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Paraquat hapten, complete antigen, nano-antibody, detection test paper, kit, preparation method and application of paraquat hapten, preparation method of nano-antibody

A complete antigen and detection test strip technology, applied in the field of chemical detection, can solve the problems of lack of immunogenicity, affecting the effect of paraquat immunodetection, and inability to activate antibodies of the immune system, achieving easy access, simple steps, and good solubility. Effect

Active Publication Date: 2020-05-29
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, paraquat is a small molecule hapten, which is not immunogenic and cannot activate the immune system to produce antibodies. Only when it is combined with a carrier to form a complete paraquat antigen can it be immunogenic
The combination of paraquat hapten and carrier determines the level of antibody, which in turn affects the effect of paraquat immunoassay

Method used

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  • Paraquat hapten, complete antigen, nano-antibody, detection test paper, kit, preparation method and application of paraquat hapten, preparation method of nano-antibody
  • Paraquat hapten, complete antigen, nano-antibody, detection test paper, kit, preparation method and application of paraquat hapten, preparation method of nano-antibody
  • Paraquat hapten, complete antigen, nano-antibody, detection test paper, kit, preparation method and application of paraquat hapten, preparation method of nano-antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0083] This embodiment provides a paraquat hapten having a structure as shown in formula (I):

[0084]

[0085] This embodiment also provides a method for preparing the above-mentioned paraquat hapten, which includes the following steps: using paraquat represented by formula (a) as a raw material, undergoing carboxylation and oximation reactions to obtain paraquat having a structure represented by formula (I) dry hapten. details as follows:

[0086] (1) Carboxylation reaction

[0087] Weigh 2.5716g (about 0.01mol) of paraquat and dissolve it in 10mL of water, then add 40-90mL of hydrogen peroxide, mix well and continue stirring at room temperature for 1-2h, after the reaction is completed, vacuum rotary evaporation removes water, and the formula (b ) The carboxylation reaction product shown.

[0088] (2) Oximation reaction

[0089] Dissolve the residue with 50-100 mL methyl ethyl ketone, then add 2.19 g (about 0.02 mol) carboxymethyl hydroxylamine hemihydrochloride, and...

Embodiment 2

[0096] This example provides a paraquat complete antigen, which has a structure as shown in formula (II):

[0097]

[0098] Wherein, the carrier protein is bovine serum albumin (BSA), and the chemical formula of the paraquat complete antigen is PQ-BSA.

[0099] This embodiment also provides a method for preparing the above paraquat complete antigen, including the following steps:

[0100] Prepare paraquat complete antigen by carbodiimide method, firstly prepare liquid A and liquid B, liquid A: take purified paraquat hapten 7mg, EDC-HCl 3.5mg, NHS 4.4mg, add to 500μL DMF, at room temperature Avoid light and shake for 1h. Liquid B: Dissolve 20 mg of BSA in 4.5 mL of PBS buffer containing 20 (v / v)% DMF, and pre-cool at 4°C.

[0101] Add solution A to solution B dropwise while stirring, and react at 4°C for 4 hours. After the reaction, the reaction solution was firstly dialyzed with PBS buffer containing 20% ​​DMF, and the content of DMF was gradually reduced, and finally di...

Embodiment 3

[0105] This example provides a paraquat-specific antibody. The paraquat-specific antibody is a paraquat nanobody obtained by immunizing alpaca with the paraquat complete antigen PQ-BSA in Example 2. The preparation steps of the paraquat nanobody include:

[0106] 1. Immunization of camelids

[0107] Healthy 18-month-old alpacas were selected, and PQ-BSA was used as the immune antigen. The first basic immunization was fully emulsified with Freund's complete adjuvant, and after the booster immunization, it was emulsified with incomplete adjuvant. The injection method adopts subcutaneous multi-point injection, and the immunization dose of each alpaca is 100 μg each time. One week before immunization, blood was collected from the jugular vein as a negative control. Seven days after the fourth immunization, blood was collected from the jugular vein to prepare serum, and the serum titer was determined by direct ELISA method with the collected serum. When the test result was positi...

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Abstract

The invention discloses a paraquat hapten. The paraquat hapten has a structure represented by formula (I) shown in the description. A group with a high electron cloud density is introduced into the paraquat hapten, and after a carrier protein is coupled, the paraquat hapten can be fully exposed to obtain a paraquat complete antigen with strong immunogenicity. The invention discloses a paraquat specific antibody based on the paraquat complete antigen. The paraquat specific antibody has the advantages of high titer, strong specificity and high sensitivity. The paraquat detection test paper comprises a chromatographic membrane and a colloidal gold module, and the colloidal gold module comprises a colloidal gold labeled paraquat specific antibody; and a detection line and a quality control line are sequentially arranged on the chromatographic membrane along the liquid chromatography direction, the detection line is coated with the paraquat complete antigen, and the quality control line iscoated with a binding antibody of the paraquat specific antibody. The paraquat detection test paper can realize sensitive and rapid detection of paraquat residues, is simple in sample pretreatment, and is suitable for large-scale detection.

Description

technical field [0001] The invention relates to the technical field of chemical detection, in particular to a paraquat hapten, a paraquat complete antigen, a paraquat-specific antibody, a paraquat nanobody, a paraquat detection test paper, a paraquat detection kit, a preparation method, and an application. Background technique [0002] Paraquat (PQ), also known as Paraquat and Gramoxone, has a chemical composition of 1,1'-dimethyl-4,4'bipyridyl dichloride and is a quick-acting contact herbicide. It is highly efficient, economical, and has little environmental pollution and is widely used. At present, paraquat has been used in more than 130 countries around the world, and its consumption is second only to the herbicide glyphosate. my country is the largest producer and user of this herbicide. However, paraquat is extremely toxic to human organs, and there is no specific antidote available, and the mortality rate of oral poisoning can reach more than 99%. Because paraquat ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D213/20C07D213/22C07K14/795C07K14/77C07K14/765C07K14/47C07K16/44C12N15/70G01N33/558G01N33/543
CPCC07D213/20C07D213/22C07K14/765C07K14/77C07K14/795C07K14/47C07K16/44C12N15/70G01N33/558G01N33/54313G01N33/184
Inventor 何庆华任萍萍欧阳子程黄林丽魏焘王梓莹冯婉滢蒲一涛游新奎伍力刘兆金
Owner SHENZHEN UNIV
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