Semicarbazide derivative, monoclonal antibody thereof and application

A monoclonal antibody and semicarbazide technology, applied in the field of immunochemical detection, can solve the problems of lack of rapid detection methods and products of detection SEM, and small molecular weight of SEM, and achieve high sensitivity, high specificity, important economic and social benefits. Effect

Inactive Publication Date: 2007-08-08
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports of SEM enzyme-linked immunosorbent assay (ELISA) at home and abroad. The main reason is that the molecular weight of SEM is very small, and it needs to be derivatized into haptens (such as CPSEM), and then the corresponding high-quality antigens can be obtained by preparing whole antigens and immunization. Antibodies, meanwhile, lack rapid assays and products for the detection of residual SEM in edible animal tissue samples

Method used

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  • Semicarbazide derivative, monoclonal antibody thereof and application
  • Semicarbazide derivative, monoclonal antibody thereof and application
  • Semicarbazide derivative, monoclonal antibody thereof and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, synthesis of artificial whole antigen (immune antigen, coating antigen) and preparation of polyclonal antibody (1) derivation of semicarbazide and synthesis of artificial whole antigen

[0039] The chemical structure and synthetic route of semicarbazide (SEM)-derived artificial immune antigen and coated antigen synthesized with bovine serum albumin (BSA) and ovalbumin (OVA) as carriers are shown in Figure 1. As shown in the figure, semicarbazide After reacting with CBA through an amide bond, its derivative CPSEM is formed, and after identification by mass spectrometry, it is coupled and purified with protein carriers BSA and OVA respectively to obtain its corresponding immune antigen and coating antigen.

[0040] The specific method process is as follows:

[0041] The specific process of the synthesis of SEM derivatives CPSEM is as follows: Take 6g of SEM and 9g of CBA, dissolve them in pH7.4 PBS and react overnight at 4°C, adjust the pH value to 3 with 2M...

Embodiment 2

[0048] Embodiment 2, the preparation of CPSEM monoclonal antibody

[0049] (1) Animal immunity

[0050] Immunize 5 female Balb / C mice, and take another 5 as negative control. Mice were immunized with 50 μg / mouse of CPSEM-BSA for each immunization.

[0051] The immunization program adopts two basic immunizations and three booster immunizations, and the immunization time is as follows: 0d: basic immunization; 15d: basic immunization; 30d: booster immunization; 45d: booster immunization; 60d: booster immunization. The immune antigen was diluted with normal saline, and the antigen emulsified with Freund's complete adjuvant was injected subcutaneously (multiple points) or intraperitoneally on the back of the neck of the mouse for the first basic immunization, and Freund's incomplete adjuvant was used for the second basic immunization. The emulsified antigen was injected subcutaneously (multiple points) or intraperitoneally on the neck and back of the mouse, and the next two boost...

Embodiment 3

[0073] Embodiment 3 Establishment of a kind of ELISA method suitable for SEM detection

[0074] 3.1 Determination of ELISA plate

[0075] Detect the absorbance of 4 kinds of blank enzyme-labeled plates randomly selected from different domestic manufacturers at a wavelength of 450nm, and then directly add enzyme-labeled secondary antibodies directly on the four types of enzyme-labeled plates, 100 μl per well, incubate at 37°C for 1 hour, wash thoroughly and add the bottom Add 100 μl of the substance solution, develop the color in the dark for 10 minutes, add 50 μl of 2mol / L sulfuric acid solution to stop, measure the absorbance at a wavelength of 450nm, and select an ELISA plate with better uniformity. The results showed that the ELISA plate purchased from Shenzhen Jincanhua Company was more suitable for the establishment of the detection method.

[0076] 3.2 Determination of the optimal reaction volume of the antibody

[0077] Set 5 different reaction volume ratios (80μl: 20...

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Abstract

The invention discloses a making method of hapten and holoantigen of semicarbazide derivant, which is characterized by the following: establishing immunity method to detect SEM in the food through ELISA method; testing SEM qualitatively and quantitatively; fitting for SEM agent box (ELISA agent box, colloidal gold test paper and so on) of edible animal tissue.

Description

technical field [0001] The invention belongs to the field of immunochemical detection, and relates to a semicarbazide derivative, its synthesis as a hapten and the preparation method of its whole antigen, as well as the preparation of its antibody (monoclonal antibody, polyclonal antibody), and a method suitable for nitrofurazone residues Immunological method and application of marker semicarbazid (SEM) residue detection. Background technique [0002] Nitrofurazone belongs to nitrofuran drugs and is a broad-spectrum antibacterial drug. Because of its low price and good effect, it is used as a therapeutic drug and feed additive to control diseases in livestock, poultry and aquaculture. Because the pharmacophore in the nitrofurazone molecule—the nitro group, is the main cause of its pharmacological action and toxicity, making nitrofurazone have side effects such as teratogenicity, carcinogenicity and induced mammalian cell chromosome damage (Fu Guo, Li Ningyi, Research progre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C281/14G01N33/577
Inventor 曾令文陈巧林高爱中雷静李想程宇
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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