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Pb<2+> antigen and corresponding monoclonal antibody and preparation method thereof

A technology of monoclonal antibody and immune antigen, applied in chemical instruments and methods, decapeptides, peptides, etc., to achieve good economic value, broad social benefits, and good stability

Inactive Publication Date: 2010-12-15
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] No bifunctional chelating agent p-NH 2 -Bn-CHX-A″-DTPA to prepare Pb 2+ Complete antigen, corresponding specific monoclonal antibody and the basis for detecting Pb 2+ ELISA kits or test strips reported

Method used

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  • Pb&lt;2+&gt; antigen and corresponding monoclonal antibody and preparation method thereof
  • Pb&lt;2+&gt; antigen and corresponding monoclonal antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Preparation and detection of immune antigen, detection antigen and control detection antigen

[0024] (1) Preparation of immune antigen KLH-NH-Bn-CHX-A″-DTPA-Pb

[0025] Weigh 1.7mg chelating agent p-NH 2 -Bn-CHX-A″-DTPA dissolved in 1.0ml 1mg / ml, pH5.5 Pb(NO 3 ) 2 Into the solution, stirred and reacted at 37°C for 1 hour, then added 1 ml of a pH 5.5, 1% glutaraldehyde solution, and stirred and reacted at 37°C for 0.5 hour. The above-mentioned reacted mixed solution was added dropwise to 2 ml of KLH solution with a concentration of 5 mg / ml, and stirred while adding. Adjust the pH value to 5.5, stir and react at 37°C for 12 hours, select an ultrafiltration centrifuge tube with a molecular weight cut-off of 30,000dal, ultrafilter at 7,500g for 6 times, wash with Hepes solution of 0.1M, pH 5.5, and centrifuge for 15min each time. After the ultrafiltration is completed, 0.1 M Hepes solution with pH 5.5 is added to make the final concentration of KLH 1 mg / ml. Immuniz...

Embodiment 2

[0069] 1. Preparation of immune antigen, detection antigen and control detection antigen

[0070] (1) Preparation of immune antigen KLH-NH-Bn-CHX-A″-DTPA-Pb

[0071] Weigh 1.7mg chelating agent p-NH 2 -Bn-CHX-A″-DTPA dissolved in 1.1ml 1mg / ml, pH5.0 Pb(NO 3 ) 2 In the solution, stirred and reacted at 37°C for 1 hour, then added 1 ml of 1% glutaraldehyde solution with pH 5.0, and stirred and reacted at 37°C for 0.5 hour. The above-mentioned reacted mixed solution was added dropwise to 2 ml of KLH solution with a concentration of 5 mg / ml, and stirred while adding. Adjust the pH value to 5.0, stir and react at 37°C for 12 hours, select an ultrafiltration centrifuge tube with a molecular weight cut-off of 30,000dal, ultrafilter at 7,500g for 6 times, wash with Hepes solution of 0.1M, pH 5.0, and centrifuge for 15min each time. After the ultrafiltration is completed, 0.1 M Hepes solution with pH 5.0 is added to make the final concentration of KLH 1 mg / ml. Immunization antigens...

Embodiment 3

[0112] 1. Preparation and detection of immune antigen, detection antigen and control detection antigen

[0113] (1) Preparation of immune antigen KLH-NH-Bn-CHX-A″-DTPA-Pb

[0114] Weigh 1.7mg chelating agent p-NH 2 -Bn-CHX-A″-DTPA dissolved in 0.9ml of 1mg / ml, pH 6.0 Pb(NO 3 ) 2 In the solution, stirred and reacted at 37°C for 1 hour, then added 1 ml of 1% glutaraldehyde solution with pH 6.0, and stirred and reacted at 37°C for 0.5 hour. The above-mentioned reacted mixed solution was added dropwise to 2 ml of KLH solution with a concentration of 5 mg / ml, and stirred while adding. Adjust the pH value to 6.0, stir and react at 37°C for 12 hours, select an ultrafiltration centrifuge tube with a molecular weight cut-off of 30,000dal, ultrafiltration at 7,500g for 6 times, and wash with 0.1M Hepes solution with a pH of 6.0, and centrifuge for 15 minutes each time. After the ultrafiltration is completed, 0.1 M Hepes solution with pH 6.0 is added to make the final concentration o...

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Abstract

The invention discloses a Pb<2+> antigen and a corresponding monoclonal antibody, and also provides a method for preparing the same. The preparation method comprises the following steps of: chelating Pb<2+> with a chelating agent p-NH2-Bn-CHX-A''-DTPA; coupling glutaraldehyde with hemocyanin (KLH); performing ultra-filtration to obtain a full immunizing antigen (KLH-NH-Bn-CHX-A''-DTPA-Pb); merging splenic cells and myeloma cells of a mouse immunizing BALB / c; and screening a specific monoclonal antibody which has strong positive for BSA-NH-Bn-CHX-A''-DTPA-Pb and no cross reaction with BSA-NH-Bn-CHX-A''-DTPA and other various metal ions. The antibody can be used for developing ELISA kits and test paper for detecting the Pb<2+> and has great application prospect.

Description

technical field [0001] The invention discloses a Pb 2+ The antigen and the corresponding monoclonal antibody, and the preparation method of the antigen and the monoclonal antibody are also provided, belonging to the technical field of immunological method detection. Background technique [0002] Heavy metal pollution is a worldwide problem. At present, more than 20 kinds of heavy metals are known to be highly toxic, among which lead (Pb), cadmium (Cd), mercury (Hg), copper (Cu), chromium, cobalt, nickel, tin, vanadium, etc. are known as "industrial wastes" It enters into the natural environment in the form of natural environment, which not only causes serious pollution to the natural environment, but also affects the quality of agricultural products, and can pose a great threat to human health through the food chain and bioaccumulation. At present, although the techniques such as atomic absorption method, stripping voltammetry, inductive coupling, and ion plasma spectrosco...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K16/44
Inventor 张燚王哲刘国文李小兵张鹏李鹏孔涛唐佳佳扬威李东娜
Owner JILIN UNIV
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