Preparation method of heavy metal mercury monoclonal antibody

A technology for cloning antibodies and mercury monoclonal antibodies, which is applied in the preparation of heavy metal mercury monoclonal antibodies, large-scale sample detection and on-site monitoring, can solve the problems of no research reports and late start, and achieve good repeatability, high accuracy, The effect of strong antigen availability

Inactive Publication Date: 2008-03-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Our country started relatively late in this area, and only in recent years have units and researchers paid attention to and engaged in the research of heavy metal immune rapid detection technology. At present, there is no research report on this kind of method in China.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Detection of mercury standard solution (sigma Co.)

[0048] The indirect competitive ELISA method is used to make a preliminary exploration of the detection curve, and the method is as follows:

[0049] Coating: Dilute the coating antigen Hg-glutathione-bovine serum albumin (9mg / ml) 10000 times with 10mM, pH7.4N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer, Add 50μl / well to a 96-well enzyme-linked microanalysis plate and place overnight at 4°C;

[0050] Washing: Wash 3 times with PBST;

[0051] Blocking: PBST diluted gelatin to 1% (m / v), 100μl / well, 37°C for 1.5 hours;

[0052] Washing: Wash 3 times with PBST;

[0053] Dosing: Dilute the mercury nitrate standard solution with 5mM glutathione phosphate buffer solution to 10 -6 mM, 10 -5 mM, 10 -4 mM, 10 -3 mM, 10 -2 mM, 10 -1 mM, 1mM series concentration, the diluted solution is placed at room temperature for 30 minutes, and then mixed with an equal volume of the mercury antibody diluted to a certain mul...

Embodiment 2

[0060] Example 2 Detection of residual mercury in tap water samples

[0061] Hg-glutathione-bovine serum albumin 0.9μg / ml is coated with 50μl / well in the microanalysis plate, the mercury antibody supernatant is diluted 2 times as the working concentration, 1% (m / v) gelatin is used as the blocking material, and the antigen antibody The pH of the reaction matrix (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer) is 5.5, the ionic strength is 0.15 mol / L, and the microanalysis plate is 50 μl / well.

[0062] Preparation of samples to be tested:

[0063] Tap water sample: Take 996μl tap water, add 4μl mercury nitrate ion standard solution (1g / L) to make 2×10 -2 Liquid samples of mmol / L.

[0064] The mercury ELISA detection method is used to detect the above samples, and the operation is as follows:

[0065] 1) Coating:

[0066] Add 50μl of Hg-glutathione-bovine serum albumin 0.9μg / ml to each well of the microanalysis plate, and place in a refrigerator overnight at 4°C or 2 hours...

Embodiment 3

[0086] Example 3 Detection of mercury residues in small green vegetables samples

[0087] The main conditions of the mercury ELISA reaction system are the same as in Example 2;

[0088] Preparation of samples to be tested:

[0089] Small green cabbage sample: Weigh 2g of dried small green cabbage, reflux and digest it (for details, please refer to the "Method for Determination of Mercury in Vegetables" drafted by the Agricultural Products Quality Supervision and Inspection Center of the Ministry of Agriculture (Hangzhou) and Zhejiang Standardization Researcher), and transfer to 100mL In the volumetric flask, cancel the solution solution 996μl, add 4μl mercury nitrate ion standard solution (1g / L) to make 2×10 -2 Liquid samples of mmol / L;

[0090] The mercury ELISA detection method is used to detect the above samples, and the operation is as follows:

[0091] Steps (1) and (2) are the same as in embodiment 2;

[0092] (3) Add antibody and the mixture of antibody and sample to be tes...

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Abstract

The present invention relates to a preparation method of heavy metal mercury monoclonal antibody, which belongs to the field of biotechnology. The present invention is particularly used in the preparation of specificity-recognition heavy metal mercury monoclonal antibody and in the fast mercury high-sensitivity measurement remained in the agricultural production and the agricultural production environment. The heavy metal mercury ion and the carrier albumen are coupled into full antigens through bifunctional metal chelate 1-(4-separate-cyano phenyl)-EDTA; the full antigens are used on Balb/C mouse and the spleen cells and the Sp2/0 myeloma cells are used to prepare hybridoma cells through the hybridoma technology. And monoclonal antibody which can stably excrete anti-Hg-EDTA is generated. The preparation technology in the present invention is easy and practical: the whole preparation process of the antigen requires no special instrument. The present invention is suitable for factory-scale production.

Description

(1) Technical field [0001] The invention is a preparation method of a heavy metal mercury monoclonal antibody, which belongs to the field of biotechnology. It is dedicated to the preparation of monoclonal antibodies that specifically recognize heavy metal mercury, and the highly sensitive and rapid detection of heavy metal residues in agricultural products and agricultural production environments, and is especially suitable for large-scale sample detection and on-site monitoring. (2) Background technology [0002] The heavy metal mercury is an important metal element that exists in nature. Because of its many excellent characteristics, mercury and its compounds are widely used in industrial and agricultural production. However, mercury is a metal pollutant that is highly toxic to humans and higher organisms. As early as the 1950s after the outbreak of Minamata disease in Kumamoto, Japan, the problem of mercury pollution attracted people's attention. In recent years, with the cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/84G01N33/577
Inventor 刘凤权杨凤丽娄旸朱晓霞胡白石
Owner NANJING AGRICULTURAL UNIVERSITY
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