Quantum-dot immunofluorescence kit for simultaneously detecting gentamycin and quinolone medicines

Abstract

The invention discloses a quantum-dot immunofluorescence kit for simultaneously detecting gentamycin and quinolone medicines, and at the same time, the invention also discloses a preparation method of ciprofloxacin hapten, corresponding artificial antigen and a quantum dot-labelled monoclonal antibody. The kit takes a conjugate of gentamycin semiantigen and carrier protein as well as the conjugate of the ciprofloxacin semiantigen and carrier protein as a primordial covering, and takes a mixture of a quantum dot-labelled gentamycin specific antibody and a ciprofloxacin specific antibody as a detection antibody. The preparation method is simple and feasible, has the advantages of low cost and high semiantigen yield, and the quantum-dot immunofluorescence kit has the advantages of simpleness, rapidity, large sample processing amount, high sensitivity, and strong specificity. The quantum-dot immunofluorescence kit can be used for self-control of quality and food safety supervision of a food production enterprise, and routine screening of a detection mechanism, and can be used as an auxiliary means for animal pharmacological and toxicologic researches in scientific research.

Description

technical field

[0001] The invention belongs to the field of food safety detection, and in particular relates to a quantum dot immunofluorescence kit for simultaneously detecting residues of gentamicin and quinolones in animal-derived foods. Background technique

[0002] Gentamycin (Gentamycin, GM) is a kind of aminoglycoside antibiotics. Gentamycin is alkaline, soluble in water, and stable in properties. It is combined with sulfuric acid to form gentamycin sulfate for clinical use. It has a wide antibacterial spectrum, against Pseudomonas aeruginosa, E. It has antibacterial effect on Gram-negative bacteria, and it also has strong antibacterial effect on Staphylococcus aureus in Gram-positive bacteria; its antibacterial mechanism is directly combined with the A part of the decoding region of 30S ribosomal subunit 16rRNA , resulting in cell death. However, with the widespread use of gentamicin, some bacteria have gradually developed resistance to it. Although gentamicin ha...

Examples

Embodiment 1

[0049] Example 1, Preparation of Conjugates, Specific Antibodies and Quantum Dot Labels of Specific Antibodies

[0050] 1. Antigen preparation

[0051] 1. Preparation of gentamicin-carrier protein conjugates

[0052] (1) Take 26mg of gentamicin API, dissolve in 4ml of carbonate buffer (take 63.6mg of anhydrous NaCO 3 and 117.6 mg NaHCO 3 , and dilute to 20 mL with pure water).

[0053] (2) Take 60mg of BSA and dissolve it in 6ml of the carbonate buffer.

[0054] (3) Add the gentamicin solution obtained in step (1) dropwise to the BSA solution obtained in step (2) and mix at room temperature for 10 minutes, then add 487 μl of 1% (volume ratio) glutaraldehyde aqueous solution and stir at room temperature (400rpm ) overnight, then dropwise added NaBH4 solution (5mgNaBH4 dissolved in 500μl pure water, freshly prepared) and stirred at room temperature for 1h, then placed in a dialysis bag and dialyzed with pure water (dialysis parameters: 4°C, 100rpm stirring, 2 days, every day...

Embodiment 2

[0087] Embodiment 2, the assembly of the quantum dot immunofluorescence kit that simultaneously detects gentamicin and quinolones

[0088] Quantum dots were purchased from Shenzhen Thales Technology Co., Ltd., the product catalog number is TLS?LumiQDTM20. The characterization of the quantum dots is as follows: the particle size is 20nm, the CV of the particle size is 15%, the quantum yield is 60%, the surface carboxyl content is 5×10-3mmol / mg, water solubility, CdSe / ZnS core-shell structure, excitation Light wavelength is 345nm, emission wavelength is 620nm; red fluorescent quantum dots. The carboxyl groups on the quantum dots form peptide bonds with the amino groups on the protein and connect them.

[0089] Quantum dot immunofluorescence detection kit includes the following components:

[0090] 1. Microwell plate coated with the original coating

[0091]Take the original coating solution prepared in Example 1 and dilute it with the coating buffer (the coating buffer is pH9...

Embodiment 3

[0110] Embodiment 3, the usage method of the quantum dot immunofluorescence kit that simultaneously detects gentamicin and quinolones

[0111] 1. Sample pretreatment method

[0112] 1. Obtaining pork or chicken test sample solution: Accurately weigh 1±0.01g homogenized pork or chicken sample into a 50mL centrifuge tube; add 20mL sample extract; at room temperature (23-27°C), vortex at high speed 1min; above 4000g, centrifuge for 10min; take 50μL supernatant for analysis.

