Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for detecting content of avian interleukin 10 and a special kit thereof

A content and auxiliary detection technology, applied in the biological field, can solve the problems of cumbersome fluorescent quantitative PCR technology, failure to reflect chicken IL-10 protein, and high professional skills requirements, and achieve fast detection methods, low instrument requirements, and solve operational problems. cumbersome effect

Inactive Publication Date: 2017-10-20
CHINA AGRI UNIV
View PDF1 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, there are commercial kits for detecting human and mouse IL-10 at the protein level, but since the homology between chIL-10 and human and mouse IL-10 is only 45% and 42%, respectively, the Domestically, whether in academic research or clinical disease analysis, the relative content of chIL-10 is mainly detected by fluorescent quantitative PCR
However, since the mRNA level and the protein level do not have an absolute linear relationship, the application of fluorescent quantitative PCR cannot reflect the actual level of chicken IL-10 protein, so it cannot be used as an experimental technique for detecting the amount of chicken IL-10 protein. At the same time, 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting content of avian interleukin 10 and a special kit thereof
  • Method for detecting content of avian interleukin 10 and a special kit thereof
  • Method for detecting content of avian interleukin 10 and a special kit thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0070] The buffer solution in the following examples is prepared as follows:

[0071] 1. 0.05M glycine-hydrochloric acid buffer (pH 2.8)

[0072] 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.

[0073] 2. 0.05M citric acid-sodium citrate buffer (pH 3.0-5.0)

[0074] 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

[0087] Example 1. Establishment of an enzyme-linked immunoassay kit for detecting poultry interleukin 10 (IL-10) and a double-antibody sandwich ELISA detection method and optimization of conditions

[0088] One, the preparation of the enzyme-linked immunoassay kit for detecting poultry interleukin 10 (IL-10)

[0089] The enzyme-linked immunoassay kit for detecting poultry interleukin 10 (IL-10) of the present invention comprises polystyrene enzyme-labeled reaction plate, IL-10 standard product (recombinant protein His-chIL-10, sandwich protein), Anti-IL-10 monoclonal antibody 1G11 (coating antibody), anti-IL-10 monoclonal antibody 1D6 (detection antibody), 0.05M carbonate buffer (pH9.6, coating solution), PBST (pH7.4, 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).

[0090] 1. Preparation of immunogen

[0091] (1) Construction of chicken IL-10 prokaryo...

Embodiment 2

[0149] Example 2. Sensitivity detection of the enzyme-linked immunoassay kit for detecting poultry IL-10

[0150] 1. Sensitivity detection of the ELISA kit for detecting poultry IL-10 of the present invention

[0151] According to the optimal conditions of the double-antibody sandwich ELISA detection method determined in Implementation 1, the 1G11 antibody was used as the coating antibody and the biotinylated antibody 1D6 was used as the detection antibody, and the sandwich protein was diluted to different concentrations before the ELISA test was performed. Sandwich protein concentration is the abscissa, in OD 450 Create a standard curve for the ordinate. Specific steps are as follows:

[0152] 1. Dilute the 1G11 antibody to 4 μg / mL with carbonate buffer (pH9.6), 100 μL / well, coat at 4 °C for 12 hours, wash with washing buffer 3 times, 300 μL / well, and dry the residual liquid in the well ;

[0153] 2. 5% skimmed milk, 300 μL / well, after blocking for 3 hours at 37°C, wash a...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
wavelengthaaaaaaaaaa
quality scoreaaaaaaaaaa
Login to View More

Abstract

The invention discloses a method for detecting a content of avian interleukin 10 and a special kit thereof. A double-sandwich ELISA detection method for chIL-10 on a protein level is established by utilizing a chIL-10 monoclonal antibody. An experiment proves that the anti-chIL-10 monoclonal antibody of the invention has good specificity and affinity, and can accurately reflect the content of chIL-10 in serum or cellular supernatant; and moreover, the detection method of the invention is rapid, efficient and accurate, can solve the problems in the prior art that a fluorescent quantitative PCR detection method is complex in operation, time-consuming, labor-consuming, susceptible to be influenced by the operation and other external conditions and the like, not only aids in evaluating a dynamic variation level of chIL-10 in the body, provides good reference for preventing and treating diseases, but also aids in knowing the occurrence, development, regression conditions of avian infectious diseases and the dynamic state of the protective immunity reaction of the body.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting poultry interleukin 10 (IL-10) content and a special kit thereof. Background technique [0002] In 1989, Fiorentino et al. first described interleukin 10 as a new immune mediator secreted by type 2 helper T cells (Th2) and inhibiting the synthesis of IL-2 and IFN-γ in Th1 cells. In 2004, Lisa Rothwell et al. extracted total RNA from chicken cecum tonsils infected with Eimeria, and obtained the full-length cDNA sequence of chIL-10 by RT-PCR, which encoded a polypeptide of 175 amino acids, of which 21 amino acids constituted the signal Peptide, the mature peptide is 154 amino acids long. The amino acid sequence homology of chicken IL-10 with human IL-10 and mouse IL-10 is 45% and 42%, respectively. Chicken IL-10 gene contains 5 exons and 4 introns. [0003] Cells such as dendritic cells, macrophages, mast cells, natural killer cells, eosinophils, a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N5/20G01N33/68G01N33/577
CPCC07K16/244G01N33/577G01N33/6869G01N2333/5428
Inventor 郑世军高俊峰王永强李晓齐曹红
Owner CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com