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Magnetic binding assays method utilizing time-resolved up-converting luminescence detection

A measurement method and magnetic technology, applied in the field of detection, can solve the problems of reducing the accuracy of results

Active Publication Date: 2013-09-11
何爱民
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method for detecting the presence and content of an analyte in a sample, which solves the problem that the excitation light in the prior art is absorbed by the analyte itself and the sample medium in the near ultraviolet region, resulting in a significant reduction The accuracy of the results, etc.

Method used

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  • Magnetic binding assays method utilizing time-resolved up-converting luminescence detection
  • Magnetic binding assays method utilizing time-resolved up-converting luminescence detection
  • Magnetic binding assays method utilizing time-resolved up-converting luminescence detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Preparation of up-conversion luminescent probes

[0086] To 500 μl of aqueous solution containing 50 mg of carboxylic acid-functionalized polymethacrylic acid latex particles PP02N (0.33 μm in diameter, supplied by Bangs Laboratories) was added 2 ml of ethanol while stirring. An appropriate amount (eg 1% by weight of the latex particles) of vinyl chloride containing europium chelate (eg 3% by weight of the total solvent) is slowly added to the particle suspension while stirring. The mixture was stirred for half an hour. An appropriate amount of water (eg, four times the total volume of the initial solvent) is then slowly added to the stirred mixture over a certain period of time (eg, 2 hours). After the addition of water was complete, most of the ethanol was removed from the mixture by rotary evaporator. The particles were then washed twice by centrifugation using 90% ethanol. The particles were then washed twice with water. The washed particles were then...

Embodiment 2

[0087] Example 2: Binding antibodies to upconverting luminescent probes to prepare probe conjugates

[0088]200 µl of the probe particles prepared in Example 1 were washed once with 1.5 ml of carbonate buffer and twice with Mes buffer (PH=4.3) by centrifugation. The washed probe particles were resuspended in 0.1 ml of Mes buffer, and 0.1 ml of Mes buffer dissolved with 6.2 mg of carbodiimide (provided by Polysciences) was added to the suspension for activation treatment. The mixture was allowed to react on a shaker at room temperature for 30 minutes. The activated probe particles were then washed twice with borate buffer. The activated probe particles were resuspended in 0.185 ml of borate buffer, and 15 μl of LHα monoclonal antibody (LHαMab, 9.8 mg / ml, supplied by Fitzgerald Industrial International) was added thereto. The reaction mixture was allowed to react overnight on a shaker. The particles were then collected and incubated in 0.2 ml 0.1 M ethanolamine for 15 minutes...

Embodiment 3

[0089] Example 3: Binding antibodies to magnetic beads to produce magnetic bead conjugates

[0090] 100 μl of 10% carboxylated magnetic beads (1.5 μm, provided by Bangs Laboratories) were washed once with 1.5 ml of carbonate buffer and washed twice with Mes buffer (PH=4.3) by passing through the magnetic separator. The washed particles were resuspended in 0.1 ml of Mes buffer, and 0.1 ml of Mes buffer dissolved with 6.2 mg of carbodiimide (provided by Polysciences) was added to the suspended particles for activation treatment. The mixture was allowed to react on a shaker at room temperature for 30 minutes. The activated magnetic beads were then washed twice with borate buffer. The activated magnetic beads were resuspended in 0.185 ml of borate buffer, and 15 μl of LH β monoclonal antibody (LH βMab, provided by Fitzgerald Industrial International) was added thereto. The reaction mixture was allowed to react overnight on a shaker. The bead conjugates (pellets) were then colle...

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Abstract

This invention describes a general magnetic binding assay method to detect the presence and quantity of analyte in a sample. The method uses magnetic particles for separating and concentrating analytes of interest from complex samples and use time-resolved up-converting fluorescence detection technique to provide highly sensitive detection without using expensive optical components such as band-pass filters. The method uses pulsed long wavelength light for excitation and time-delayed luminescence detection, resulting in little interferences from sample matrices. Furthermore, the usage of long wavelength excitation light requires simpler sample preparation and clean-up such as removal of red blood cells, which otherwise will significantly interfere with excitation efficiency of the fluorescence probes.

Description

technical field [0001] The invention belongs to the technical field of detection, and specifically relates to a method for measuring magnetic binding by using a time-resolved up-conversion luminescence detection technology, and more specifically relates to a method for detecting the presence and content of an analyte in a sample. Background technique [0002] Magnetic beads have been widely used as a carrier for analyte separation and enrichment because they can be easily separated by a magnetic field without requiring special and expensive equipment. Complex samples can be captured by labeling the surface of magnetic beads with a specific adhesive. Species to be studied (such as proteins, nucleic acids (DNA or RNA), cells, and microorganisms) in the Captured species are easily separated from the rest of the sample by a magnet. The captured species can then be lysed and released from the bead surface by various means. To detect the species of interest, a detection probe la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/63G01N21/64
CPCG01N33/54326G01N33/582G01N2458/40
Inventor 何爱民
Owner 何爱民
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