Method for preparing micro-cantilever beam modified with antibody

A micro-cantilever and antibody technology, applied in chemical instruments and methods, carrier binding/immobilization of peptides, peptides, etc., can solve problems such as destroying antibody structure and affecting antibody activity.

Inactive Publication Date: 2009-04-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The principle of this method is to form a disulfide bond after the antibody reacts with a thiol reagent, and then add dithiothreitol (DTT) to reduce the free sulfhydryl group. The disadvantage is that the heavy chain of the antibody and the heavy chain There are many disulfide bonds between the light chain and the reduction with DTT may destroy the structure of the antibody and affect the activity of the antibody and the detection limit of the micro-cantilever sensor

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1, preparation is modified with the microcantilever of antibody

[0016] 1. Using thiolimine hydrochloride to thiolate the antibody

[0017] (1) Accurately weigh 10.0 mg of anti-clenbuterol monoclonal antibody (purchased from US Biological Company) and dissolve it in 1.0 mL of PBS to obtain an antibody solution with a concentration of 10 g / L;

[0018] (2) Weigh 2.0 mg of thiol imine hydrochloride (purchased from Sigma, USA) and dissolve it in 1.0 mL of distilled water to obtain a mercaptolation reagent with a concentration of 2.0 g / L (prepared and used now);

[0019] (3) 1.0 mL of the antibody solution of the above step (1) is mixed with 45.8 μl of the thiol reagent of the above step (2), so that the molar ratio of the anti-clenbuterol monoclonal antibody and thiolimine hydrochloride in the mixed solution is 1:3, react with slight stirring at room temperature for 1 h;

[0020] (4) Dialyze the reaction solution of the above step (3) with 20mM PBS buffer solu...

Embodiment 2

[0049] Embodiment 2, preparation is modified with the microcantilever of antibody

[0050] 1. Using thiolimine hydrochloride to thiolate the antibody

[0051] (1) Accurately weigh 10.0 mg of anti-clenbuterol monoclonal antibody (purchased from US Biological Company) and dissolve it in 1.0 mL of PBS to obtain an antibody solution with a concentration of 10 g / L;

[0052] (2) Weigh 2.0 mg of thiol imine hydrochloride (purchased from Sigma, USA) and dissolve it in 1.0 mL of distilled water to obtain a mercaptolation reagent with a concentration of 2.0 g / L (prepared and used now);

[0053] (3) 1.0 mL of the antibody solution of the above step (1) is mixed with 91.6 μL of the thiol reagent of the above step (2), so that the molar ratio of the anti-clenbuterol monoclonal antibody and thiolimine hydrochloride in the mixed solution is 1:6, gently stirred at room temperature for 2 hours;

[0054] (4) Dialyze the reaction solution of the above step (3) with 20mM PBS buffer solution (co...

Embodiment 3

[0083] Example 3, preparation of microcantilever modified with antibody

[0084] 1. Using thiolimine hydrochloride to thiolate the antibody

[0085] (1) Accurately weigh 10.0 mg of anti-clenbuterol monoclonal antibody (purchased from US Biological Company) and dissolve it in 1.0 mL of PBS to obtain an antibody solution with a concentration of 10 g / L;

[0086] (2) Weigh 2.0 mg of thiol imine hydrochloride (purchased from Sigma, USA) and dissolve it in 1.0 mL of distilled water to obtain a mercaptolation reagent with a concentration of 2.0 g / L (prepared and used now);

[0087] (3) Mix 1.0 mL of the antibody solution of the above step (1) with 15.3 μL of the thiol reagent of the above step (2), so that the molar ratio of the anti-clenbuterol monoclonal antibody and thiolimine hydrochloride in the mixed solution is 1:1, react with slight stirring at room temperature for 0.5h;

[0088] (4) Dialyze the reaction solution of the above step (3) with 20mM PBS buffer solution (containi...

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Abstract

The invention discloses a method for preparing a micro-cantilever decorated with an antibody, which uses hydrochloride mercaptan imine as a sulfhydrylation agent, and the antibody is sulfhydrylized, and the sulfhydrylation antibody is fixed on a gold membrane of the micro-cantilever to obtain the micro-cantilever decorated with the antibody. The result shows that by introducing the sulfhydrylation agent-hydrochloride mercaptan imine, the method for preparing the micro-cantilever decorated with the antibody has the advantages of short reaction time, simple operation, few loss of antibody activity and the like, compared with the existing method.

Description

technical field [0001] The invention relates to a method for preparing a microcantilever beam modified with an antibody. Background technique [0002] With the advent of atomic force microscopy (AFM) and microelectromechanical systems (MEMS), microcantilever sensing technology has rapidly developed into a new sensing method. The principle is that when biochemical reactions occur on the surface of the microcantilever, surface The change of stress will lead to the bending deformation of the micro-cantilever beam, and the information of the whole biochemical reaction process can be known by combining optical or electrical methods to monitor this deformation. Compared with the traditional enzyme-linked immunosorbent assay (ELISA), the micro-cantilever immunosensing technology established by relying on immune-specific recognition does not need to label the sample to be tested, and eliminates the possibility of labeling. Additional effects, and high sensitivity, can realize real-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/14
Inventor 王保民谭伟明南铁贵何素平刘威赵洪伟高巍李召虎
Owner CHINA AGRI UNIV
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