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Immunochip test method of staphylococcus enterotoxins and fumonisin

A staphylococcal intestinal and immune chip technology, applied in measurement devices, instruments, fluorescence/phosphorescence, etc., can solve the problems of long detection time and cumbersome steps, and achieve the effects of saving experimental costs, wide application range and high stability

Inactive Publication Date: 2012-05-16
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The technical problem to be solved by the present invention is: to overcome the shortcomings of the existing biological toxins Staphylococcus enterotoxin and fumonisin detection time-consuming, cumbersome steps, first fix the antigen or antibody on the surface of the aldylated glass slide, and use The glass sheet with aldehyde group on the surface treated by aldehyde and other reagents is used as a carrier, and the antigen or antibody is fixed by combining the aldehyde group on the surface of the carrier with the free amino group of the antigen or antibody, and the biological toxin to be tested and the labeled toxin complete antigen Competes binding to antibodies immobilized on glass slides and provides sensitive, rapid detection

Method used

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  • Immunochip test method of staphylococcus enterotoxins and fumonisin
  • Immunochip test method of staphylococcus enterotoxins and fumonisin
  • Immunochip test method of staphylococcus enterotoxins and fumonisin

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Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Preparation of an immune protein chip that can simultaneously detect SEA and SEB

[0028] The experimental method is as follows:

[0029] (1) According to figure 1 Aldehydated slides were activated.

[0030] (2) Extraction and concentration determination of anti-SEA and SEB antibodies in 2 rabbit serum

[0031] 2.1 Weigh 400g of analytically pure (AR) crystalline ammonium sulfate, dissolve it in 500ml of distilled water preheated to 70-80°C, stir for about 2 hours, cool to room temperature, crystals will precipitate at the bottom, and this solution is saturated ammonium sulfate solution . Adjust pH to 7.0 with 28% ammonia water.

[0032] 2.2 Take 10ml of the above-mentioned saturated ammonium sulfate solution, adjust the pH value to 5-6, take 7ml after filtration, add 3ml of distilled water, and prepare an ammonium sulfate solution with a saturation of 70%.

[0033] 2.3 Add 0.5 ml of 70% ammonium sulfate solution dropwise into 0.5 ml of rabbit serum with...

Embodiment 2

[0049] Embodiment 2: Immunochip detection method of staphylococcal enterotoxin

[0050] (1) Add standard

[0051] Add 20 μL of different concentrations of SEA and SEB toxin solutions dropwise to each detection cell of the chip, incubate at 37°C for 2 hours, wash as above, then add Cy3-labeled rabbit anti-SEA and SEB antibodies, incubate at 37°C for 2 hours, press Method flushing, scanner scanning.

[0052] (2) Signal fluorescence detection and data processing

[0053] After the antigen-antibody reaction or antibody competition reaction is completed, use the laser confocal scanner GenePix TM 4000B detection, fluorescence signal quantification was analyzed by software GenePix Pro 4.0. Scan results such as figure 2 and image 3 shown.

Embodiment 3

[0054] Example 3: p-fumonisin FB 1 immunochip detection method

[0055] (1) Put the object under test FB 1 (respectively take different concentrations of FB 1 : 1, 10, 50, 100, 200μg / ml), with a certain amount of Cy3-labeled FB 1 -BSA was mixed in equal amounts, and 22 μl of the mixture was added dropwise to the immobilized anti-FB 1 Place the antibody on a glass grid at 37°C for 2 hours at saturated humidity, then rinse with PBS washing solution for 5 times, rinse with distilled water for 3 times, and dry in the air.

[0056] (2) Signal fluorescence detection and data processing

[0057] After the antigen-antibody reaction or antibody competition reaction is completed, use the laser confocal scanner GenePix TM 4000B detection, fluorescence signal quantification was analyzed by software GenePix Pro 4.0. Scan results such as Figure 4 shown.

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Abstract

The invention relates to an immunochip test method of staphylococcus enterotoxins (SEA and SEB) and fumonisin (AFB1), so as to realize the simultaneous test on the same chip. According to the invention, for the test of AFB1, the monoclonal antibody of AFB1 is immobilized on the surface of a piece of aldehyde glass and then added to the chip together with a complete antigen labeled by a certain amount of Cy3, and the technical research on the immunochip can be carried out by using a competition method. For the test of SEA and SEB, a double-antibody sandwich method is adopted and the optimization of experiment conditions is carried out; in the test, SEA and SEB are added to a reaction tank, SE which is not combined is washed off after reaction, and then a corresponding labeled antibody is added so as to form an immobilized antibody- objected to be determinand-labeled antibody sandwich structure, and the intensity of a fluorescence signal is increased with increase of the concentration of the objected to be determinand. The method disclosed by the invention is simple in operation, low in cost, sensitive and rapid, the result can be stably and reliably obtained, and the whole test process needs 1-2 hours. A new method for rapid test of various biotoxins is provided.

Description

technical field [0001] The invention relates to an immune chip detection method for staphylococcus enterotoxin and fumonisin in food. Background technique [0002] Food safety has become the focus of global attention, and there are severe and prominent problems in our country. Staphylococcal enterotoxin is an important biological toxin that causes food poisoning, and it is also a commonly used biological warfare agent; the contamination of corn and its products by fumonisins may be related to the high incidence of human esophageal cancer. Mycotoxin of major hygiene importance. Currently, there is no simple and reliable way to detect it. Commonly used biotoxin detection methods include HPLC, GC-MS, ELISA and colloidal gold immunochromatography. Because HPLC and GC-MS are laboratory confirmation methods, they have the advantages of accurate quantitative detection, good repeatability, and high sensitivity, but the operation is complicated and requires professional and techni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/552G01N21/64
Inventor 高志贤刘楠左小霞
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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