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Method for detecting activity of lectin of beans

A technology for lectins and beans, which is applied in the field of measuring the activity of bean lectins, can solve the problems of large error in results, difficult operation, high subjectivity, etc., and achieves high stability and accuracy, simple operation method, and high measurement results. accurate effect

Inactive Publication Date: 2009-03-11
CHINA AGRI UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing methods for measuring lectin activity mainly have the following disadvantages: (1) the equipment used is expensive, the operation steps are cumbersome, and the operation is difficult; (3) only qualitative or semi-quantitative assays can be performed; (4) standard cell suspensions are prepared The methods are different, the reproducibility is poor, and many methods require fresh blood to prepare red blood cell suspension on the same day; secondly, (5) the determination of the reading end point time is relatively random, and some results are observed with the naked eye, which is highly subjective, resulting in The error of the result is large, and the accuracy is reduced; (6) There are many measurement methods, and it is difficult to compare the measured results

Method used

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  • Method for detecting activity of lectin of beans
  • Method for detecting activity of lectin of beans
  • Method for detecting activity of lectin of beans

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Determination of Mung Bean Lectin Activity

[0040] (1) Preparation of mung bean lectin solution

[0041] Take 25g mung beans, crush them and pass through a 40-mesh sieve, soak them in 100ml of PBS buffer solution (0.05M, pH7.0, containing 0.15M NaCl), stir at 0°C for 6h, then centrifuge at 4°C and 16000×g for 15min, Collect the supernatant, soak the resulting precipitate with PBS buffer solution (0.05M, pH7.0, containing 0.15M NaCl), and stir at 0°C for 3 hours, then centrifuge at 4°C, 16000×g for 15min, collect The two supernatants were combined to obtain a mung bean lectin solution.

[0042] (2) Preparation of standard red blood cell solution

[0043]Take fresh rabbit blood added with 10% sodium citrate, centrifuge at 1277×g for 15 minutes, then suspend the collected red blood cell pellet in normal saline, and then centrifuge, repeat this 5 times until the supernatant has no obvious red color; Add potassium phosphate saline buffer solution (pH8.5, 5mM, add 25ml pe...

Embodiment 2

[0053] Comparative experiment of different lectin determination methods

[0054] Various measurement methods are as follows:

[0055] (1) Spectrophotometry: The preparation of the mung bean agglutinin solution and the standard erythrocyte suspension are the same as the enzyme standard method (see Example 1). The mung bean agglutinin solution prepared in Example 1 was serially diluted to two times, followed by the following operations: draw 1 ml of each of the above solutions into 15 test tubes of 0.5×8 cm, and then take 1 ml of the standard erythrocyte solution prepared in Example 1 and add Each test tube is mixed with a vortex mixer, then put into a colorimetric cell with an optical diameter of 0.5cm and left for 90 to 120 minutes, and then carefully (minimize the shaking) into the spectrophotometer to measure A 620nm . Then calculate according to the following formula

[0056] log x = log A + ...

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Abstract

The invention discloses a method for determining the activity of bean agglutinin, which mainly comprises the steps that: bean agglutinin solution is prepared, standard erythrocyte solution is prepared, then the bean agglutinin solution is carried out series doubling dilution on the 96-hole elisa plate and the standard erythrocyte solution with the same volume is added to each hole, finally, the light absorption value under 620nm is determined by using an eliasa and the activity of the agglutinin is calculated according to the formula. The invention has accurate determination result, high stability and precision, and strong comparability, establishes a method for preparing standard red blood cells which can be stored and placed for a long time; the tested samples and standard erythrocyte suspension required by the method are few, and a plurality of samples can be determined simultaneously: in addition, the invention has simple operation method, can directly read the data by the eliasa, thus being applicable to determining the activity of the agglutinin of big samples.

Description

technical field [0001] The invention belongs to a method for measuring the activity of bean lectin. Background technique [0002] Lectin is a protein that can be synthesized and secreted by both animal cells and plant cells and can bind to sugar. It is named lectin because it can agglutinate red blood cells. Lectin has more than one sugar-binding site, so it can participate in cell recognition and adhesion, and connect different cells. [0003] Lectins are mainly used in the following aspects: (1) separation and purification of sugar-containing polymers and cells; (2) identification of microorganisms; (3) research on the distribution of glycoproteins and glycolipids on the cell surface; (4) identification of sugar chain structures (5) diagnosis of malignant tumor diseases; (6) identification of blood types; (7) directional delivery of drugs, etc. [0004] Currently, methods for measuring lectin activity include cell agglutination, Mancini precipitation, Kocourek affinity e...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N1/28
Inventor 沈群郇美丽
Owner CHINA AGRI UNIV
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