Method for fast detecting beta-lactamase contained in milk product by optical fiber biosensor

A biosensor, lactamase technology, applied in the biological field, achieves high sensitivity, fast response time, and small size

Inactive Publication Date: 2010-01-13
WUXI YIDA BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Using biosensors, especially optical fiber biosensors to detec

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] β-lactamase antibody preparation

[0028] Take 100 μg of β-lactamase and 5 mg of BCG, grind it with an equal volume of Freund’s complete adjuvant, and inject it into the peritoneal cavity of 6-week-old female BALBPC mice. Afterwards, Freund’s incomplete adjuvant is used for intraperitoneal injection 5 times every other week. On the first day, 50 μg β-lactamase standard solution was injected intraperitoneally, with 50% PEG4000 as the fusion agent, the mouse splenocytes were fused with NS1 cells, IMDM2HAT was selected for culture, and HT culture medium was changed after 2 weeks. Using β-lactamase as an antigen, the positive cloned hybridoma cells were screened by indirect ELISA, and the obtained cell lines continued to be amplified and cultured. The positive cell line was injected into the peritoneal cavity of BALBPC mice to induce ascites, and the β-lactamase antibody was detected by rabbit anti-mouse immunoglobulin and agar double diffusion method to prepare the β-lacta...

Embodiment 2

[0030] (1) Preparation of coating antibody

[0031] Dilute the β-lactamase antibody to a certain concentration with the coating solution and put it into a capillary, insert the optical fiber at 4°C and incubate for 12-16 hours, and then it can be used for the corresponding immune reaction. Store at 4°C when not in use.

[0032] (2) Preparation of labeled antibody

[0033] Dilute the antibody of the tested specimen to a certain concentration with diluent, combine with the fluorescein dye Cy5 in an alkaline environment, and separate the Cy5-labeled monoclonal antibody by gel filtration.

[0034] (3) Production of antibody immobilization

[0035] The surface of the optical fiber is silanized by silane substances such as triaminopropyltriethylsilane, and then the β-lactamase monoclonal antibody labeled with Cy5 is immobilized by the bifunctional cross-linking agent glutaraldehyde.

(4) specific implementation

[0036] Fix the Cy5-labeled β-lactamase monoclonal antibody on the ...

Embodiment 3

[0038] (1) Preparation of standard curve

[0039] Take a certain amount of β-lactamase standard solution of different concentrations and drop it on the sensitive membrane of the sensor, then insert the sensor into the system interface, press the test button, and respond for a period of time. After about 300s, the detection result will be displayed and displayed. save. Record the light signal values ​​corresponding to different concentrations of β-lactamase standard solutions to make a standard curve.

[0040] (2) Sample pretreatment and detection

[0041] Weigh 1 g of milk powder without β-lactamase, add 5 times the volume of DMSO to shake for 1 h, and centrifuge to obtain 2 μl of supernatant. Add β-lactamase with a final concentration of 50 μg / L to the above serum, dilute it 10 times as a detection control sample, and use an optical fiber biosensor for testing.

[0042] Take a 20ul sample, drop it on the sensitive membrane of the fiber optic sensor, then insert the sensor ...

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PUM

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Abstract

The invention discloses a method for fast detecting beta-lactamase contained in milk products by an optical fiber biosensor, belonging to the field of the optical fiber biosensor and comprising an optical system, an electronic system, a data collection system, a control and processing system, optical fibers and a sensor, wherein optical fiber material is polystyrene; the sensor is provided with a sensitive film, the sensitive film contains a beta-lactamase monoclonal antibody fixed by glutaraldehyde and marked by Cy5, and the monoclonal antibody is prepared by taking the beta-lactamase as an antigenic and immunized mouse, fusing cells, and the like. The invention has the advantages of high sensitivity, electromagnetic radiation resistance, electronic frequency interference resistance, corrosion resistance, small instrument volume, portability, few needed detection sample, rapid response time, easy operation, low cost, and the like and can be used for detecting the beta-lactamase contained in the milk products, such as liquid milk, powdered milk, solid samples, and the like.

Description

technical field [0001] The invention relates to a method for rapidly detecting beta-lactamases of dairy products by using an optical fiber biosensor, and belongs to the field of biotechnology. Background technique [0002] The scientific name of "antibiotic" is β-lactamase, which is a chemical extracted from β-lactam antibiotics. It is an antibiotic decomposing agent, which can effectively decompose the antibiotics left in milk. β-lactamase is a food additive that is not allowed in my country. The use of this enzyme conceals the antibiotics actually contained in milk. However, in order to pursue profits, some vendors use it to degrade the antibiotics left in milk and produce so-called "artificial antibiotic-free milk", which causes the residues of antibiotics to enter dairy products and cause harm to human health. At the same time, it can Destroy the structure of penicillin lactam and lose its activity, leading to increased resistance to antibiotics such as penicillin and c...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/544G01N33/532G01N21/64C12N15/06C12P21/08
Inventor 张建中赵晓联赵春城徐祖奇杨婷婷蔡建荣马祯婉
Owner WUXI YIDA BIO TECH
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