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Beta-fructofuranosidase enzyme-linked immunoassay detection kit

A technology of fructofuranosidase and fructofuranose, which is applied in measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the problems that are not suitable for high-throughput, rapid detection, complicated pre-treatment, long detection time, etc., and achieve determination Stable results, simple sample processing, and rapid detection

Active Publication Date: 2012-06-13
济宁高新科达科技项目服务有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the detection method of β-fructofuranosidase is mainly high-performance liquid chromatography, which is expensive, has a long detection time and complicated pretreatment, and is not suitable for high-throughput and rapid detection.

Method used

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  • Beta-fructofuranosidase enzyme-linked immunoassay detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation and purification of antibodies

[0022] 1.1 Preparation of mouse monoclonal antibody

[0023] Healthy 8-week-old female BALB / C mice were selected for β-fructofuranosidase immunization, and the immunization dose was 50-100 μg / mouse based on β-fructofuranosidase, with an interval of two weeks between each immunization and five times of immunization. For the first immunization, the immunogen β-fructofuranosidase was mixed with the same amount of Freund's complete adjuvant to make an emulsified injection, and the neck and back were injected subcutaneously at multiple points. For 2-4 immunizations, the immunogen was mixed with the same amount of Freund's Incomplete adjuvant was mixed with emulsifier injection, and multi-point subcutaneous injection was used on the back of the neck. The last time, abdominal booster immunization was carried out without adjuvant. Three days after the last immunization, mouse splenocytes were fused with Sp2 / 0 myeloma cell...

Embodiment 2

[0026] Embodiment 2: the formation of kit

[0027] The ELISA kit of the present invention comprises: mouse anti-β-fructofuranosidase monoclonal antibody-coated ELISA plate, rabbit anti-β-fructofuranosidase polyclonal antibody, enzyme-labeled secondary antibody working solution, β-fructofuranoside Enzyme Standards, Sample Diluent, Substrate Developing Solution, Concentrated Wash and Stop Solution.

[0028] 2.1 Coating of ELISA plate

[0029] Use the checkerboard method to determine the optimal coating concentration, dilute the monoclonal antibody obtained in step 1.1 to 2-20 μg / ml with the coating solution (5-50mmol / L carbonic acid buffer), mix well, add to the 96-well microtiter plate, 100μl per well, 14-16 hours at 4°C. Pour off the solution in the well, wash the plate 4 times with concentrated washing solution, and pat dry. Add 150 μl of blocking solution to each well, hold at 37°C for 1 hour, pour off the solution in the well, and wash the plate 4 times with the concentr...

Embodiment 3

[0038] Example 3 Detection of β-fructofuranosidase in honey samples

[0039] 3.1. Sample pretreatment

[0040] Accurately weigh a certain amount of honey sample, add 3 times the weight sample diluent, bathe in a constant temperature water bath at 50-80°C for 5-10 minutes, take it out, and cool it to room temperature for later use.

[0041] 3.2 Kit detection

[0042] (1) Take out the coated ELISA plate, add 100 μl standard or sample to each well, and incubate at 37°C for 40 minutes.

[0043] (2) Plate washing: Pour off the solution in the wells, add 200-300 μl of washing solution to each well, pour off the washing solution, repeat 3-4 times, and pat dry the microplate.

[0044] (3) Add 100 μl polyclonal antibody working solution and incubate at 37°C for 40 minutes.

[0045] (4) Wash the plate with concentrated washing solution.

[0046] (5) Add 100 μl enzyme-labeled secondary antibody working solution and incubate at 37°C for 40 minutes.

[0047] (6) Wash the plate with co...

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Abstract

The invention relates to a beta-fructofuranosidase enzyme-linked immunoassay detection kit which comprises a beta-fructofuranosidase standard enzyme solution, an enzyme label plate enveloped with a beta-fructofuranosidase monoclonal antibody, a sample diluent, a beta-fructofuranosidase rabbit polyclonal antibody working solution, an enzyme labelled secondary antibody working solution, a substrate coloured solution, a washing concentrate and a stop solution. For the kit disclosed by the invention, the content of beta-fructofuranosidase in a honey sample is measured by adopting a double antibodies sandwich ELISA (enzyme-linked immuno sorbent assay) method; a linearly dependent coefficient of a dosage-reaction curve is more than 0.980; R2 is equal to 0.9993; the sensitivity is 10U / KG; and the national ELISA detection quality requirement is met; and an intra-plate variation coefficient (CV%) of the kit is less than 10 percent. The kit has stable measurement result and good precision. The kit disclosed by the invention has the advantages of simpleness in processing the sample, high detection speed, short detection time of only 2.5 hours, sensitivity, accuracy and high throughput, and provides an effective means for detecting counterfeiting of honey.

Description

technical field [0001] The invention relates to an ELISA detection kit for β-fructofuranosidase. technical background [0002] β-fructofuranosidase (EC 3.2.1.26), also known as invertase, is mainly derived from yeast. Catalyze the following reaction, [0003] Sucrose + water glucose + fructose [0004] The enzyme is the same as the catalyzed product of sucrose invertase naturally present in honey. Therefore, criminals often illegally use β-fructofuranosidase in the honey production and processing process, and add this enzyme to fake honey with cheap syrup. If this enzyme is added to artificial syrup, it can be detected by isotope analysis, but if it is added to C3 sugar (such as beet sugar), it is difficult to detect by isotope analysis. [0005] Honey is a natural product. According to GB / T 18796---2005 National Standard for Honey, no sugar or sugar substitutes shall be added or mixed into honey. Fake honey has caused great harm to society. First of all, honey fraud ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/31
Inventor 陈英珠齐颖颖李君华史延茂闫静辉吴萌
Owner 济宁高新科达科技项目服务有限公司
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