spore @zr 4+ Preparation of functional microspheres and their application in immunoassay

A microsphere and functional technology, applied in measuring devices, instruments, scientific instruments, etc., can solve problems such as hazards, time-consuming environment, complex chemical modification process, etc.

Inactive Publication Date: 2020-06-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, commercial microspheres are pre-modified with some functional groups such as amino, carboxyl and phosphoric acid groups on their surface as chelating agents to immobilize metal ions during the synthesis process. 18-20 ; however, the chemical modification process is often complex, time-consuming, and environmentally hazardous

Method used

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  • spore @zr <sup>4+</sup> Preparation of functional microspheres and their application in immunoassay
  • spore @zr <sup>4+</sup> Preparation of functional microspheres and their application in immunoassay
  • spore @zr <sup>4+</sup> Preparation of functional microspheres and their application in immunoassay

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Experimental program
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Embodiment 1

[0046] Embodiment 1 (preparation embodiment)

[0047] In this embodiment, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a bacterial strain publicly distributed by the China Type Culture Collection, and its preservation number is CCTCC AB 2013062) and the bacterial strain Bacillus megaterium (Bacillus megaterium) publicly distributed by the China Type Culture Collection , whose preservation number is CCTCC AB 92052) as the starting material for preparing functional microspheres.

[0048] Specific steps are as follows:

[0049] (1) Cultivation of Bacillus and collection of spores: Take 200 μL of Bacillus amyloliquefaciens or Bacillus megaterium strains stored at –20°C with 50% glycerol, respectively, and add 5 mL of sterilized In LB liquid culture medium, culture overnight at 180rpm in a temperature-controlled shaker at 37°C to activate the strains; when OD 600 =0.6, draw 200 μL and add it to the cooled LB solid medium plate, spread evenly with a glass rod, collec...

Embodiment 2( application Embodiment 1

[0055] The spores@Zr prepared by the present invention 4+ Application of microspheres in the detection of serum antibodies against Mycobacterium bovis infection

[0056] Specific steps are as follows:

[0057] (1) Take 100μL 1.2×10 10 particles mL –1 The spore @Zr 4+ Microspheres with 10 μg mL -1 The MPB83 antigen protein was reacted at 37 ℃ for 20min, so that the MPB83 antigen protein was coupled to the spore@Zr 4+ on the surface of the microspheres;

[0058] (2) Take out the reaction centrifuge tube, add 500 μL PBST to each tube after centrifugation to elute the immune microspheres, mix well and let stand for 3 minutes, centrifuge to remove the eluent, and repeat washing 3 times;

[0059] (3) Using 15% sheep serum solution as a blocking agent, the spore @Zr coupled with MPB83 antigen protein 4+ Microspheres are sealed;

[0060] (4) Take out the reaction centrifuge tube and repeat step (2).

[0061] (5) Add 200uL of the serum to be tested to make the antibody in the ...

Embodiment 3( application Embodiment 2

[0070] spore@Zr 4+ Application of Microspheres in Fluorescent Immunoassay of Bovine IFN-γ

[0071] 1. Whole Blood Culture

[0072] The blood samples of 54 dairy cows were collected from a dairy farm in Huanggang City, Hubei Province, and heparin was added for anticoagulation. Transported to the laboratory at room temperature and cultured within 30 hours after blood collection. Add anticoagulant blood to a 24-well tissue culture plate, add 2 tubes of 1.5mL anticoagulant blood to each cow, then add 100 μL poultry PPD and bovine PPD to the corresponding wells respectively, and place in a 37°C humid-temperature incubator Incubate for 16-24h. Carefully draw about 400 μL of the upper layer of plasma with a pipette for subsequent testing.

[0073] 2. Bovine IFN-γ fluorescent immunoassay steps

[0074] (1) Take 1mL concentration as 3.30×10 9 particles / mL of spores@Zr 4+ After the microspheres were centrifuged to remove the supernatant, 5ug / mL of anti-bovine IFN-γ monoclonal ant...

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Abstract

The invention belongs to the field of biological material preparation, and particularly relates to preparation of spore@Zr<4+> as functional microspheres and applications of spore@Zr<4+> in immunoassay. According to the present invention, spores are treated by using a biochemical method to prepare suspension microspheres with high stability and good dispersion, Zr<4+> ions are adsorbed onto the surfaces of the spores, and the obtained spore@Zr<4+> is used in the field of immunoassay; bacterial spores cultured for 7 days are collected, centrifugation washing and ultrasonic treatment are performed, treatment is performed with a trypsin solution and a spore coat removing treatment solution to obtain bacterial spore microspheres with characteristics of smooth surface, spore wall removing and spore coat removing, and the bacterial spore microspheres adsorb Zr<4+> ions to prepare the spore@Zr<4+> functional microspheres; the prepared spore@Zr<4+> functional microspheres have characteristicsof good mechanical strength, uniform particle size, proper specific gravity, simple preparation process, low cost, high-throughput detection and environmental protection; and the invention further discloses the applications of the spore@Zr<4+> microspheres in the immunoassay of Mycobacterium bovis infection.

Description

technical field [0001] The invention belongs to the field of preparation of immunoassay materials, in particular to a spore@Zr 4+ Preparation method of functional microsphere and application in immunoassay. Background technique [0002] Immunoassay technology is an analysis method based on the specific reaction of antigen and antibody. Due to the advantages of high specificity, high sensitivity, and low cost, this technique has become an important technique for analyzing specific proteins or other substances. However, the traditional immunoassay methods still have some defects. [0003] Taking enzyme-linked immunosorbent assay (ELISA) as an example, this method is costly and labor-intensive, and at the same time, the detection dynamic range and detection sensitivity are obviously limited. As a new immunoassay method, immunomicrosphere detection technology breaks through the inherent limitations of traditional methods and develops rapidly 1-8 . Compared with traditional ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/531
CPCG01N33/531G01N33/54313G01N33/54353G01N33/5695
Inventor 胡涌刚夏苗苗李政
Owner HUAZHONG AGRI UNIV
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