[0113] 2. Obtaining the milk test sample solution: Take a milk sample of more than 3000g and centrifuge it for 10 minutes, lightly suck the middle layer, dilute it 5 times with the sample diluent, and take 50 μL for analysis.

[0114] 2. Application of Quantum Dot Immunofluorescence Kit for Detection

[0115] Add 50 μL of standard solution or sample solution to be tested, 50 μL of primary antibody working solution, and react at room temperature for 30 minutes, discard the supernatant, and wash 3 times (the...

technical field

[0001] The invention belongs to the field of food safety detection, and in particular relates to a quantum dot immunofluorescence kit for simultaneously detecting residues of gentamicin and quinolones in animal-derived foods. Background technique

[0002] Gentamycin (Gentamycin, GM) is a kind of aminoglycoside antibiotics. Gentamycin is alkaline, soluble in water, and stable in properties. It is combined with sulfuric acid to form gentamycin sulfate for clinical use. It has a wide antibacterial spectrum, against Pseudomonas aeruginosa, E. It has antibacterial effect on Gram-negative bacteria, and it also has strong antibacterial effect on Staphylococcus aureus in Gram-positive bacteria; its antibacterial mechanism is directly combined with the A part of the decoding region of 30S ribosomal subunit 16rRNA , resulting in cell death. However, with the widespread use of gentamicin, some bacteria have gradually developed resistance to it. Although gentamicin ha...

Examples

Embodiment 1

[0049] Example 1, Preparation of Conjugates, Specific Antibodies and Quantum Dot Labels of Specific Antibodies

[0050] 1. Antigen preparation

[0051] 1. Preparation of gentamicin-carrier protein conjugates

[0052] (1) Take 26mg of gentamicin API, dissolve in 4ml of carbonate buffer (take 63.6mg of anhydrous NaCO 3 and 117.6 mg NaHCO 3 , and dilute to 20 mL with pure water).

[0053] (2) Take 60mg of BSA and dissolve it in 6ml of the carbonate buffer.

[0054] (3) Add the gentamicin solution obtained in step (1) dropwise to the BSA solution obtained in step (2) and mix at room temperature for 10 minutes, then add 487 μl of 1% (volume ratio) glutaraldehyde aqueous solution and stir at room temperature (400rpm ) overnight, then dropwise added NaBH4 solution (5mgNaBH4 dissolved in 500μl pure water, freshly prepared) and stirred at room temperature for 1h, then placed in a dialysis bag and dialyzed with pure water (dialysis parameters: 4°C, 100rpm stirring, 2 days, every day...

Embodiment 2

[0087] Embodiment 2, the assembly of the quantum dot immunofluorescence kit that simultaneously detects gentamicin and quinolones

[0088] Quantum dots were purchased from Shenzhen Thales Technology Co., Ltd., the product catalog number is TLS?LumiQDTM20. The characterization of the quantum dots is as follows: the particle size is 20nm, the CV of the particle size is 15%, the quantum yield is 60%, the surface carboxyl content is 5×10-3mmol / mg, water solubility, CdSe / ZnS core-shell structure, excitation Light wavelength is 345nm, emission wavelength is 620nm; red fluorescent quantum dots. The carboxyl groups on the quantum dots form peptide bonds with the amino groups on the protein and connect them.

[0089] Quantum dot immunofluorescence detection kit includes the following components:

[0090] 1. Microwell plate coated with the original coating

[0091]Take the original coating solution prepared in Example 1 and dilute it with the coating buffer (the coating buffer is pH9...

Embodiment 3

[0110] Embodiment 3, the usage method of the quantum dot immunofluorescence kit that simultaneously detects gentamicin and quinolones

[0111] 1. Sample pretreatment method

[0112] 1. Obtaining pork or chicken test sample solution: Accurately weigh 1±0.01g homogenized pork or chicken sample into a 50mL centrifuge tube; add 20mL sample extract; at room temperature (23-27°C), vortex at high speed 1min; above 4000g, centrifuge for 10min; take 50μL supernatant for analysis.

[0113] 2. Obtaining the milk test sample solution: Take a milk sample of more than 3000g and centrifuge it for 10 minutes, lightly suck the middle layer, dilute it 5 times with the sample diluent, and take 50 μL for analysis.

[0114] 2. Application of Quantum Dot Immunofluorescence Kit for Detection

[0115] Add 50 μL of standard solution or sample solution to be tested, 50 μL of primary antibody working solution, and react at room temperature for 30 minutes, discard the supernatant, and wash 3 times (the...

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