Gold-labeled kit of beta-lactamase in dairy product and preparation method thereof

A technology of lactamase and colloidal gold test paper, applied in the biological field, can solve the problems of human health injury and bacterial resistance, and achieve the effects of short reaction time and simple operation steps.

Inactive Publication Date: 2010-01-06
WUXI YIDA BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some unscrupulous manufacturers use it to degrade the antibiotics left in milk to produce the so-called "artificial antibiotic-free milk", which causes the re

Method used

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  • Gold-labeled kit of beta-lactamase in dairy product and preparation method thereof
  • Gold-labeled kit of beta-lactamase in dairy product and preparation method thereof
  • Gold-labeled kit of beta-lactamase in dairy product and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0025] Example 1

[0026] Preparation of β-lactamase monoclonal antibody

[0027] Mix 0.5 mL of 1 mg / mL β-lactamase solution with an equal volume of complete Freund’s adjuvant, and fully emulsify it for the first immunization. Choose 8-week-old female BABL / C mice as immunized animals, and the immunization dose is 300 μg Intraperitoneal injection and booster immunization every 2 weeks thereafter. The booster immunization is by tail vein injection with a dose of 250 μg per mouse. The last immunization is by intrasplenic injection. After 3 to 4 days, the spleen will be fused and the separated immunity will be small. Mouse spleen cells and myeloma cells SP-2 / o in the logarithmic growth phase were mixed at a ratio of 10:1, centrifuged, the supernatant was removed, and 1 mL 50% polyethylene glycol (molecular weight 1500) was added within 50S~90S Into the cells, mix well and make them fuse. After 1 min, add 20mL DMEM medium (Dulbecco Modified Eagle Medium) to terminate the fusion. After ...

Example Embodiment

[0028] Example 2

[0029] The production process of test strips:

[0030] 1. Preparation of colloidal gold solution and gold-labeled antibody

[0031] Prepare 100mL 0.01% chloroauric acid solution with fresh double-distilled water, place it in an Erlenmeyer flask, heat to boiling with a constant temperature electromagnetic stirrer, and quickly add 1% citric acid preheated at 37°C under 100r / min magnetic stirring The trisodium solution is 4.5mL, keeping the temperature and rotating speed unchanged, and stirring and heating is continued for about 15 minutes until the solution shows a clear wine-red color and the size of the colloidal gold is 20-30nm. . Cool at room temperature, keep in refrigerator at 4℃ for later use

[0032] Take 30mL colloidal gold solution and add it to a 50mL centrifuge tube, use 0.1M K 2 CO 3 Or adjust the pH to 7.0~7.5 with 0.1M HCl, wrap the centrifuge tube with aluminum foil, and shake gently; while shaking, add 3mL of 250μg β-lactamase monoclonal antibody so...

Example Embodiment

[0038] Example 3

[0039] Detection method of β-lactamase kit

[0040] 1. Sample pretreatment

[0041] Take 10ml sample and centrifuge at 12000rpm for 10 minutes. Use a plastic pipette to carefully suck the upper layer of liquid, accurately pipette 1ml of the supernatant into a test tube with a stopper, add 4ml of 20mM PBS solution, close the lid, and mix thoroughly. This diluted sample is the sample to be tested and can be used directly in the test.

[0042] 2. Test kit

[0043] (1) Take out the test card from the aluminum foil bag and place it on a clean horizontal table;

[0044] (2) Add 100μl of the sample to be tested into the sample hole;

[0045] (3) The result can be judged within 3-15 minutes.

[0046] Result judgment: positive (+): only a purple-red band appears on the quality control line, and no purple-red band appears on the test line. At this time, the result indicates that the β-lactamase content is above 50ppm; negative (-): quality Both the control line and the detectio...

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Abstract

The invention provides a colloidal gold test strip for semiquantitatively and rapidly detecting beta-lactamase and a preparation method thereof. A anti-beta-lactamase monoclonal antibody is marked by colloidal gold according to immunology principles of antigen-antibody specific binding and is coated on a glass fiber film to be served as a gold-labeled pad; a beta-lactamase antigen and goat anti-mouse IgG are respectively coated on a pyroxylin film ( NC film) to be served as a detection line and a quality control line. When a detected sample contains beta-lactamase, an antigen-antibody combo can be formed on the detection line, and a mauve strip is presented on the NC film, and thereby, beta-lactamase contained in dairy products, such as liquid milk, milk powder, solid samples and the like can be simply and rapidly detected by naked eyes.

Description

technical field [0001] The invention relates to a gold label kit for detecting β-lactamase in dairy products and a preparation method thereof, belonging 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. Some unscrupulous manufacturers use it to degrade the antibiotics left in milk to produce the so-called "artificial antibiotic-free milk", which causes the residues after the enzymatic hydrolysis of antibiotics to enter the dairy products, causing harm to human health, and may also cause drug resistance of bacteria . Therefore, there is an urgent need for a rapid detection method, so as to quickly and accurately detect β-lactamase in dairy products. [0003] The gold labeling method, also known as the "colloidal gold method", is a new type of immunolabeling...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558G01N33/532
Inventor 张建中赵晓联赵春城徐祖奇蔡建荣马祯婉
Owner WUXI YIDA BIO TECH
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