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An Immunoassay Method Based on Differential Impedance Particle Counting

A particle counting and immune analysis technology, which is applied in the fields of environmental monitoring, in vitro diagnosis, and food safety, can solve the problems of high cost of particle counting instruments, interference with test results, and difficulty in cleaning and removing blockages, so as to improve detection accuracy and anti-interference ability, The effect of low detection cost and high degree of automation

Active Publication Date: 2022-04-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has good stability and high particle identification efficiency, but particle counting instruments based on the Coulter counting method are expensive and inconvenient to carry. During use, repeated cleaning is required to eliminate background interference. When the pore size is small, blockage will occur and it is not easy to clean and remove the blockage, which greatly interferes with the detection results and reduces the detection efficiency

Method used

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  • An Immunoassay Method Based on Differential Impedance Particle Counting
  • An Immunoassay Method Based on Differential Impedance Particle Counting
  • An Immunoassay Method Based on Differential Impedance Particle Counting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Detection of procalcitonin (PCT) in whole blood by double-antibody sandwich method based on magnetic separation

[0052] (1) Preparation of magnetic particle-procalcitonin capture antibody conjugate

[0053] Take 2 mg of carboxyl-coupled magnetic nanoparticles (1 μm, 10 mg / mL), wash twice with MES buffer (pH=6.0), resuspend in MES buffer, add 50 μL of EDC (10 mg / mL) and 25 μL of NHS (10 mg / mL), mix well and activate at room temperature for 15 minutes.

[0054] After activation, wash twice with PBS buffer, resuspend in PBS buffer, add 0.2mg procalcitonin capture antibody, react at room temperature for 2-4 hours; after the reaction, block with 1% BSA solution for 30 minutes, block After completion, wash twice with PBST buffer, resuspend the magnetic particle-capture antibody conjugate with PBS buffer and store at 4°C until use.

[0055] (2) Preparation of polystyrene microspheres-procalcitonin detection antibody conjugates

[0056] Take 2 mg of carboxyl-coup...

Embodiment 2

[0070] Example 2 Detection of pathogenic bacteria Salmonella by double-antibody sandwich method based on magnetic separation

[0071] (1) Preparation of magnetic particle-Salmonella capture antibody conjugate

[0072] Take 5 mg of carboxyl-coupled magnetic nanoparticles (1 μm, 10 mg / mL), wash twice with MES buffer (pH=6.0), resuspend in MES buffer, add 100 μL of EDC (10 mg / mL) and 50 μL of NHS (10 mg / mL), mix well and activate at room temperature for 15 minutes.

[0073] After activation, wash twice with PBS buffer, resuspend in PBS buffer, add 0.2 mg Salmonella capture antibody, and react at room temperature for 2-4 hours; after the reaction, block with 1% BSA solution for 30 minutes, and use The PBST buffer was washed twice, and the magnetic particle-capture antibody conjugate was resuspended in PBS buffer and stored at 4°C until use.

[0074] (2) Preparation of polystyrene microsphere-Salmonella detection antibody conjugate

[0075] Take 2 mg of carboxyl-coupled polysty...

Embodiment 3

[0079] Example 3 Detection of Pesticide Molecule Chlorpyrifos by Competitive Method Based on Magnetic Separation

[0080] (1) Preparation of magnetic particle-chlorpyrifos antibody conjugate

[0081] Take 2 mg of carboxyl-coupled magnetic nanoparticles (1 μm, 10 mg / mL), wash twice with MES buffer (pH=6.0), resuspend in MES buffer, add 50 μL of EDC (10 mg / mL) and 25 μL of NHS (10 mg / mL), mix well and activate at room temperature for 15 minutes.

[0082] After activation, wash twice with PBS buffer, resuspend in PBS buffer, add 0.1 mg chlorpyrifos antibody, and react at room temperature for 2-4 hours; after the reaction, block with 1% BSA solution for 30 minutes, and use PBST after blocking The buffer was washed twice, and the magnetic particle-antibody conjugate was resuspended in PBS buffer and stored at 4°C until use.

[0083] (2) Preparation of polystyrene microspheres-chlorpyrifos complete antigen conjugate

[0084] Take 2 mg of carboxyl-coupled polystyrene microspheres...

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Abstract

The invention discloses an immunoassay method based on differential impedance particle counting, comprising the following steps: 1) using polymer insulating microspheres as signal probes, coupling biorecognition molecules with insulating microspheres, and then subjecting the target object to be tested Perform an immune reaction with biorecognition molecules to change the particle size and quantity of insulating microspheres; 2) inject the reaction solution into the particle counting channel, and at the same time inject buffer into the channel at high speed, forming a laminar flow based on the principle of fluid mechanics, so that the reaction solution The insulating microspheres pass through the counting channel stably and orderly; 3) Set positive and negative electrodes at both ends of the particle counting channel to collect potential signals, and analyze the particle size and quantity of the insulating microspheres after amplification and filtering. Then the content of the target substance to be measured is obtained. The invention has the advantages of low detection cost, convenient operation, no channel blockage, no need for repeated cleaning and blockage removal, greatly improves detection efficiency, and has better accuracy and anti-interference ability.

Description

technical field [0001] The invention belongs to the fields of food safety, in vitro diagnosis and environmental monitoring, and relates to an immune analysis method, in particular to an immune analysis method based on differential impedance particle counting. Background technique [0002] Food safety, in vitro diagnostics and environmental monitoring are closely related to people's livelihood and living standards. Therefore, it is of great practical significance to improve the detection methods and means in the fields of food safety, in vitro diagnostics and environmental testing. Traditional food safety, in vitro diagnostics and environmental monitoring instruments have the characteristics of high precision and good accuracy, but most of the instruments and equipment are expensive and inconvenient to carry. Before testing, complex pretreatment of samples is required. Equipment personnel also need professional training. Therefore, traditional instrument detection methods ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/546G01N15/10G01N15/02
CPCG01N33/54313G01N33/54326G01N33/54346G01N15/1031G01N15/0266
Inventor 陈翊平周阳王知龙
Owner HUAZHONG AGRI UNIV