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Method and special kit for detecting content of avian gamma-interferon (IFN-gamma)

A content and auxiliary detection technology, applied in the method and its special kit to detect the content of poultry γ-interferon, can solve the problem that the actual level of chicken IFN-γ protein cannot be reflected, the fluorescence quantitative PCR operation is cumbersome, and the professional skills are required to be high. problems, to achieve the effect of fast detection method, good specificity and affinity, and low instrument requirements

Inactive Publication Date: 2017-10-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, the main experimental technique used to detect changes in chicken IFN-γ is fluorescent quantitative PCR. Fluorescent quantitative detection is the change of mRNA level. PCR cannot reflect the actual level of chicken IFN-γ protein, so it cannot be used as an experimental technique to detect the amount of chicken IFN-γ protein. At the same time, fluorescent quantitative PCR technology is cumbersome to operate, requires high professional skills and is expensive , not suitable for actual production

Method used

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  • Method and special kit for detecting content of avian gamma-interferon (IFN-gamma)
  • Method and special kit for detecting content of avian gamma-interferon (IFN-gamma)
  • Method and special kit for detecting content of avian gamma-interferon (IFN-gamma)

Examples

Experimental program
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Effect test

preparation example Construction

[0067] The buffer solution in the following examples is prepared as follows:

[0068] 1. 0.05M glycine-hydrochloric acid buffer (pH 2.8)

[0069] Solution A: Weigh 1.5g of glycine and dissolve it in 100mL of distilled water to prepare 0.2M glycine as mother liquor for later use. Liquid B: Measure 1.65mL of concentrated hydrochloric acid, dilute to 100mL with distilled water, and prepare 0.2M hydrochloric acid as mother liquor for later use. Take 25mL of solution A and 8.4mL of solution B, add distilled water to make up to 100mL.

[0070] 2. 0.05M citric acid-sodium citrate buffer (pH 3.0-5.0)

[0071] Solution A: Weigh 10.5g of citric acid and dissolve it in 500ml of distilled water to prepare 0.05M citric acid as mother liquor for later use. Solution B: Weigh 14.7g of sodium citrate and dissolve it in 500ml of distilled water to prepare 0.05M sodium citrate as mother liquor for later use. Mix 93ml of solution A and 7ml of solution B to obtain a 0.05M citric acid-sodium ci...

Embodiment 1

[0083] Example 1. Establishment of an enzyme-linked immunoassay kit for detecting poultry gamma-interferon (IFN-γ) and a double-antibody sandwich ELISA detection method and optimization of conditions

[0084] 1. Preparation of an enzyme-linked immunoassay kit for detecting poultry gamma-interferon (IFN-γ)

[0085] The enzyme-linked immunoassay kit for detecting poultry gamma-interferon (IFN-γ) of the present invention comprises polystyrene enzyme-labeled reaction plate, chIFN-γ standard product (recombinant protein His-chIFN-γ, sandwich protein), Anti-chIFN-γ monoclonal antibody LYN1 (coating antibody, the present invention selects the antibody secreted by the LYN1 hybridoma cell line as the coating antibody), anti-chIFN-γ monoclonal antibody LYN2 (detection antibody, the present invention selects the LYN2 hybridoma cell line Secreted antibody as detection antibody), 0.05M citric acid-sodium citrate buffer (pH5.0, coating solution), PBST (pH7.4, washing buffer), 5% skimmed mil...

Embodiment 2

[0151] Example 2. Sensitivity detection of the enzyme-linked immunoassay kit for detecting poultry gamma-interferon

[0152] According to the optimal conditions of the double-antibody sandwich ELISA detection method determined in Implementation 1, the sandwich protein (Recombinant protein His-chIFN-γ) was diluted to different concentrations and then ELISA test was performed, and the concentration of the sandwich protein was taken as the abscissa, and the OD 450 Create a standard curve for the ordinate. Specific steps are as follows:

[0153] 1. Dilute the coating antibody anti-chIFN-γ monoclonal antibody LYN1 to 4 μg / mL with 0.05M citric acid-sodium citrate buffer (pH5.0), 100 μL / well, after coating at 4°C for 12 hours, wash buffer Wash 6 times, 300 μL / well, and dry the residual liquid in the well;

[0154] 2. 5% skimmed milk, 300 μL / well, after blocking for 12 hours at 4°C, wash as above;

[0155] 3. Dilute the sandwich protein (recombinant protein His-chIFN-γ) to differe...

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Abstract

The invention discloses a method and a special kit for detecting content of avian gamma-interferon (IFN-gamma). A double-sandwich ELISA detection method of chIFN-gamma on the protein level is established by application of a chIFN-gamma monoclonal antibody. Experiments prove that the anti-chlFN-gamma monoclonal antibody has good specificity and affinity and can accurately reflect the content of chIFN-gamma in serum or cell supernatant; besides, the detection method is quick, efficient and accurate, and problems of being cumbersome, time-consuming and labor-consuming to operate, susceptible to operation and other external conditions and the like of a fluorescence quantitative PCR (polymerase chain reaction) detection method in the prior art are solved; evaluation for the dynamic change level of chIFN-gamma in vivo is facilitated, quite good reference is provided for prevention and treatment of diseases, and occurrence, development and regression conditions of avian infectious diseases and the dynamic condition of protective immune responses of organisms are understood.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting poultry gamma-interferon (IFN-gamma) content and a special kit thereof. Background technique [0002] Gamma-interferon (IFN-γ) is produced by activated T lymphocytes and NK cells, belongs to type II interferon, and plays an important role in anti-virus, anti-tumor and immune regulation. IFN-γ belongs to TH1 type cytokines, which can promote cellular immune response. IFN-γ has the function of anti-infection and enhancing the immune effect of vaccines, and has potential value as an immune adjuvant. [0003] At present, there is no kit capable of detecting chicken IFN-γ protein in domestic or foreign markets, but similar kits capable of detecting IFN-γ protein in human or mouse serum or cell supernatant have been widely used. For chicken The research on IFN-γ is still in its infancy. Therefore, the establishment of a detection method for chicken IFN-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N5/20G01N33/68G01N33/577G01N33/532C12R1/91
CPCC07K16/249C07K2317/33G01N33/532G01N33/577G01N33/6866G01N2333/57
Inventor 郑世军柳亚楠王永强李晓齐曹红
Owner CHINA AGRI UNIV
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