Method for carrying out enzyme-linked immunoadsorption detection on integral zebra fish and application thereof

Abstract

The invention relates to a method for carrying out enzyme-linked immunoadsorption detection on an integral zebra fish and application of the method in drugs for identifying genotoxic compounds, screening anti-tumor drugs and repairing DNA damage. On one hand, the invention can be used for evaluating the genotoxicity of the compounds including environmental compounds, drugs, cosmetics, food additives and the like, and on the other hand, the invention can be used for screening new drugs, such as the anti-tumor drugs, antioxidants, anti-aging drugs, anti-neurodegenerative disease drugs and the like. The invention has very important guiding significance on diagnosing and treating certain diseases and novel drug research and development and compound toxicity screening. By applying the zebra fish to evaluate the compound toxicity and screen the novel drugs, the advantages of low cost, low use level of samples, automatic operation and the like can be achieved, and an in vivo physiological environment of a live animal can be provided, therefore, the goals of simple and convenient evaluating and screening works, rapidness, economy, high efficiency and high flux can be achieved.

Description

technical field

[0001] The present invention relates to an enzyme-linked immunosorbent assay, more specifically, to a method for performing enzyme-linked immunosorbent assay on whole zebrafish, and the method is useful in identifying genotoxic compounds, screening anti-tumor drugs, and repairing DNA damage application in medicines. Background technique

[0002] Genotoxicity refers to direct or indirect cellular DNA damage. DNA is an important biomacromolecule that has the function of transmitting genetic information in cells, and is an important research object of biochemistry and molecular biology. The DNA molecule is composed of two antiparallel polynucleotide chains through the principle of base pairing to form a double helix structure, and the genetic information carried by it is determined by the type, quantity and arrangement order of the bases on the polynucleotide chain. The sequence is hidden deep inside the DNA double helix. The double helix structure composed of...

Examples

Embodiment 1

[0172] Example 1 Determination of the optimal developmental stage of zebrafish during compound treatment

[0173] Embryo collection

[0174] In the early morning, 5 pairs of zebrafish were taken, freely combined and divided into 5 groups to mate and hatch embryos, and 1200 zebrafish were collected, the sediment was sucked out, put into the incubation solution, and incubated in a 28°C incubator. Take out the white embryos (dead) in time to prevent the water quality from deteriorating. During this period, the zebrafish obtains nutrients through the yolk, so no other nutrients are needed.

[0175] drug treatment

[0176] The zebrafish were divided into 7 experimental groups, which were 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days after fertilization. Each experimental group was further divided into three groups, blank group, 0.1% dimethyl sulfoxide (DMSO) negative control group and experimental treatment group (50 μM staurosporine, 0.1% dimethyl sulfoxide (DMSO) D...

Embodiment 2

[0184] Example 2 Determination of optimal time length for compound treatment

[0185] Embryo collection

[0186] In the early morning, 5 pairs of zebrafish were taken, freely combined and divided into 5 groups to mate and hatch embryos, and 1200 zebrafish were collected, the sediment was sucked out, put into the incubation solution, and incubated in a 28°C incubator. Take out the white embryos (dead) in time to prevent the water quality from deteriorating. During this period, the zebrafish obtains nutrients through the yolk, so no other nutrients are needed.

[0187] drug treatment

[0188] The zebrafish were divided into five experimental groups, each group including blank group, treatment group (50 μM staurosporine (staurosporine), dissolved in 0.1% dimethyl sulfoxide (DMSO)) and negative control group (0.1% dimethyl sulfoxide (DMSO) sulfoxide (DMSO)). The experimental groups were treated 2 days after zebrafish fertilization, and the treatment time was 6 hours, 12 hours, ...

Embodiment 3

[0196] Example 3 Detection of drug genotoxicity

[0197] Embryo collection

[0198] In the early morning, 10 pairs of zebrafish were taken, freely combined and divided into 10 groups to mate and hatch embryos, 1200 zebrafish were collected, the sediment was sucked out, put into the incubation solution, and incubated in a 28°C incubator. Take out the white embryos (dead) in time to prevent the water quality from deteriorating. During this period, the zebrafish obtains nutrients through the yolk, so no other nutrients are needed.

