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Double-antibody sandwich ELISA detection kit for alicyclobacillus acidoterrestris in fruit juice

A double-antibody sandwich and detection method technology, which is applied in the direction of testing food, measuring devices, testing beverages, etc., can solve the problems of long detection time and achieve the effects of short detection time, improved detection efficiency, and stable detection results

Inactive Publication Date: 2015-04-08
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when this method is used for the ELISA detection of heat-resistant bacteria, it is first necessary to prepare antibodies and coat the microtiter plate, and the detection time is relatively long.

Method used

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  • Double-antibody sandwich ELISA detection kit for alicyclobacillus acidoterrestris in fruit juice
  • Double-antibody sandwich ELISA detection kit for alicyclobacillus acidoterrestris in fruit juice
  • Double-antibody sandwich ELISA detection kit for alicyclobacillus acidoterrestris in fruit juice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The heat-resistant bacteria rabbit-derived polyclonal antibody was used as a detection antibody to prepare a double-antibody sandwich ELISA detection kit for heat-resistant bacteria in fruit juice, and the preparation method was as follows:

[0035] (1) Thermostable bacteria rabbit-derived polyclonal antibody-coated microtiter plate

[0036]Dilute the heat-resistant bacteria rabbit-derived polyclonal antibody solution with a concentration of 10 μg / mL to a concentration of 5 μg / mL with carbonic acid buffer solution with a concentration of 0.05 mol / L and a pH value of 9.6, and add it to the wells of the microtiter plate. 100 μL, placed in a constant temperature incubator at 37 ° C for 2 hours, 1 mL of Tween-20 was dissolved in 2 L of phosphate buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 to prepare a washing solution with a volume ratio of 1:2000. Wash the microplate with the lotion for 2.5 minutes, repeat the washing 4 times, shake it off gent...

Embodiment 2

[0055] The heat-resistant bacteria rabbit-derived polyclonal antibody was used as a detection antibody to prepare a double-antibody sandwich ELISA detection kit for heat-resistant bacteria in fruit juice, and the preparation method was as follows:

[0056] (1) Thermostable bacteria rabbit-derived polyclonal antibody-coated microtiter plate

[0057] Dilute the heat-resistant bacteria rabbit-derived polyclonal antibody solution with a concentration of 0.05 mol / L and a pH value of 9.6 to a concentration of 4.8 μg / mL, and add it to the wells of the microtiter plate. Add 100 μL, place in a constant temperature incubator at 37°C and incubate for 1.9 hours, mix Tween-20 with 0.01 mol / L phosphate buffer solution with a pH value of 7.4 according to the volume ratio of 1:1900, and prepare washing Wash the microplate with the lotion for 3 minutes, repeat the washing 3 times, shake it off gently, and pat the microplate dry on the gauze upside down.

[0058] (2) Sealed microtiter plate

...

Embodiment 3

[0069] The heat-resistant bacteria rabbit-derived polyclonal antibody was used as a detection antibody to prepare a double-antibody sandwich ELISA detection kit for heat-resistant bacteria in fruit juice, and the preparation method was as follows:

[0070] (1) Thermostable bacteria rabbit-derived polyclonal antibody-coated microtiter plate

[0071] Dilute the heat-resistant bacteria rabbit-derived polyclonal antibody solution with a concentration of 10 μg / mL to a concentration of 5.2 μg / mL with carbonic acid buffer solution with a concentration of 0.05 mol / L and a pH value of 9.6, and add it to the wells of the microtiter plate. Add 100 μL, place in a constant temperature incubator at 37°C and incubate for 2.1 hours, mix Tween-20 with a phosphate buffer solution with a concentration of 0.01 mol / L and a pH value of 7.4 to prepare a washing solution at a volume ratio of 1:2200, and use Rinse the microplate with this lotion for 2 minutes, repeat the rinse 5 times, shake it off ge...

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Abstract

The invention relates to a preparation method and application of a double-antibody sandwich ELISA (enzyme-linked immunosorbet assay) detection kit for alicyclobacillus acidoterrestris in fruit juice. According to the invention, a rabbit-derived polyclonal antibody is coated on an enzyme label plate in advance, then a sealing solution is added, and an aluminum foil bag is adopted to conduct vacuum packaging so as to obtain a standard kit, so that standardization of an ELISA rapid detection method can be realized. During detection, an alicyclobacillus acidoterrestris antigen captured in a sample is added into the kit, then an alicyclobacillus acidoterrestris mouse-derived polyclonal antibody is added so as to form an antibody-antigen-antibody double-antibody complex, and an enzyme labelled antibody and a substrate working solution are prepared. The enzyme on the double-antibody complex is made to catalyze the substrate. Then a microplate reader is employed to determine a spectrophotometric value of the enzyme label plate, and the spectrophotometric value is compared with a cut off value of a negative control sample so as to judge whether a sample is negative or positive, thus realizing a double-antibody sandwich ELISA detection method of the alicyclobacillus acidoterrestris. The detection method has the characteristics of short detection time, stable detection result, and greatly improved detection efficiency.

Description

technical field [0001] The invention relates to the field of food microorganism detection, in particular to a preparation method or application of a kit used for rapid detection of double-antibody sandwich ELISA in fruit juice. Background technique [0002] The heat-resistant bacteria (Alicyclobacillus acidoterrestris) can form spores at high temperature or in an unfavorable environment. One of the main microorganisms that deteriorate, heat-resistant bacteria exceeding the standard is also the most urgent problem that apple juice manufacturers need to solve. [0003] At present, manufacturers mostly use the traditional plate culture counting method to detect heat-resistant bacteria, and the plate culture counting method mainly includes the American KFL method (the American Cook Laboratory method) and the Australian Berri company method. The KFL method mainly adopts filter membrane filtration, traps heat-resistant bacteria on the filter membrane, and then puts the filter mem...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N21/31G01N33/14
Inventor 李建科夏凯黄瑞蕊
Owner SHAANXI NORMAL UNIV
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