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Chip detection method of visual protein in residual gentamicin in animal derived food

A gentamicin, animal-derived technology, applied in the field of visual protein chip detection, can solve the problems of being unsuitable for on-site monitoring and screening of a large number of samples, high requirements for technicians, and high equipment requirements, to achieve visualization of test results, reduce Operation steps, high-sensitivity effects

Active Publication Date: 2012-11-21
NANJING XIANGZHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The physical and chemical detection methods are mainly HPLC and HPLC-MS, but this method has high requirements for equipment, high technical difficulty, high requirements for technicians, cumbersome operation, and is not suitable for on-site monitoring and screening of a large number of samples

Method used

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  • Chip detection method of visual protein in residual gentamicin in animal derived food
  • Chip detection method of visual protein in residual gentamicin in animal derived food
  • Chip detection method of visual protein in residual gentamicin in animal derived food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of gentamicin-coated antigen.

[0049] 1) Dissolve 5 mg of GM in 10 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution I, and stir in an ice-water bath for 10 min.

[0050] 2) Dissolve 20 mg of carbodiimide in 10 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution II, and stir in an ice-water bath for 10 min.

[0051] 3) Weigh 10 mg of carrier protein and dissolve in 20 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution III, and stir in an ice-water bath for 10 min.

[0052] 4) Add solution I to solution III and stir on a magnetic stirrer, then slowly add solution II to the above mixture drop by drop in an ice-water bath, stir for 1 hour in the dark, and react for 3 hours at 4°C to synthesize gentamicin Protein-carrier protein coupling product.

[0053] 5) Put the gentamicin-carrier protein coupling product into a dialysis bag, dialyze with PBS buffer (0.1M, pH7.4) at 4°C for 3 days, and change the dialysate every 8 hours.

[0054] 6) The cou...

Embodiment 2

[0055] Example 2: Preparation of gentamicin-coated antigen.

[0056] 1) Dissolve 200mg GM in 50mL PBS buffer (0.1M, pH7.4) to obtain solution I, and stir in an ice-water bath for 10min.

[0057] 2) Dissolve 100 mg of carbodiimide in 50 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution II, and stir in an ice-water bath for 10 min.

[0058] 3) Weigh 50 mg of carrier protein and dissolve in 100 mL of PBS buffer (0.1 M, pH 7.4) to obtain solution III, and stir in an ice-water bath for 10 min.

[0059] 4) Add solution I to solution III and stir on a magnetic stirrer, then slowly add solution II to the above mixture drop by drop in an ice-water bath, stir for 1 hour in the dark, and react for 3 hours at 4°C to synthesize gentamicin Protein-carrier protein coupling product.

[0060] 5) Put the gentamicin-carrier protein coupling product into a dialysis bag, dialyze with PBS buffer (0.1M, pH7.4) at 4°C for 3 days, and change the dialysate every 8 hours.

[0061] 6) The coupled pr...

Embodiment 3

[0062] Example 3: Method 1 for the quantitative detection of gentamicin content by visual protein chip method.

[0063] (1) Using bovine serum albumin (BSA) as a blank control, the bovine serum albumin and gentamicin-carrier protein conjugates were spotted by a biochip spotter according to different specifications of the matrix, and the wells of the spot plate were Add 30 μL of bovine serum albumin and 30 μL of gentamicin-carrier protein conjugate, and place the spotted slides in a 4°C refrigerator to fix overnight;

[0064] (2) After fixation, add 20 μL of 10 mg / mL bovine serum albumin buffer solution to each reaction well to block the chip for 1 hour, then wash with PBST 3 times for 5 minutes each time; The area is blocked to prevent non-specific binding of proteins in the sample to be tested and other active groups, resulting in high background or false positives;

[0065] (3) Add 20 μL of gentamicin antibody and different concentrations of free gentamicin standard solutio...

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Abstract

The invention discloses a chip detection method of visual protein in residual gentamicin in animal derived food and belongs to indirect competitive immune method. The chip detection method is characterized by utilizing a carbodiimide method to further combine gentamicin envelope antigen and create gentamicin in animal derived food. The method includes fixing the gentamicin envelope antigen and an internal standard substance on a chip carrier, then adding free gentamicin and gentamicin monoclonal antibody into a reaction region, conducting competitive reaction between the free gentamicin and conjugate fixed on a chip, conducting washing after reaction is finished, then adding secondary antibody marked by collaurum, conducting washing after reaction is complete, adding silver enhancement chromogenic agent, enabling the collaurum to catalyze silver atoms into visual black silver, scanning images through a scanner and finally achieving in-hole semi-quantitative and quantitative detection simultaneously. By means of the chip detection method, quantity of the residual gentamicin in the animal derived food can be quickly and accurately detected, and the detection method is high in sensitivity, short in detection time and simple and easy to operate.

Description

technical field [0001] The invention relates to a visual protein chip detection method for residual gentamycin in animal-derived food, and belongs to the technical field of food safety supervision or immune analysis of food analysis. Background technique [0002] Gentamicin is an aminoglycoside antibiotic. Antibiotics are widely used as veterinary drugs and feed additives, which may cause antibiotic residues in animal-derived foods. For dairy products such as milk, antibiotic residues will not only affect the quality of dairy products, such as seriously affecting the production of fermented dairy products, reducing production and destroying the flavor of later products, causing economic losses to producers, but also causing great harm to the human body. Such as causing allergic reactions in the human body (such as skin rashes, anaphylactic shock, etc.), inhibiting the growth of normal sensitive bacteria in the intestinal tract, causing a large number of pathogenic bacteria a...

Claims

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Application Information

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IPC IPC(8): G01N33/552G01N33/531
Inventor 李周敏孙思云
Owner NANJING XIANGZHONG BIOTECH
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