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Aromatic hydrocarbon receptor and immune detection method of antibody mediated therewith

An immunodetection method and an aromatic hydrocarbon receptor technology, which can be used in biological testing, material inspection products, etc., and can solve the problems of environmental persistence, inability to degrade easily, and toxic reactions.

Active Publication Date: 2017-07-04
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PAHs are a class of highly inert hydrocarbons that cannot be easily degraded, can exist stably and accumulate in various media, cause persistent harm to the environment, and can pass through respiration, diet (bioaccumulation and food chain) The high fat solubility of PAHs makes it easy to accumulate and enrich in the body, especially in fat, which can lead to adverse or toxic reactions in the body for a long time

Method used

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  • Aromatic hydrocarbon receptor and immune detection method of antibody mediated therewith
  • Aromatic hydrocarbon receptor and immune detection method of antibody mediated therewith
  • Aromatic hydrocarbon receptor and immune detection method of antibody mediated therewith

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Isolation and purification of aryl hydrocarbon receptor (AhR) in carp

[0032] 1. Extraction of AhR-Containing Cytosol

[0033] Take the carp liver domesticated in the water containing Ant, first wash the blood on the surface of the liver with normal saline, then dry the surface water, weigh, and then add HEDG buffer at a mass-volume ratio of 1g:4ml for grinding. Liver tissue homogenate was obtained (all operations were performed on ice). The homogenate was centrifuged at 13000 rmp at 4°C for 1 h, and the supernatant was aspirated, which was the cytosol containing the receptor. It can be stored at -80°C and can be used for further experiments.

[0034] 2. Separation and purification of AhR

[0035] The protein was purified using ion exchange chromatography. The sample was equilibrated with the starting buffer overnight. The sample was carefully added to the surface of the resin along the tube wall with a dropper, and the lower nozzle valve was opened. When the sa...

Embodiment 2

[0039] 1. Drawing of standard curve and detection limit of indirect competitive ELISA method

[0040] Add 100 μL of coated antigen diluted 2000 times to each well of the 96-well ELISA plate, and coat at 4°C for 12 hours overnight. Pour out the liquid in the well, shake and wash with washing solution PBS-T for 3 min, repeat 3 times, pat dry for use; add 200 μL of blocking liquid gel to each well, seal at 37 °C for 60 min, pour out the liquid in the well, repeat the above washing steps, Pat dry until use; dilute primary antibody to 0.275 μg mL -1 , add 50 μL to each well, and dilute Ant with 10% volume of acetone solution to prepare a series of concentrations of 10, 20, 50, 100, 200, 500, 1000, 10000ng / mL, add 50 μL to each well, and set a blank control at the same time. Incubate at °C for 60 min. Repeat the above washing steps, pat dry for use; dilute the enzyme-labeled secondary antibody to 0.1 μg·mL -1 , add 100 μL to each well, incubate at 37°C for 60 min, repeat the abov...

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Abstract

The invention relates to an aromatic hydrocarbon receptor (AhR) and an immune detection method of an antibody mediated therewith. The method includes the steps of: 1) separating and purifying the AhR from livers of carps, and combining the AhR with PAHs to prepare a coated antigen and adsorbing the coated antigen on a solid phase carrier; 2) adding a to-be-tested PAHs sample, the AhR and a corresponding antibody, so that the PAHs-AhR on the solid phase and the to-be-tested PAHs-AhR are subjected to a competitive reaction with the antibody; 3) after the reaction, adding enzyme-labeled secondary antibody, and finally performing detection in a substrate color developing manner; 4) according to standard series of known PAHs and OD value of the to-be-tested sample, calculating inhibition rate, and according to the logarithmic relationship between the inhibition rate and concentration of the PAHs, drawing a graph to obtain a standard curve, and calculating the concentration of the to-be-tested PAHs. The method is used for performing trace-amount PAHs in water body by means of the AhR antibody, is high in detection specificity and simple in operations, is high in sensitivity, is less in usage amount of sample, and can be used for in-situ detection.

Description

technical field [0001] The invention belongs to the field of detecting the interaction between receptors and antibodies, in particular to an immunological detection method for aromatic hydrocarbon receptors and antibodies mediated by them. Background technique [0002] Polycyclic aromatic hydrocarbons (PAHs) are defined as a class of hydrocarbons containing two or more benzene rings, and there are many types. U.S. Environmental Protection Agency (USEPA) puts naphthalene (NAP), acenaphthene (ANA), acenaphthylene (Acenaphthylene, NY), fluorene (Fluorene, FLU), anthracene (Anthracene, ANT), phenanthrene (Phenanthrene, PHE), Fluoranthene (Fluoranthene, FLT), pyrene (Pyrene, PYR), benzo [a] anthracene [Benzo (a) anthracene, BaA], (Chrysene, CHR), benzo (a) pyrene [Benzo (a) pyrene, BaP], benzo (b) fluoranthene [Benzo (b) fluoranthene, BbF], benzo (k) fluoranthene [Benzo (k) )fluoranthene, BKF], benzo (g, hi) perylene [Benzo (g, hi) perylene, BPE], dibenzo (a, h) anthracene [Di...

Claims

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Application Information

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IPC IPC(8): G01N33/53
CPCG01N33/53
Inventor 赵晓祥吕晏锋杨莲张弦白玉杰
Owner DONGHUA UNIV
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