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Preparation method of yolk antibody IgY of high-abundance protein in anti-human serum/blood plasma and application thereof

A technology of egg yolk antibody and anti-human serum, which is applied to the preparation method of peptides, anti-animal/human immunoglobulin, chemical instruments and methods, etc., can solve the complex steps of antibody preparation, non-specific antibody binding, the influence of mixed signals, etc. problem, to achieve the effect of low feeding cost, strong specificity and low cost

Active Publication Date: 2013-03-06
XIAN GOLDMAG NANOBIOTECH
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AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a preparation method and application thereof for anti-human serum / plasma high-abundance protein antibody, which solves the problem of using rabbits, rats and other mammals as the anti-serum / plasma high-abundance protein antibody in the prior art. The use of mammalian serum as the source of antibody extraction leads to complex antibody preparation steps, low yield, high cost, non-specific binding of antibodies, low titer and instability; and solves the problem of mammalian-derived antibodies in the protein Separation, non-specific separation problems caused by extraction
Solve the problem of heterogeneous signals caused by non-specific adsorption of mammalian antibodies in immunological detection

Method used

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  • Preparation method of yolk antibody IgY of high-abundance protein in anti-human serum/blood plasma and application thereof

Examples

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Effect test

Embodiment 1

[0026] Embodiment 1 is the IgY preparation process of anti-transferrin chicken:

[0027] 1. Detect the eggs of hens immunized with transferrin to obtain eggs containing high-titer antibodies. The specific steps are:

[0028] (1) Use the indirect ELISA method to detect the titer of the immune serum, and use a microplate reader to detect the light absorption value at 450 nm. The lowest dilution of the serum when the light absorption value of the positive serum at 450 nm is greater than 2.1 times the average value of the negative control is the ELISA titer.

[0029] (2) Indirect ELISA was used to detect the titer of the immune serum. After the titer of the ELISA reached 1:10000, the eggs were collected and stored at 4°C.

[0030] 2. Separation and extraction of high-abundance egg yolk antibody IgY from high-titer antibody eggs: dissolving the yolk liquid of high-titer antibody eggs in acidic water, freezing and thawing, filtering, ammonium sulfate precipitation and DEAE-fast-flo...

Embodiment 2

[0035] Embodiment 2 is the IgY preparation process of anti-albumin chicken:

[0036] 1. To detect the titer of the hen's serum immunized with albumin, and obtain eggs containing high-titer antibodies. The specific steps are:

[0037] (1) Use the indirect ELISA method to detect the titer of the immune serum, and use a microplate reader to detect the light absorption value at 450 nm. The lowest dilution of the serum when the light absorption value of the positive serum at 450 nm is greater than 2.1 times the average value of the negative control is the ELISA titer.

[0038] (2) Indirect ELISA was used to detect the titer of the immune serum. After the titer of the ELISA reached 1:10000, the eggs were collected and stored at 4°C.

[0039] 2. Separation and extraction of high-abundance egg yolk antibody IgY from high-titer antibody eggs: dissolving the yolk liquid of the high-titer antibody eggs in acidic water, freezing and thawing, filtering, ammonium sulfate precipitation and ...

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Abstract

The invention relates to a preparation method of a yolk antibody IgY of high-abundance protein in anti-human serum / blood plasma and application thereof; and eggs of a hen immunized by high-abundance protein are used as a source for extracting antibody. The preparation method comprises the steps of: detecting the serum potency of the immunized hen through an indirect ELISA method, obtaining an egg containing antibody with high potency after the potency meets the standard; acid hydrolyzing, freeze thawing and filtering the egg yolk liquid, and depositing by using ammonium sulphate; and further purifying through DEAE ion exchange layer chromatograph to obtain the yolk antibody IgY. The high-abundance protein of human serum / blood plasma is a high-abundance protein with albumin, IgG, transferrin, C3 alexin, IgA, IgM, trypsin, globin, lipoprotein, F factor, C9 alexin, C4 alexin, ceruloplasmin, apolipoprotein B, apolipoprotein A-1, alpha-1-acid glycoprotein, H factor, alexin factor B, prealbumin and C1q alexin, and the like, and the concentration of which are higher than 10 mug / ml.

Description

technical field [0001] The invention relates to a preparation method and application of an anti-high-abundance egg yolk antibody IgY in human serum / serum. Background technique [0002] Biological samples such as human serum / plasma contain a variety of low-abundance proteins secreted by tissue cells and shed into the blood, which may be key proteins in signal transduction and regulation. For example, during necrosis, apoptosis or hemolysis, some intracellular proteins are released into the blood. Carrying out serum proteomics research on diseases and looking for low-abundance proteins will help clarify the pathogenesis, and may discover some marker proteins associated with specific states of the disease, thereby providing new methods for disease diagnosis. However, the presence of some high-abundance proteins, especially albumin and antibodies, seriously affects the identification and detection of low-abundance proteins. [0003] In proteomics research, antibody affinity ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K16/02C07K1/22G01N33/53
Inventor 崔亚丽胡佳陈超
Owner XIAN GOLDMAG NANOBIOTECH
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