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A double-antibody sandwich ELISA method for detecting transgenic plant PMI and a kit

A double-antibody sandwich and antibody detection technology, which is applied in biological testing, measuring devices, material inspection products, etc., to achieve reliable detection data, easy operation, and accurate results

Inactive Publication Date: 2016-03-02
BEIJING PROTEIN INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no PMI protein detection kit for grains with PMI as a genetically modified label, especially food crops and food products

Method used

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  • A double-antibody sandwich ELISA method for detecting transgenic plant PMI and a kit
  • A double-antibody sandwich ELISA method for detecting transgenic plant PMI and a kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 plant protein extraction

[0022] Weigh 20-200 mg of rice roots, stems, leaves and dry seeds at the seedling stage, grind them into powder after cooling in liquid nitrogen, add 1 mL of 0.01mol / L, pH7.4 phosphate buffer solution to lyse and extract protein, and place on ice for 30 minutes , centrifuged at 4 degrees for 15 min, and the supernatant was taken to obtain the protein extract to be detected. Total protein was quantified with the ThermoScientific BCA Kit.

Embodiment 2

[0023] The assembly of embodiment 2ELISA kit

[0024] In the assembly of the double antibody sandwich kit, the 60728-35 monoclonal antibody was used as the coating antibody, and the coating buffer (Na 2 CO 3 1.5gNaHCO 3 2.9g, add 1000ml of double distilled water) to dilute the antibody to 1-10μg / mL, the typical concentration is 2μg / mL, add 100μL of coated antibody to each well of a 96-well microtiter plate, and discard it after coating for 12 hours at 4°C Coating antibody. After each well was washed 3 times with 200 μL of PBS (pH 7.4) buffer solution, 300 μL of 2% bovine serum albumin was added to each well as a blocking solution, incubated at 37°C for 3 hours, washed 3 times with PBS, and dried at 25°C Box dry for 12 hours. Put the dried microplate plate into a sealable plastic bag for vacuuming, add a desiccant, and press and seal it for later use. The enzyme-labeled detection antibody in the kit is 60728-44, which is labeled with horseradish peroxidase or biotin. When ...

Embodiment 3E

[0025] Embodiment 3ELISA detects

[0026] Add the protein extract solution to be tested (with the volume ratio of the sample extract to be tested and the PBS buffer as the dilution factor) and the PMI recombinant protein standard (5 μg / mL per hole, to 1:3 serial dilution to 7ng / mL, each sample was repeated three times), 100μL / well, incubated at 37°C for 1h. The PMI protein that may exist in the sample was specifically bound to the antibody to form a conjugate; after washing with PBS, 100 μL / well of HRP-labeled detection antibody was added and incubated at 37°C for 1 h. Make it form a complex with the PMI protein captured by the coated antibody; after washing with PBS, add the chromogenic reagent TMB solution, 100 μL per well, incubate at room temperature for 3 minutes, add 2M H 2 SO 4 , 50 μL per well to terminate the reaction. read OD 450 Value, to determine the presence and concentration of PMI protein in the sample. Judgment of the result: take the logarithm of the con...

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Abstract

The invention relates to a preparing method of a double-antibody sandwich enzyme linked immunosorbent assay (ELISA) kit detecting the content of phosphomannose isomerase (PMI) in transgenic plant and uses of the kit. The objective of the invention is to provide a detecting method for detecting whether a non-antibiotic selection marker that is the PMI exits in transgenic plant (corn, rice, cotton, and the like) or not and for quantifying the content of the PMI in the plant. A monoclonal antibody used in the detecting method is specifically described in the patent for invention 201410387508.1. ELISA detection on transgenic rice materials shows that the detecting method is high in sensitivity, high in specificity, simple and convenient in operation, rapid in qualitative detection and accurate in quantification, the lowest detectable limit for the PMI is 1.18 ng / mL, the linear detection range is 21-560 ng / mL, and the detecting method can be used for detection of transgenic materials.

Description

technical field [0001] The invention relates to a double-antibody sandwich ELISA method and a kit for detecting transgenic PMI protein in natural plants. Background technique [0002] With the continuous emergence of genetically modified products, the following problem is the safety evaluation and determination of genetically modified products. In the cultivation and later identification of transgenic varieties, selection markers and reporter genes for transformation are inseparable. The most commonly used selection markers include antibiotic resistance selection marker genes and herbicide resistance genes. Such resistance selection marker genes may be Gene pollution will be caused by gene drift, which will have adverse effects on the environment (Wei Wei, Ma Keping. How to face gene flow and gene pollution [J]. China Agricultural Science and Technology Herald, 2002, 4(4): 10-15.). In addition, the genetically modified food containing this marker gene may not be absorbed an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
Inventor 尹长城武鹏程郝育杰刘国振吴琳刘斯奇
Owner BEIJING PROTEIN INNOVATION
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