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Tagged molecule based method for detecting protein-microsphere chemical coupling efficiency

A chemical coupling and protein technology, applied in the field of immunochemistry, can solve the problems of uncoupling reaction result analysis, many experimental steps, and unable to determine the specific cause of the error due to chemical reagents or reaction conditions, etc.

Inactive Publication Date: 2012-12-12
TIANJIN NIUSAI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty in the implementation of this technical solution is that there are many experimental steps and a large amount of serum from different allergic patients is required
[0004] In addition, when there is a problem with the coupling reaction, due to the lack of internal reference in the reaction, the above two technical solutions cannot directly analyze the results of the coupling reaction
For example, when the efficiency of the coupling reaction is determined to be lower than expected by the above two schemes, neither of the two schemes can determine whether the specific reason for the failure is due to the problem of chemical reagents or reaction conditions, or the quality of the allergen extract

Method used

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  • Tagged molecule based method for detecting protein-microsphere chemical coupling efficiency
  • Tagged molecule based method for detecting protein-microsphere chemical coupling efficiency
  • Tagged molecule based method for detecting protein-microsphere chemical coupling efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A detection method for chemical coupling efficiency (conditions for optimizing the coupling reaction) between protein-carboxylated polystyrene microspheres based on exogenous tracer molecules, the method comprises the following specific steps:

[0044] (1) Activation of carboxylated polystyrene microspheres

[0045] Take 5×10 6 Carboxylated polystyrene microspheres were washed with water, centrifuged and the supernatant was removed, and then 100 μL of activation solution (0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, pH 6.0) was added, Vortex for 30 seconds, then sonicate for 30 seconds, add 10 μL each of freshly prepared 50 mg / ml nitrogen hydroxysuccinimide sulfonate (sulfo-NHS) and water-soluble carbodiimide (EDC-HCl) aqueous solutions, and vortex for 10 seconds Afterwards, shake and incubate in the dark at room temperature for 20min, centrifuge the activated microspheres to remove the supernatant, wash twice with 0.5mL phosphate buffer solution (0....

Embodiment 2

[0051] Using exogenous tracer molecules to determine the relative concentration of effective molecules in the solution, the method comprises the following steps:

[0052] (1) Activation of carboxylated polystyrene microspheres

[0053] Take 5×10 6 Carboxylated polystyrene microspheres, washed with water, centrifuged and removed the supernatant, added 100 μL activation solution (0.1M 2-(N-morpholino)ethanesulfonic acid / MES buffer solution, pH6.0), vortexed Mix for 30 seconds, then sonicate for 30 seconds, add 10 μL each of freshly prepared 50 mg / ml nitrogen hydroxysuccinimide sulfonate (sulfo-NHS) and water-soluble carbodiimide (EDC-HCl) aqueous solutions, and vortex for 10 seconds , shake and incubate in the dark at room temperature for 20min, centrifuge the activated microspheres to remove the supernatant, and wash twice with 0.5mL phosphate buffer solution (0.1M phosphate, 150mM sodium chloride, pH7.2) to obtain the surface activated microspheres;

[0054] (2) Chemical co...

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Abstract

The invention relates to a tagged molecule based method for detecting protein-microsphere chemical coupling efficiency. The method comprises the following steps: (1) mixing protein with exogenous tagged molecules according to a certain mass ratio or molar ratio; (2) under specific coupling reaction conditions, carrying out chemical coupling on carboxylated polystyrene microspheres and a protein-exogenous tagged molecule solution, and further optimizing the chemical coupling reaction by changing coupling reaction conditions and using protein-exogenous tagged molecule solution with different concentration; and (3) quantitating the exogenous tagged molecules on the surface of the carboxylated polystyrene microspheres. According to the present invention, a small amount of exogenous tagged molecules are added to the amino-carboxyl coupling reaction, and tracked, so as to effectively monitor chemical coupling efficiency of protein and polystyrene microspheres. The method can rapidly, reasonably and effectively monitor the coupling reaction.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and relates to a method for detecting chemical coupling of protein-solid support medium, in particular to a method for detecting chemical coupling efficiency of protein-carboxylated polystyrene microspheres, and is especially suitable for variable Detection and evaluation of chemical coupling efficiency between allergen extracts and carboxylated polystyrene microspheres. Background technique [0002] During the development of immunochemical products, it is often necessary to couple proteins such as antibodies to solid-phase support media in a covalent manner. In coupling reactions, high-purity protein solutions are usually used as coupling substrates. But in some cases, high-purity protein is difficult to obtain, and a large amount of impurities are mixed in the protein solution. Taking the development of serum allergen-specific IgE or IgG4 detection products as an example, it is necess...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/546G01N33/532
Inventor 程爱阳
Owner TIANJIN NIUSAI BIOTECH CO LTD