[0199] drug treatment

[0200] The zebrafish were divided into eight experimental groups, respectively 0.1, 1, 10, 100, 1000 μM genotoxic drug cisplatin (cisplatin), and then dissolved in the incubation solution according to the above concentration; the blank group was no treatment, 0.1% Dimethyl sulfoxide (DMSO) negative control group, 50μM staurosporine (staurosporine) positive control group that can cause genotoxicity. Processing starts at 2dpf ...

technical field

[0001] The present invention relates to an enzyme-linked immunosorbent assay, more specifically, to a method for performing enzyme-linked immunosorbent assay on whole zebrafish, and the method is useful in identifying genotoxic compounds, screening anti-tumor drugs, and repairing DNA damage application in medicines. Background technique

[0002] Genotoxicity refers to direct or indirect cellular DNA damage. DNA is an important biomacromolecule that has the function of transmitting genetic information in cells, and is an important research object of biochemistry and molecular biology. The DNA molecule is composed of two antiparallel polynucleotide chains through the principle of base pairing to form a double helix structure, and the genetic information carried by it is determined by the type, quantity and arrangement order of the bases on the polynucleotide chain. The sequence is hidden deep inside the DNA double helix. The double helix structure composed of...

Examples

Embodiment 1

[0172] Example 1 Determination of the optimal developmental stage of zebrafish during compound treatment

[0173] Embryo collection

[0174] In the early morning, 5 pairs of zebrafish were taken, freely combined and divided into 5 groups to mate and hatch embryos, and 1200 zebrafish were collected, the sediment was sucked out, put into the incubation solution, and incubated in a 28°C incubator. Take out the white embryos (dead) in time to prevent the water quality from deteriorating. During this period, the zebrafish obtains nutrients through the yolk, so no other nutrients are needed.

[0175] drug treatment

[0176] The zebrafish were divided into 7 experimental groups, which were 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days after fertilization. Each experimental group was further divided into three groups, blank group, 0.1% dimethyl sulfoxide (DMSO) negative control group and experimental treatment group (50 μM staurosporine, 0.1% dimethyl sulfoxide (DMSO) D...

Embodiment 2

[0184] Example 2 Determination of optimal time length for compound treatment

[0185] Embryo collection

[0186] In the early morning, 5 pairs of zebrafish were taken, freely combined and divided into 5 groups to mate and hatch embryos, and 1200 zebrafish were collected, the sediment was sucked out, put into the incubation solution, and incubated in a 28°C incubator. Take out the white embryos (dead) in time to prevent the water quality from deteriorating. During this period, the zebrafish obtains nutrients through the yolk, so no other nutrients are needed.

[0187] drug treatment

[0188] The zebrafish were divided into five experimental groups, each group including blank group, treatment group (50 μM staurosporine (staurosporine), dissolved in 0.1% dimethyl sulfoxide (DMSO)) and negative control group (0.1% dimethyl sulfoxide (DMSO) sulfoxide (DMSO)). The experimental groups were treated 2 days after zebrafish fertilization, and the treatment time was 6 hours, 12 hours, ...

Embodiment 3

[0196] Example 3 Detection of drug genotoxicity

[0197] Embryo collection

[0198] In the early morning, 10 pairs of zebrafish were taken, freely combined and divided into 10 groups to mate and hatch embryos, 1200 zebrafish were collected, the sediment was sucked out, put into the incubation solution, and incubated in a 28°C incubator. Take out the white embryos (dead) in time to prevent the water quality from deteriorating. During this period, the zebrafish obtains nutrients through the yolk, so no other nutrients are needed.

[0199] drug treatment

[0200] The zebrafish were divided into eight experimental groups, respectively 0.1, 1, 10, 100, 1000 μM genotoxic drug cisplatin (cisplatin), and then dissolved in the incubation solution according to the above concentration; the blank group was no treatment, 0.1% Dimethyl sulfoxide (DMSO) negative control group, 50μM staurosporine (staurosporine) positive control group that can cause genotoxicity. Processing starts at 2dpf ...

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