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Synchronous and indirect competitive immunological detection method and kit of plural small molecular compounds

A technology for simultaneous detection of small molecular compounds, applied in measurement devices, analytical materials, material excitation analysis, etc., to achieve the effect of large flux and high sensitivity

Inactive Publication Date: 2010-10-27
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention uses phycoerythrin for fluorescent labeling and applies it to a fluorescent immunoassay for specific competitive reactions of antigens and antibodies. Since the microspheres are coded according to the ratio of embedded fluorescent dyes, microspheres with different codes are selected as the immobilization method. Phase carrier, connect different capture antigens, then mix and react on a 96-well filter plate, which can overcome the disadvantage that ELISA can only detect a single target, and can detect multiple small molecule antigens in the same sample at the same time, or Detect single or multiple small molecule antigens to be tested in different samples in different microwell plates, aiming to improve detection throughput and detection speed

Method used

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  • Synchronous and indirect competitive immunological detection method and kit of plural small molecular compounds
  • Synchronous and indirect competitive immunological detection method and kit of plural small molecular compounds
  • Synchronous and indirect competitive immunological detection method and kit of plural small molecular compounds

Examples

Experimental program
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Effect test

Embodiment 1

[0037] The microsphere indirect competitive immunofluorescence detection of gentamicin includes the following specific steps:

[0038] 1. Microsphere-capture antigen coupling:

[0039] Take 1.25×10 6 Each microsphere is a batch. In a 1.5mL centrifuge tube, use EDC and sulfo-NHS solution to activate the carboxyl groups on the surface of the microspheres at pH 6.2 for 20 minutes at room temperature, and then replace the microspheres with phosphate buffer ( In PBS, 0.01M, PH7.4), add an optimized amount of gentamicin-BSA-coupled capture antigen, shake for 2 hours at room temperature in the dark, centrifuge to wash off unbound capture antigen, and wash with 1% BSA Resuspend in PBS, avoid light and shake at room temperature for 0.5 hours to block the remaining sites on the surface of the microspheres that are not bound to the capture antigen. Finally, store the microsphere-capture antigen conjugate in PBS containing 0.1% BSA.

[0040] 2. Phycoerythrin-secondary antibody labeling:...

Embodiment 2

[0047] The microsphere indirect competitive immunofluorescence detection of ractopamine includes the following specific steps:

[0048] The operations were the same as those in Example 1, except that the capture antigen and detection antibody were ractopamine-BSA conjugate and anti-ractopamine monoclonal antibody, and the small molecule antigen to be detected was ractopamine. Similarly, the sample to be tested containing ractopamine is added to the reaction system, and the concentration of ractopamine in the sample is obtained by comparing the fluorescence intensity with the standard curve. Taking the logarithm of the concentration of ractopamine as the abscissa, MFI / MFI 0 The ratio of is the ordinate, draw the standard curve, see image 3 .

Embodiment 3

[0050] The microsphere indirect competitive immunofluorescence detection of simultaneous detection of kanamycin and gentamicin includes the following specific steps:

[0051] 1. Microsphere-capture antigen coupling:

[0052] Take the microspheres coded as 20 and 30 respectively 1.25×10 6 Each is a batch, and in two 1.5mL centrifuge tubes, use EDC, sulfo-NHS solution to activate the carboxyl groups on the surface of the microspheres at room temperature for 20 minutes under the condition of pH6.2, and then replace the microspheres with phosphate buffer ( In PBS, 0.01M, pH7.4), add the optimized amount of kanamycin-BSA and gentamycin-BSA-coupled capture antigen respectively, shake and react at room temperature for 2 hours in the dark, and centrifuge to wash off the unbound antigen. Capture the antigen, resuspend it with PBS containing 1% BSA, and shake it in the dark to react at room temperature for 0.5 hours. Block the remaining sites on the surface of the microspheres that are...

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Abstract

The invention provides a synchronous and indirect competitive immunological detection method and a kit of plural small molecular compounds. The immunological detection method is a liquid phase immunological detection method which uses an encoded microsphere as a solid phase carrier and phycoerythrin as a fluorescent marker and is used for competitive specificity reactions of a small molecular compound antigen. Firstly, a captured antigen is covalently bound to the surface of the encoded microsphere, an indirect competitive fluorescent immune complex of microsphere-captured antigen-detected antibody-second antibody-phycoerythrin is formed in a filter film plate through capturing the antigen, detecting the antibody and binding the second antibody specificity, and then the indirect competitive fluorescent immune complex flows across a suspension chip or a detection region of a flow analysis system one by one, recognizes different encoded microspheres and detects the fluorescent intensity of the phycoerythrin to complete the detection. Different captured antigens are connected by using the different encoded microspheres, a plurality of small molecule antigens in the same sample can be synchronously detected, and one or more small molecule antigens to be detected in different samples can also be detected. The invention has the advantages of fast speed and high flux.

Description

technical field [0001] The invention relates to a microsphere-based small molecule multi-residue fluorescence immunoassay method, in particular to an indirect competitive immunoassay method using phycoerythrin as a fluorescent marker and polystyrene fluorescently encoded microspheres as a solid-phase carrier and Kit used. Background technique [0002] Small molecule residues in food such as veterinary drugs, pesticides, and additives are a major issue affecting people's livelihood, and have become one of the food safety issues that have attracted widespread attention at home and abroad. At present, the commonly used detection methods mainly include: microbiological method, chromatography (such as high performance liquid chromatography, HPLC) or chromatography-mass spectrometry (such as liquid chromatography-mass spectrometry tandem method, LC-MS); enzyme-linked immunosorbent assay (ELISA). The microbiological detection method is simple to operate, but the detection cycle i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/533G01N21/64
Inventor 邹明强王燕飞刘天龙
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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