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Diazepam monoclonal antibody screening and application

A technology of monoclonal antibody and clonal antibody, which is applied in the direction of instruments, analytical materials, peptides, etc., can solve the problems of cumbersome pretreatment, expensive instruments, and inapplicability to rapid and large-scale detection

Inactive Publication Date: 2015-04-15
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sample pretreatment is cumbersome, the equipment is expensive, the detection cost is high, professional and technical personnel are required to operate, and the operation is time-consuming, the analysis efficiency is low, and the technology is difficult to popularize. Most of them are used for forensic identification of poisoning symptoms and are not suitable for edible animals. The detection of diazepam residues in tissues is also not suitable for on-site rapid and large-scale detection

Method used

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  • Diazepam monoclonal antibody screening and application
  • Diazepam monoclonal antibody screening and application
  • Diazepam monoclonal antibody screening and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0020] Embodiment 1: Identification of complete antigen of diazepam

[0021] Identification by UV Spectroscopy

[0022] Prepare 1mg / mL BSA, OVA and appropriate concentration of coupling products with PBS, and prepare 0.02mg / mL DZP small molecule solution with 10% methanol as solvent. Scan the absorbance value in the wavelength range of 200-400nm, and judge the change of the ultraviolet absorption peak shape before and after coupling. Depend on figure 1 It can be seen from the figure that there are two main changes in the UV absorption spectrum before and after coupling. One is the change of the characteristic absorption peak. Coupling pre-carrier proteins (BSA, OVA) have characteristic absorption peaks at around λ=280nm, and after coupling, the characteristic absorption peaks shift slightly, or the characteristic absorption peaks disappear. The second is the change of the ultraviolet absorption peak shape. It may be due to the fact that some functional groups (benzene ri...

Embodiment approach 2

[0025] Embodiment 2: Detection of diazepam by indirect competitive ELISA

[0026] DZP-OVA was used as the coating source, the concentration of the coating buffer was diluted to 1.25 μg / mL, and the coating volume was 100 μL / well, added to the enzyme-linked plate, refrigerated at 4°C overnight, washed 4 times with PBST, 1% OVA After washing, add 50 μL diazepam monoclonal antibody and serially diluted diazepam standard or test substance at a ratio of 1:16000, incubate at 37°C for 1 hour, add horseradish peroxidase labeling after washing goat anti-mouse IgG, incubated at 37°C for 45 minutes; after washing 5 times, add TMB substrate color development solution, 100 μL / well, develop color at 37°C in the dark for 3-10 minutes, add 50 μL / well stop solution for microplate reader Measure the absorbance of each well at 450nm and 630nm. Depend on image 3 It can be seen that the regression curve of this method is I=28.72641LgC+94.76472, R ２ =0.9555, sensitivity IC50=0.0277μg / mL, linear ...

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Abstract

Belonging to the field of biotechnologies, the invention discloses a preparation method of a diazepam monoclonal antibody, and especially discloses application of the diazepam monoclonal antibody in the rapid detection method of ELISA. The method includes: coupling carboxylation reformed diazepam with carrier protein by an EDC technique, immunizing 5-6-week-old Balb / c mice by intraperitoneal injection, taking spleen from the mouse with positive binding activity and competitive activity to conduct cell fusion and carrying out screening to prepare the monoclonal antibody, and optimizing reaction conditions, determining the concentration of optimal coating antigen DZP-OVA at 1.25 microgram / mL and the optimal antibody dilution factor at 1:16000, thus establishing the ELISA method for the rapid detection of diazepam. The lowest detection limit IC10 of the method is 0.0011 microgram / mL. The invention provides the basic reagent antibody for immunoassay of diazepam and the detection method.

Description

technical field [0001] The invention relates to a preparation method of a diazepam monoclonal antibody, in particular to the application of a diazepam monoclonal antibody in a rapid detection ELISA method, and belongs to the field of biotechnology. Background technique [0002] Diazepam (Diazepam, DZP), alias stability or benzodiazepine, chemical name 7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepam Azepine-2-one, molecular formula C 16 h 13 C1N 2 O, molecular weight 284.76, DZP is white or off-white crystalline powder, melting point 125-126 ℃, odorless, bitter taste; easily soluble in chloroform, acetone, ethanol, almost insoluble in water. DZP belongs to the long-acting benzodiazepines (see Figure 1-2 ) anti-anxiety drugs have anti-anxiety, sedative, hypnotic, anticonvulsant, antiepileptic, skeletal muscle relaxation and memory elimination effects. Widely used in clinical medicine. Studies have shown that taking a large amount of diazepam is harmful to hum...

Claims

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Application Information

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IPC IPC(8): C07K16/44G01N33/577
Inventor 高志贤宁保安彭媛白家磊李桂敏孙思明
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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