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Preparation method of antibody orientation modified fluorescent microsphere probe and application thereof in immunochromatography

A fluorescent microsphere and antibody technology, applied in the field of nanomaterials and immunoassays, can solve the problems of antibody shedding and probe activity reduction, and achieve the effects of avoiding occupation, improving sensitivity and improving efficiency.

Inactive Publication Date: 2019-03-19
NANJING NANOEAST BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Otherwise, extreme conditions such as repeated washing, spray drying, and long-term storage will easily lead to antibody shedding and decreased probe activity

Method used

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  • Preparation method of antibody orientation modified fluorescent microsphere probe and application thereof in immunochromatography
  • Preparation method of antibody orientation modified fluorescent microsphere probe and application thereof in immunochromatography
  • Preparation method of antibody orientation modified fluorescent microsphere probe and application thereof in immunochromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1-Preparation of fluorescent microsphere probes targeting cTnI (cardiac troponin I) antibody orientation modification

[0030] Protein G was coupled to EDC / NHS activated carboxyl microspheres, and then incubated with anti-cTnI antibody and fixed with EDC, and finally coupled with fluorescent molecules.

[0031] Proceed as follows:

[0032] 1) Disperse 1mg of carboxyl microspheres with a particle size of 150nm in 1mL pH6 PBS buffer, add 50μg EDC and 50μg NHS, and activate at 25°C for 30 minutes;

[0033] 2) Centrifuge to remove excess cross-linking agent, disperse the precipitate in 1mL pH7 PBS buffer, add 200μg protein G, and incubate at 25°C for 1h to couple protein G to the microspheres;

[0034] 3) Wash by centrifugation, disperse the precipitate in 200μL pH7 PBS buffer, add 200μg of anti-cTnI monoclonal antibody (mouse source), and incubate at 25°C for 1h;

[0035] 4) Add 50μg EDC to the reaction system, and continue coupling at 25°C for 2h, so that the antibody is fix...

Embodiment 2

[0039] Example 2-Preparation of lateral immunochromatographic fluorescent test strips for detecting cTnI

[0040] The basic structure of the test strip is as follows figure 1 Shown. It is composed of a PVC bottom plate (1), a sample pad (2), a bonding pad (3), a nitrocellulose membrane (4) and absorbent paper (5) which are sequentially overlapped on the bottom plate (1). The adjacent components of each overlapping part need to overlap by about 2mm to ensure the smooth chromatography of the sample on the test strip. The assembled and chopped test strips need to be put into a card case, sealed in a tin foil bag containing a desiccant, and stored in a cool, dry environment.

[0041] Specific steps are as follows:

[0042] 1) Treat sample pads and binding pads: add 0.5% inert protein (such as BSA), 0.05% (such as Tween-20) and 0.05% high molecular polymer (such as PEG2K) to Tris buffer, and mix well. Adjust the pH to 7-8 to obtain the treatment solution; each mat (specification 20cm×3...

Embodiment 3

[0052] Example 3-Detection of cTnI in samples by lateral immunochromatographic test strips

[0053] Specific steps are as follows:

[0054] 1) Restore the serum, plasma or whole blood sample to room temperature;

[0055] 2) Take the test strip out of the tin foil bag;

[0056] 3) Take 100μL of sample and drop it into the sample hole;

[0057] After 15 minutes of chromatography, insert the test strip into the fluorescence quantitative immunoassay analyzer to read the test results.

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Abstract

The invention discloses a preparation method of an antibody orientation modified fluorescent microsphere probe. Antibodies can be efficiently and firmly fixed on the surfaces of microspheres in an oriented manner, and thus, Fab ends are fully exposed and keep bioactivity. The preparation method of the antibody orientation modified fluorescent microsphere probe comprises the following steps: activating carboxyl groups on the surfaces of carboxyl microspheres, fixing protein A or protein G by chemical coupling, specifically combining Fc ends of the antibodies, then irreversibly fixing the antibodies on the substrate of the protein A or the protein G by chemical bonds, and finally coupling fluorescent molecules. The invention further provides application of the fluorescent microsphere probe in immunochromatography, and the detection sensitivity and the stability of a test strip can be improved remarkably.

Description

Technical field [0001] The invention relates to a preparation method of a fluorescent microsphere probe modified by antibody orientation and its application in immunochromatography, belonging to the field of nanomaterials and immunoanalysis. Background technique [0002] Immunochromatography technology is a new type of rapid immunoassay technology, which is widely used in the identification and detection of markers in the fields of medical diagnosis, environmental monitoring, and food safety due to its convenience, speed, accuracy and pollution-free characteristics. The fluorescence method has gradually replaced the traditional colloidal gold method due to its high sensitivity, good stability, and easy quantification. Among them, the construction method of the fluorescent probe often determines the technical level of the test strip. Loading antibodies and fluorescent molecules on microsphere carriers is the current mainstream construction method. Compared with the method of dire...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/558G01N33/58G01N33/68C09K11/06
CPCC09K11/06G01N33/533G01N33/558G01N33/582G01N33/585G01N33/68
Inventor 张宇娄豆豆韩国志
Owner NANJING NANOEAST BIOTECH
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