel sepharose4B with coupling buffer (0.1M NaHCO 3 , 0.8M NaCl, pH8.2) and washed 3 times. Add 20mol / L amino-modified vitamin B12 aptamer, and couple at room temperature for 8 hours

[0047] 3. Wash the coupled vitamin B12 aptamer-agarose carrier 3 times with 20mM, pH7.4 phosphate buffer PBS

[0048] 4. closed

[0049] Add 100mmol / L Tris.Cl pH8.0 to the coupled vitamin B12 aptamer-agarose carrier, and react at room temperature for 2 hours

[0050] 5. Wash the blocked vitamin B12 aptamer-agarose carrier 3 t...

Embodiment 2

[0060] Example 2: Preparation of an affinity column using a biotin-modified vitamin B12 aptamer

[0061] A preferred embodiment of the present invention to prepare vitamin B12 aptamer affinity column is as follows:

[0062] 1. Take 1ml streptavidin (SA)-modified agarose gel sepharose4B, wash 3 times with 10ml ultrapure water

[0063] 2. Wash the (SA) modified sepharose4B three times with coupling buffer (0.02M PBS, 0.8M NaCl, pH7.4). Add 20mol / L biotin-modified vitamin B12 aptamer, and couple at room temperature for 4 hours

[0064] 3. Wash the coupled vitamin B12 aptamer-agarose carrier 3 times with 20mM, pH7.4 phosphate buffer PBS

[0065] 4. Column packing

[0066] Put the vitamin B12-agarose carrier into the chromatographic column according to the need, and prepare vitamin B12 affinity purification columns with different capacities according to the need

[0067] 1) Take the empty cylinder, add the lower sieve plate, add 1ml PBS, let it drain naturally

[0068] 2) Add ...

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PUM

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Abstract

The invention relates to a vitamin B12 aptamer affinity column, and a preparation method and application thereof. The preparation method comprises the following steps: by using a chemically-modified solid-phase carrier, carrying out covalent coupling on a vitamin B12 aptamer and the carrier; and filling the affinity column. The aptamer affinity column is mainly used for purifying vitamin B12 in food, milk powder, milk, beverages and many other samples, thereby facilitating the high performance liquid chromatography (HPLC) detection and mass spectrometric detection on vitamin B12 in the sample in the later period.

Description

technical field [0001] The invention relates to an aptamer affinity column for vitamin B12, a preparation method and application thereof. It belongs to the field of food testing. Background technique [0002] Vitamin B12 is the only vitamin that contains the main mineral cobalt, so it is also called cobalamin, including cyanocobalamin, hydroxocobalamin, methylcobalamin, and adenosylcobalamin. Vitamin B12 is usually referred to narrowly as cyanocobalamin, which is the main form of B12 in food and nutritional supplements. B12 is easily soluble in water and needs to be combined with calcium when absorbed. Most of it is absorbed by the small intestine. The human body can only use methylcobalamin and adenosylcobalamin. Therefore, cyanocobalamin must be converted into these two forms by cells in the body to be absorbed Human body absorbs and utilizes. [0003] Vitamin B12 deficiency in infants and young children manifests as abnormal mental mood, sluggish expression, unresponsi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/38G01N30/06
CPCB01D15/3819G01N30/06
Inventor 张彦明
Owner BEIJING MEIZHENG BIOTECHNOLOGY CO LTD
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