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Enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10

An ELISA and interleukin technology, which is applied in the direction of interleukin, anti-cytokine/lymphokine/interferon immunoglobulin, cytokine/lymphokine/interferon, etc., can solve the bottleneck of fine identification, No establishment, large differences in molecular structure, etc., to achieve rapid and efficient detection methods and low cost effects

Inactive Publication Date: 2015-04-08
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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Problems solved by technology

However, at present, the method for detecting the secretion of fish IL-10 has not been established, mainly because the molecular structure of IL-10 of teleost fish is quite different from that of mammals and amphibians, and the IL-10 antigen of other races and its antigens cannot be used. Detection of IL-10 in teleost fish by antibody substitution assay
At the same time, the lack of IL-10 antibody in fish makes the secretion detection and fine identification of IL-10 a bottleneck

Method used

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  • Enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10
  • Enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10
  • Enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10

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Embodiment Construction

[0023] Below, the present invention will be described in detail in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0024] One: Reagents:

[0025] Maleimide-activated horseradish peroxidase kit was purchased from Thermo Scientific

[0026] Tetramethylbenzidine (TMB) was purchased from Tiangen Biotechnology Company

[0027] Other reagents are analytical reagents

[0028] Two: Steps:

[0029] 1. Preparation of immunogen: clone grass carp IL-10 cDNA sequence into prokaryotic expression vector pET-30a(+), construct recombinant prokaryotic expression vector, and transform this vector into Escherichia coli BL21(DE3), pass through 1mM IPTG Induced expression of recombinant protein IL-10, the protein is mainly expressed in soluble form, using Ni 2+ The recombinant grass carp IL-10 protein was purified by affinity chromatogra...

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Abstract

The invention relates to a competitive inhibition enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10 (IL-10). The method comprises the following steps of: immunizing a New Zealand white rabbit by utilizing grass carp interleukin 10 proteins which is expressed in a recombined way as an immunogen to obtain a polyclonal antibody; marking the antibody by utilizing horseradish peroxidase; and finally, establishing the competitive inhibition ELISA method by taking the recombined grass carp interleukin IL-10 protein as a covered antigen, wherein the competitive inhibition ELISA method can be used for the protection detection of the grass carp interleukin 10. The detection range of the ELISA method is 0.1-100 ng / mL. By utilizing the polyclonal antibody and the competitive inhibition ELISA method, the cost is low, and the stability and the repeatability are good. The conventional method is that the competitive inhibition ELISA method is firstly established to detect protection expression of the grass carp interleukin 10.

Description

technical field [0001] The invention belongs to the field of enzyme-linked immunoassay (ELISA) biotechnology, and in particular relates to an ELISA detection method for grass carp interleukin-10 competitive inhibition. Background technique [0002] Interleukin-10 (IL-10) is an important cytokine. In 1989, Fiorentino et al. discovered that mouse Th2 cells could secrete a substance that inhibits Th1 cells from secreting interferon and other cytokines, so they named this substance cytokine synthesis inhibiting factor (CSIF), and later called white blood cells. Interleukin 10. Subsequent studies have found that this cytokine plays an important role in inflammation, malignant tumors, and autoimmune diseases. It can inhibit the proliferation and differentiation of T cells and macrophages, and can also promote the stable differentiation of B cells. Its immune diversity makes it play an important role in the immune system. Current research also shows that interleukin 10 has immun...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68C12N15/24C12N15/70C07K14/54C07K16/24
Inventor 周红韦鹤赵太强杨牧汪新艳
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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