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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 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

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

Embodiment 1

[0026] Preparation of β-lactamase monoclonal antibody

[0027] Mix 0.5mL of β-lactamase solution containing 1mg / mL with an equal volume of complete Freund's adjuvant and fully emulsify it for the first immunization. Select 8-week-old female BABL / C mice as immunized animals, and the immunization dose is 300μg Intraperitoneal injection per mouse, followed by booster immunization once every 2 weeks, tail vein injection for booster immunization, immunization dose 250 μg per mouse, intrasplenic injection for the last immunization, spleen fusion after 3 to 4 days, and isolated immune small Rat splenocytes and myeloma cells SP-2 / o in the logarithmic growth phase were mixed at a ratio of 10:1, centrifuged, and the supernatant was removed, and 1 mL of 50% polyethylene glycol (molecular weight: 1500) was added within 50S to 90S. After 1 min, add 20 mL of DMEM culture medium (Dulbecco Modified Eagle Medium) to stop the fusion, and centrifuge after 10 min in water bath to remove the super...

Embodiment 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 freshly prepared double distilled water, place it in a conical flask, heat it to boiling with a constant temperature electromagnetic stirrer, and quickly add 1% citric acid that has been kept at 37°C under magnetic stirring at 100r / min 4.5 mL of trisodium solution, keep the temperature and rotating speed constant, continue to stir and heat for about 15 minutes, until the solution is clear wine red, and the size of colloidal gold is 20-30nm. . Cool at room temperature, store in refrigerator at 4°C for later use

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

Embodiment 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 straw to carefully absorb the upper liquid, accurately absorb 1ml of the supernatant into a stoppered test tube, add 4ml of 20mM PBS solution, cover the lid, and mix well. The diluted sample is the sample to be tested and can be directly used in the test.

[0042] 2. Kit detection

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

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

[0045] (3) The result can be judged within 3 to 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 detection line. At this time, the result indicates that the content of β-lactamase is above 50ppm; Negative (-): quality Purple-red bands appeared in both the control line an...

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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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