Immunoassay kit and special antibody for detecting fumonisins

A fumonisin and single-chain antibody technology, which is applied in biological testing, immunoglobulin, and the use of carriers to introduce foreign genetic materials, can solve the problems of long time consumption, complicated production process, and high cost, and achieve cost reduction and easy production Effect

Inactive Publication Date: 2011-01-26
北京维德维康生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of monoclonal antibodies or polyclonal antibodies must be obtained through cell cultu

Method used

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  • Immunoassay kit and special antibody for detecting fumonisins
  • Immunoassay kit and special antibody for detecting fumonisins
  • Immunoassay kit and special antibody for detecting fumonisins

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Embodiment 1, preparation and function test of antibody

[0060] 1. Preparation of fumonisin single-chain antibody

[0061] (1) Antibody screening

[0062] Six-month-old male Balb / c mice were taken, and the total RNA of splenocytes was extracted by Trizol one-step method, purified to obtain mRNA, and then reverse-transcribed to obtain cDNA. Using a complete set of primers, the heavy chain variable region (VH) and light chain variable region (VL) genes were respectively amplified using cDNA as a template. Splicing into a single-chain antibody gene (ScFv), then linking the ScFv with the vector pCANTAB5E to obtain pCANTAB5E / ScFv, and transforming Escherichia coli TG1 to obtain a mouse-derived non-immune single-chain antibody library. Phage particles with specific anti-fumonin single-chain antibody were screened by ELISA method, and the nucleotide sequence of the antibody was obtained by sequencing.

[0063] The coding gene sequence of the single-chain antibody is shown ...

Embodiment 2

[0093] Embodiment 2, chemiluminescent immunoassay kit and its preparation and application

[0094] 1. The chemiluminescence immunoassay kit consists of the following materials:

[0095] 1. A microtiter plate coated with a fumarin hapten and carrier protein conjugate;

[0096] 2. Fumonisin single-chain antibody: the antibody described in Example 1. The concentration of the antibody working solution is 5 ng / mL, and the antibody working solution is obtained by diluting the purified antibody in Example 1 with the sample diluent;

[0097] 3. Fumonisin standard substance: the concentration of the standard solution is 0ng / L, 50ng / L, 150ng / L, 450ng / L, 1350ng / L and 4050ng / L respectively; the standard substance of fumonisin is Fumonisin B1 (Fumonisin B1), purchased from Sigma-Aldrich Company, the product catalog number is 32936; Dilute to the above-mentioned concentrations with sample diluent;

[0098] 4. Enzyme-labeled secondary antibody: horseradish peroxidase (HRP)-labeled mouse a...

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Abstract

The invention discloses an immunoassay kit and a special antibody for detecting fumonisins. A single-chain antibody is prepared by sequentially connecting a heavy chain variable region, short peptide connecting the heavy chain variable region and a light chain variable region, and the light chain variable region, wherein the amino acid sequence of the heavy chain variable region is represented by 1st to 118th amino acid residues of a sequence 1 from an N end; and the amino acid sequence of the light chain variable region is represented by 134th to 246th amino acid residues of the sequence 1 from the N end. The affinity constant of the antibody is 1.73*10<9>L/ mol, and the half abatement (IC50) is 0.15 ng/mL. In the invention, antibody sources with high valence and high specificity are provided for establishing a method for detecting fumonisins residues in food. The kit has the advantages of high sensitivity, high accuracy, high precision, high specificity for fumonisins B1 and low cost. Therefore, the antibody, the kit and the detection method play an important role in detecting the fumonisins B1.

Description

technical field [0001] The invention relates to an immune kit for detecting fumonisin and a special antibody thereof. Background technique [0002] Fumonisins (FB) are a group of structurally related mycotoxins mainly produced by Fusarium moniliforme. Fumonin mostly exists in corn and corn products, and its content generally exceeds 1mg / kg. There are also low concentrations of fumonin in rice, noodles, condiments, sorghum, beer, and asparagus. The chemical composition of fumonin The structure is very similar to sphingosine, which can specifically interfere with the metabolism of sphingosine in the body, and then can cause diseases such as equine leukomalacia, pig pulmonary edema, and sheep nephropathy. Some experiments have shown that there is a parallel relationship between the content of fumarin in corn and the incidence of human esophageal cancer. The International Agency for Research on Cancer (IARC) classifies fumonin into Group 2B, which is a possible human carcinoge...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10G01N33/53G01N33/577
Inventor 沈建忠江海洋吴小平李杰超王战辉徐飞刘丹杨光李娜杨丽丽王富伟王进
Owner 北京维德维康生物技术有限公司
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