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Preparation and application of anthraquinone artificial hapten, immunogen, artificial coating antigen and antibody

An artificial hapten and hapten technology, applied in the preparation of organic compounds, immunoglobulin, carboxylic acid amide preparation, etc., to achieve the effect of simple and fast operation, high similarity and good detection effect

Pending Publication Date: 2021-11-30
赵颖
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is the design and synthesis of anthraquinone hapten, the preparation of anthraquinone artificial antigen and antibody

Method used

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  • Preparation and application of anthraquinone artificial hapten, immunogen, artificial coating antigen and antibody
  • Preparation and application of anthraquinone artificial hapten, immunogen, artificial coating antigen and antibody
  • Preparation and application of anthraquinone artificial hapten, immunogen, artificial coating antigen and antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] (1) Synthesis of anthraquinone hapten

[0057] Weigh 2.23g (0.01mol) of 2-aminoanthraquinone into a 100ml three-neck flask, add 20ml of glacial acetic acid, heat up to 65°C and stir to dissolve, then weigh 1.1g (0.011mol) of succinic anhydride and dissolve it in 3mL of glacial acetic acid In the three-necked flask, it was added dropwise to keep warm for 6 hours. Cool down to 5-10°C, filter with suction, dry the obtained solid, and recrystallize with 1,4-dioxane. After recrystallization, a yellow solid was precipitated, and after filtration and drying, 1.62 g of a yellow solid was obtained, with a yield of 50.1%. 1 H NMR (500MHz, DMSO-d6), δ (ppm): 12.22 (s, 1H), 10.62 (s, 1H), 8.41 (d, J = 2.15, 1H), 8.17-8.14 (m, 2H), 8.12 (d, J=8.5,1H),8.03(dd,J=2.2,2.2,1H),7.92-7.86(m,2H),2.67(t,J=6.1,6.45,2H),2.59(t,J =6.45,6.5,2H).

[0058] (2) Preparation of anthraquinone immunogen and coating original

[0059] Dissolve 0.01mmoL anthraquinone hapten in 1mL DMF, add 0.2mL DMF ...

Embodiment 2

[0065] The anthraquinone monoclonal antibody obtained in Example 1 was used for immunodetection.

[0066] (1) Coating: Dissolve anthraquinone-OVA in 0.05mol / L carbonic acid buffer (pH 9.6), coat 100μl / well on a microplate, and coat overnight at 4°C in a refrigerated environment. After taking it out, use 0.01M Wash 3 times with PBST.

[0067] (2) Sealing: After the coating is completed, prepare 2% skimmed milk powder with 0.01mol / L PBS (pH 7.4), seal the microplate with 300μl / well, and seal it in a 37°C incubator for 30 minutes. After taking it out, use 0.01mol / L Wash 2 times with L PBST.

[0068] (3) Competitive reaction: dissolve the sample to be tested in 0.01mol / L PBS solution of 30% methanol, add 50 μl / well to the microtiter plate, then add 50 μl / well of anthraquinone antibody solution, and react in a 37°C incubator for 1 hour , wash the plate 3 times after taking it out.

[0069] (4) Enzyme-labeled secondary antibody reaction: Dilute rabbit anti-mouse enzyme-labeled se...

Embodiment 3

[0074] Cross-reactivity assay was performed using the anthraquinone antibody obtained in Example 1.

[0075] Use the checkerboard test method to determine that the coating concentration of the coating source is 5 μg / L, the coating volume is 100 μL per well, the antibody reaction concentration is 1 μg / L, and the dilution factor of the rabbit anti-mouse enzyme-labeled secondary antibody is 1:40000. Antibody specificity was determined below. According to the degree of cross-reaction between antibody and anthraquinone compound, to inhibit antibody 50% (IC 50 ) Concentration of desired target analyte and approximate target analyte IC 50 Expressed as a percentage of the concentration ratio, that is, the cross-reactivity rate C.R (%). C.R(%)=S=Y / Z×100%, Y: IC 50 Concentration of target analyte at time, Z: IC 50 approximate the concentration of the target analyte. The smaller the cross-reaction rate, the higher the specificity of the antibody; the larger the cross-reaction rate, ...

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Abstract

The invention discloses design and synthesis of a hapten of an environmental pollutant anthraquinone, and preparation and application of an anthraquinone artificial antigen and an antibody. The artificial antigen with immunogenicity is prepared through the steps of anthraquinone hapten design, connecting arm introduction, carrier introduction, synthesis, purification and the like, and an anthraquinone hapten product modified by aminobutyric acid is obtained. The anthraquinone hapten with the structure is respectively coupled with bovine serum albumin and chicken ovalbumin to synthesize an immunogen and a coating antigen, and tests show that the artificial hapten has high immunocompetence, can stimulate an animal immune system to generate an anti-anthraquinone antibody, and fills the gap that no appropriate artificial antigen for preparing a specific and high-sensitivity antibody exists in anthraquinone at present. An anthraquinone monoclonal hybridoma cell strain is obtained through screening, and an anthraquinone monoclonal antibody is obtained through preparation. An enzyme-linked immunosorbent assay method established by adopting the monoclonal antibody has good specificity and sensitivity.

Description

technical field [0001] The invention belongs to the field of immune analysis and detection of small molecular compounds, and specifically relates to technologies such as organic synthesis, immunochemistry, biochemistry, and physical and chemical determination. In particular, it involves the design and synthesis of haptens and artificial antigens of environmental pollutants anthraquinone small molecule compounds with aminobutyric acid molecular structure, mouse immunization, hybridoma cell screening, preparation of specific monoclonal antibodies and establishment of immunoassay methods. Background technique [0002] Anthraquinone compounds are intermediates of advanced dyes in organic synthesis, such as indanthrene vat dyes, acid dyes, and important raw materials for some reactive dyes. Used as cooking agent for papermaking pulping and hydrogen peroxide raw material, etc., it can reduce the amount of alkali used, shorten the cooking time, increase the yield of pulp, and reduc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C233/33C07C231/02C07C231/24C07K14/77C07K14/795C07K16/44G01N33/53G01N33/577
CPCC07C233/33C07C231/02C07C231/24C07K14/795C07K14/77C07K16/44G01N33/577G01N33/5308C07C2603/24C07K2317/33
Inventor 赵颖
Owner 赵颖
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