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Preparation method of diazepam-resistant single-chain antibody, diazepam-resistant single-chain antibody product and diazepam detection method

A detection method, the technology of diazepam, which is applied in the field of biological detection, can solve the problems that limit the wide application of ELISA technology and is rare, and achieve the effects of good consistency, good accuracy and precision, and good specificity

Active Publication Date: 2021-02-19
INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulty in preparing antibodies to some psychotropic drugs, there are few studies on monoclonal and polyclonal antibodies against DZP, which limits the wide application of ELISA technology to a certain extent.

Method used

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  • Preparation method of diazepam-resistant single-chain antibody, diazepam-resistant single-chain antibody product and diazepam detection method
  • Preparation method of diazepam-resistant single-chain antibody, diazepam-resistant single-chain antibody product and diazepam detection method
  • Preparation method of diazepam-resistant single-chain antibody, diazepam-resistant single-chain antibody product and diazepam detection method

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Experimental program
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preparation example Construction

[0042] The embodiment of the present invention proposes a method for preparing an anti-diazepam single-chain antibody, which at least includes the following steps:

[0043] S1. Construction of scFv gene fragments: design and artificially synthesize the nucleotide sequences of the VH gene fragment and VL gene fragment of the anti-diazepam antibody. The nucleotide sequence of the VH gene fragment is shown in SEQ ID NO: 1, and the VL gene The nucleotide sequence of the fragment is shown in SEQ ID NO: 2, and the VH gene fragment is connected with the VL gene fragment to obtain the scFv gene fragment;

[0044] S2. Expression of scFv-Fc antibody: The scFv gene fragment was connected into the pFUSE_hIgG1e5_Fc2 plasmid (purchased from Invitrogen, USA) containing human IgG1 Fc, and the pFUSE_hIgG1e5_Fc2-scFv recombinant plasmid was constructed, and the expression cells were transfected, and the scFv-Fc antibody was expressed in fusion ;

[0045] S3. Purification: the supernatant of th...

Embodiment 1

[0085] Example 1. Construction, expression and activity detection of diazepam scFv-Fc antibody

[0086] Step 1. Construction of scFv

[0087] (1) Design of scFv antibody primers and amplification of VH and VL gene fragments

[0088] Design the nucleotide sequences of the VH gene fragment and the VL gene fragment of the anti-diazepam antibody, and carry out artificial synthesis, the nucleotide sequence of the VH gene fragment is shown in SEQ ID NO: 1, the nucleotide sequence of the VL gene fragment As shown in SEQ ID NO:2;

[0089] The designed upstream and downstream primers for the light chain and heavy chain are listed in Table 1. The primers were synthesized by Huada Genomics, and the synthesized primers were in powder form, and the primers were synthesized with ddH 2 O Dissolve each primer to 100 nM and then dilute it to 10 nM for PCR amplification.

[0090] Using the sequence of the artificially synthesized VH gene fragment and VL gene fragment as a template, amplify ...

Embodiment 2

[0131] Example 2 Identification of Competitive Activity of Anti-Diazepam scFv-Fc Antibody

[0132] (1) Indirect competitive enzyme-linked immunosorbent assay (ELISA)

[0133] The competition activity of the purified scFv-Fc antibody was identified by indirect competition ELISA. The optimal concentrations of the coating source and purified antibody were determined in advance by checkerboard titration.

[0134] The initial selection of coating concentration and purified antibody dilution was performed using the checkerboard method. Using DZP-OVA as the coating source, the coating source and antibody were serially diluted for binding ELISA experiments, the absorbance value at 450nm was measured, and the dilution gradient with better linear relationship was selected as the initial concentration. The specific operation is as follows:

[0135] 1. Coating: Dilute DZP-OVA coating material with CBS buffer to a concentration of 2 μg / mL, 100 μL per well, coat in a polystyrene microwel...

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Abstract

The invention relates to the field of biological detection, in particular to a preparation method of a diazepam-resistant single-chain antibody, a diazepam-resistant single-chain antibody product anda diazepam detection method. The preparation method disclosed by the invention comprises the following steps: constructing an scFv gene segment, and expressing and purifying an scFv-Fc antibody. Compared with a parent diazepam monoclonal antibody, for the diazepam-resistant single-chain antibody prepared by the invention, the sensitivity of is obviously improved, and the diazepam-resistant single-chain antibody has better specificity.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a preparation method and product of an anti-diazepam single-chain antibody and a method for detecting diazepam. Background technique [0002] Diazepam (Diazepam, DZP), also known as stability, belongs to benzodiazepines. It is commonly used clinically for the treatment of mental illnesses such as insomnia, convulsions, and anxiety. It is usually safe and effective for short-term use in small doses, but it can be inhibited by taking large doses. Central nervous system, inhibit breathing, cause poisoning, and long-term intake can also form dependence or addiction. Although my country has explicitly banned the use of DZP and other benzodiazepines in feed and animal drinking water, the abuse of DZP is still serious due to economic interests, its high-efficiency drug effects and other reasons. In order to pursue high profits, some companies illegally add DZP to animal drinking wat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K1/22C12N15/13C12N15/85G01N33/543G01N33/53
CPCC07K16/44C12N15/85G01N33/9486G01N33/948G01N33/54393C07K2317/622C07K2317/52C07K2317/64C07K2317/33C12N2800/107
Inventor 徐泽华孙铁强韩振宇刘肖张樱樱范龙兴白家磊宁保安
Owner INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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