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Cu2<+> specific monoclonal antibody and preparation method thereof

A monoclonal antibody and specific technology, applied in the field of bioengineering, can solve the problems of difficulty in adapting to the rapid customs clearance of product import and export, inability to use on-site detection, etc., and achieve the effects of fast detection speed, low detection cost and strong selectivity.

Inactive Publication Date: 2010-09-29
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional heavy metal copper detection methods such as: atomic absorption spectrometry, inductively coupled plasma emission spectrometry, etc. require large and expensive analytical instruments, which cannot be used for on-site detection, and are difficult to adapt to the environment and on-site random inspection of agricultural and livestock products and rapid customs clearance of product import and export. requirements

Method used

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  • Cu2&lt;+&gt; specific monoclonal antibody and preparation method thereof
  • Cu2&lt;+&gt; specific monoclonal antibody and preparation method thereof
  • Cu2&lt;+&gt; specific monoclonal antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Preparation of complete antigen:

[0037] Weigh 4mg Cu(NO 3 ) 2 Dissolve in 50 μL ultrapure water, 10 mg p-SCN-Bn-DOTA in 50 μL DMSO, 20 mg carrier protein KLH or BSA, OVA, HSA in 1 mL 100 mM HEPES buffer (pH9.5). Cu(NO 3 ) 2 The solution was added dropwise into the p-SCN-Bn-DOTA solution, and reacted for 3 h under constant stirring to form Cu 2+ - p-SCN-Bn-DOTA hapten. Will Cu 2+ -p-SCN-Bn-DOTA solution was added dropwise to 0.5 mL carrier protein solution, and reacted with shaking at room temperature for 24 hours. Transfer the antigen to an ultrafiltration tube with a molecular weight cut-off of 30000dal, centrifuge at 7500rpm for 10min, wash 6 times with 100mM HEPES buffer (pH7.5), then dilute the antigen with 100mM HEPES buffer (pH7.5), and pack to obtain Cu 2+ Complete antigen, stored at -20°C.

[0038] (2) Immunization of mice:

[0039] The immunization antigen Cu prepared by step (1) 2+ - p-SCN-Bn-DOTA-KLH immunized 6-8 week old BALB / c mice. On the 1...

Embodiment 2

[0050] (1) Preparation of complete antigen:

[0051] Weigh 4mg Cu(NO 3 ) 2 Dissolve in 50μL ultrapure water, 10mg p-SCN-Bn-DOTA in 50μL DMSO, 20mg carrier protein KLH or BSA, OVA, HSA in 1mL 50mM carbonate buffer (pH9.5). Cu(NO 3 ) 2 The solution was added dropwise into the p-SCN-Bn-DOTA solution, and reacted for 3 h under constant stirring to form Cu 2+ - p-SCN-Bn-DOTA hapten. Will Cu 2+ -p-SCN-Bn-DOTA solution was added dropwise to 0.5 mL carrier protein solution, and reacted with shaking at room temperature for 24 hours. Transfer the antigen to an ultrafiltration tube with a molecular weight cut-off of 30000dal, centrifuge at 7500rpm for 10min, wash 6 times with 15mM ammonium citrate buffer (pH7.5), then dilute the antigen with 15mM ammonium citrate buffer (pH7.5), Subpackage, get Cu 2+ Complete antigen, stored at -20°C.

[0052] (2) Immunization of mice:

[0053] The immunization antigen Cu prepared by step (1) 2+ - p-SCN-Bn-DOTA-KLH immunized 6-8 week old BALB / ...

Embodiment 3

[0064] (1) Preparation of complete antigen:

[0065] Weigh 4mg Cu(NO 3 ) 2 Dissolve in 50μL ultrapure water, 10mg p-SCN-Bn-DOTA in 50μL DMSO, 20mg carrier protein KLH or BSA, OVA, HSA in 1mL 100mM HEPES buffer (pH9.5). Cu(NO 3 ) 2 The solution was added dropwise into the p-SCN-Bn-DOTA solution, and reacted for 3 h under constant stirring to form Cu 2+ - p-SCN-Bn-DOTA hapten. Will Cu 2+ -p-SCN-Bn-DOTA solution was added dropwise to 0.5 mL carrier protein solution, and reacted with shaking at room temperature for 24 hours. Transfer the antigen to an ultrafiltration tube with a molecular weight cut-off of 30000dal, centrifuge at 7500rpm for 10min, wash 6 times with 100mM HEPES buffer (pH7.5), then dilute the antigen with 100mM HEPES buffer (pH7.5), and pack to obtain Cu 2+ Complete antigen, stored at -20°C.

[0066] (2) Immunization of mice:

[0067] The immunization antigen Cu prepared by step (1) 2+ - p-SCN-Bn-DOTA-KLH immunized 6-8 week old BALB / c mice. On the 1st, ...

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Abstract

The invention relates to a Cu2<+> specific monoclonal antibody and a preparation method thereof. The preparation method comprises the following steps of: reacting Cu2<+> with a difunctional chelating agent to form a half hapten; reacting Cu2<+>-chelating agent with carrier protein to form a complete antigen; immuning a BALB (Binaural Alternate Loudness Balance) / c mouse by using the complete antigen; and fusing a splenocyte of the mouse with an SP2 / 0 cell to prepare a hybridoma cell and obtaining a Cu2<+>-resisting monoclonal antibody. The monoclonal antibody has the specificity on the Cu2<+>, high titer and strong specificity. The invention also provides a preparation method of the Cu2<+>-resisting monoclonal antibody. The antibody and a detection technology are used for detecting residual Cu2<+> in agricultural and animal products and have the advantages of high detection speed, high sensitivity, strong selectivity and low detection cost.

Description

Technical field: [0001] The invention provides a Cu 2+ The invention discloses a specific monoclonal antibody and also discloses a preparation method of the monoclonal antibody, which belongs to the technical field of bioengineering. Background technique: [0002] Copper accumulates in the soil and crops, which will pollute grain grains, and copper will remain in agricultural and livestock products to varying degrees, resulting in excessive copper pollution in agricultural and livestock products, seriously affecting the quality and international competitiveness of my country's agricultural and livestock products, and It also seriously endangers human health. Therefore, all countries in the world attach great importance to the control and monitoring of heavy metal pollution in the environment and agricultural products. Traditional heavy metal copper detection methods such as: atomic absorption spectrometry, inductively coupled plasma emission spectrometry, etc. require large...

Claims

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Application Information

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IPC IPC(8): C07K16/44C07K14/795C07K14/765C07K14/77
Inventor 孔涛刘国文李小兵王哲张燚唐佳杨威孙佳许超张鹏
Owner JILIN UNIV
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