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Method for detecting content of avian interleukin 4 and special kit for detecting content of avian interleukin 4

A content-assisted detection technology, applied in the biological field, can solve the problems of cumbersome fluorescent quantitative PCR technology, failure to reflect chIL-4, and high professional skills requirements, and achieve the effects of fast detection methods, improved sensitivity, and cumbersome operations

Inactive Publication Date: 2017-10-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, there are commercial kits for detecting human and mouse IL-4 at the protein level, but since the homology between chIL-4 and human and mouse IL-4 is only 22.48% and 21.23%, respectively, the Domestically, whether in academic research or clinical disease analysis, the relative content of chIL-4 is mainly detected by fluorescent quantitative PCR
However, since the mRNA level does not have an absolute linear relationship with the protein level, the application of fluorescent quantitative PCR cannot reflect the actual level of chIL-4, so it cannot be used as an experimental technique for detecting the amount of chIL-4 protein. Fluorescent quantitative PCR technology is cumbersome to operate, requires high professional skills and is expensive, so it is not suitable for actual production

Method used

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  • Method for detecting content of avian interleukin 4 and special kit for detecting content of avian interleukin 4
  • Method for detecting content of avian interleukin 4 and special kit for detecting content of avian interleukin 4
  • Method for detecting content of avian interleukin 4 and special kit for detecting content of avian interleukin 4

Examples

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preparation example Construction

[0065] The buffer solution in the following examples is prepared as follows:

[0066] 1. 0.05M glycine-hydrochloric acid buffer (pH 2.8)

[0067] 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.

[0068] 2. 0.05M citric acid-sodium citrate buffer (pH 3.0-5.0)

[0069] Liquid A: Weigh 10.5 g of citric acid and dissolve in 500 mL of distilled water to prepare 0.05 M citric acid as the mother liquid 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 ...

Embodiment 1

[0084] Example 1. Establishment of an enzyme-linked immunoassay kit for detecting poultry interleukin 4 (chIL-4) and a double-antibody sandwich ELISA detection method and optimization of conditions

[0085] One, the preparation of the enzyme-linked immunoassay kit for detecting poultry interleukin 4 (chIL-4)

[0086] The enzyme-linked immunoassay kit for detecting poultry interleukin 4 (chIL-4) of the present invention comprises polystyrene enzyme-labeled reaction plate, chIL-4 standard product (recombinant protein GST-chIL-4, sandwich protein), anti- chIL-4 monoclonal antibody 1G11-7F-9E-6B (coating antibody), biotinylated anti-chIL-4 monoclonal antibody 2E5-G7-10C-7E (detection antibody), 0.05M phosphate buffer (pH 6.0 , coating solution), PBST (pH 7.4, washing buffer), 5% skimmed milk (blocking solution), 1% BSA (sample and antibody diluent), HRP-labeled streptavidin, substrate TMB, 2M h 2 SO 4 solution (stop solution).

[0087] 1. Preparation of immunogen

[0088] (1)...

Embodiment 2

[0152] Example 2. Sensitivity detection of the enzyme-linked immunoassay kit for detecting poultry IL-4

[0153] One. Sensitivity detection of the ELISA kit for detecting poultry IL-4 of the present invention

[0154] According to the optimal conditions of the double antibody sandwich ELISA detection method determined in Implementation 1, using 1G11-7F-9E-6B as the coating antibody and biotinylated antibody 2E5-G7-10C-7E as the detection antibody, the sandwich protein was doubled ELISA test was performed after diluting to different concentrations, and the abscissa was the sandwich protein concentration, and the OD was 450 Create a standard curve for the ordinate. Specific steps are as follows:

[0155] 1. Dilute the coating antibody 1G11-7F-9E-6B with pH 6.0 phosphate buffer solution to 5 μg / mL, 100 μL / well, after coating at 4°C for 12 hours, wash with washing buffer 6 times, 300 μL / well, shake Residual liquid in the dry hole;

[0156] 2. 5% skimmed milk, 300 μL / well, afte...

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Abstract

The invention discloses a method for detecting poultry interleukin-4 content and a special kit thereof. The present invention uses chIL-4 monoclonal antibody to establish a double-sandwich ELISA detection method for chIL-4 at the protein level. It is proved by experiments that the anti-chIL-4 monoclonal antibody of the present invention has good specificity and affinity, can accurately reflect the content of chIL-4 in serum or cell supernatant, and the detection method of the present invention is fast, efficient and accurate , which solves the cumbersome operation, time-consuming and labor-intensive, easily affected by operation and other external conditions brought about by the fluorescent quantitative PCR detection method in the prior art, and not only helps to evaluate the dynamic change level of chIL-4 in the body , provide a good reference for disease prevention and treatment, and help to understand the occurrence, development, outcome of poultry infectious diseases and the dynamics of the body's protective immune response.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting poultry interleukin-4 content and a special kit thereof. Background technique [0002] Interleukin 4 (IL-4) was discovered in the early 1980s. It is a cytokine with a variety of immunological regulation functions. Immunomodulation, which can regulate the host's immune response by interacting with various types of cells. In 2004, a single-copy functional chicken Interleukin 4 (chIL-4) gene was discovered, its mRNA gene sequence is 411bp in length, and its precursor is 137 peptides, including 25 amino acid residues Signal peptide, secreted mature chIL-4 has 112 amino acid residues, contains 3 intramolecular disulfide bonds and 2 possible N-terminal glycosylation sites. [0003] Currently, there are commercial kits for detecting human and mouse IL-4 at the protein level, but since the homology between chIL-4 and human and mouse IL-4 is only 22.48% an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/532
CPCG01N33/577G01N33/532G01N2333/5406
Inventor 郑世军关晓宇王永强李晓齐曹红
Owner CHINA AGRI UNIV
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