Heavy metal copper monoclonal antibody and preparation method thereof

A technology for cloning antibodies and heavy metals, applied in the fields of botanical equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problems of late start and no research reports, and achieve good repeatability and stability. , the effect of antigen practicability

Active Publication Date: 2009-10-21
NANJING AGRICULTURAL UNIVERSITY
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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 ...

Method used

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  • Heavy metal copper monoclonal antibody and preparation method thereof

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Embodiment 1

[0044] The detection of embodiment 1 copper standard solution (Sigma Co.)

[0045] The indirect competitive ELISA method was used to conduct a preliminary exploration of the detection curve. The method is as follows:

[0046] Coating: Dilute the coating antigen bovine serum albumin-1-(4-isothiocyanatophenyl)-B with 10mM, pH7.4N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer Diaminetetraacetic acid-Cu, add 50 μl / well to a 96-well enzyme-linked microanalysis plate, and place it at 4°C overnight;

[0047] Washing: wash 3 times with PBST;

[0048] Blocking: PBST diluted gelatin to 1% (m / v), 100 μl / well, 1.5 hours at 37°C;

[0049] Washing: wash 3 times with PBST;

[0050] Adding samples: dilute the copper nitrate standard solution step by step with phosphate buffer containing 2mM ethylenediaminetetraacetic acid to 10 -5 μg / ml, 10 -4 μg / ml, 10 -3 μg / ml, 10 -2 μg / ml, 10 -1 μg / ml, 1μg / ml, 10μg / ml, 100μg / ml series concentration, the diluted solut...

Embodiment 2

[0057] Detect copper residue in the tap water sample of embodiment 2

[0058] Bovine serum albumin-1-(4-isothiocyanatophenyl)-ethylenediaminetetraacetic acid-Cu 0.4 μg / ml coated microanalysis plate 50 μl / well, copper antibody supernatant diluted 4 times as the working concentration, 1% (m / v) gelatin was used as a blocker, the pH value of the antigen-antibody reaction matrix (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution) was 5.0, and the ionic strength was 0.15mol / L. 50 μl / well in microassay plate.

[0059] Sample preparation to be tested:

[0060] Tap water sample: measure 100 μl of tap water, add 0.5 μl of 1000ug / ml copper standard solution, and prepare a 5ug / ml liquid sample.

[0061] The above samples were detected by copper ELISA detection method, and the operation was as follows:

[0062] 1) Coating:

[0063] Bovine serum albumin-1-(4-isothiocyanatophenyl)-ethylenediaminetetraacetic acid-Cu 0.4μg / ml Add 50μl to each well of the mi...

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Abstract

The invention relates to a preparation method of a heavy metal copper monoclonal antibody, belonging to the field of biotechnology. The preparation method is specially used for the preparation of the specifically recognized heavy metal copper monoclonal antibody and the high-sensitivity rapid detection of copper residue in agriculture products and agriculture production environment. Heavy metal copper ions are coupled with a carrier protein by a bifunctional metal chelating agent 1-(4- isothiocyanatobenzo)-ethylenediamine tetracetic acid to form a complete antigen, the complete antigen is used for immunizing a Balb/C mouse, spleen cells and Sp2/0 myeloma cells are used for preparing hybridoma cells by the hybridoma technology, so as to obtain the monoclonal antibody with steady secretion of anti-Cu-EDTA. The preparation technology of the antibody is simple and feasible, and the whole preparation process of the antigen does not need any special instrument or equipment, thereby having easy factory-scale production.

Description

(1) Technical field [0001] The invention relates to a heavy metal copper monoclonal antibody and a preparation method thereof, belonging to the field of biotechnology. It is specially used for the preparation of monoclonal antibodies that specifically recognize heavy metal 1 copper, and the highly sensitive and rapid detection of heavy metal residues in agricultural products and agricultural production environments, especially suitable for large-scale sample detection and on-site monitoring. (2) Background technology [0002] Heavy metal copper is an important metal element existing in nature. Copper and its compounds are widely used in industrial and agricultural production due to their many excellent properties. However, copper is a metal pollutant that is highly toxic to humans and higher organisms. As early as the 1980s after the outbreak of the "green oyster" incident in Taiwan, the problem of copper pollution has attracted people's attention. In recent years, with t...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N15/06C12P21/08C12N5/20
Inventor 刘凤权娄旸刘艳徐丹
Owner NANJING AGRICULTURAL UNIVERSITY
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