Method for detecting content of avian interleukin 2 and special kit thereof

A content and auxiliary detection technology, applied in the biological field, can solve the problems of not reflecting the actual level of chicken IL-2 protein, cumbersome fluorescent quantitative PCR operation, and limiting the in-depth research of chicken IL-2, so as to achieve fast detection method and good specificity Sex and affinity, the effect of less demanding equipment

Inactive Publication Date: 2017-10-20
CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

However, because IL-2 has species specificity, the homology of chicken IL-2 and mammalian IL-2 is quite different, and there is no cross-reactivity. The detection kit cannot be used for the research of chicken IL-2, which greatly limits the in-depth research on chicken IL-2
[0006] In the prior art, the experimental technology mainly used to detect changes in chicken interleukin 2 (IL-2) is fluorescent quantitative PCR, which detects changes in mRNA levels, but since mRNA levels and protein levels are not absolutely linear Therefore, the application of fluorescent quantitative PCR cannot reflect the actual level of chicken IL-2 protein, so it cannot be used as an experimental technique for detecting the amount of chicken IL-2 protein. The requirements are high and the price is expensive, so it is not suitable for actual production

Method used

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  • Method for detecting content of avian interleukin 2 and special kit thereof
  • Method for detecting content of avian interleukin 2 and special kit thereof
  • Method for detecting content of avian interleukin 2 and special kit thereof

Examples

Experimental program
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preparation example Construction

[0071] The buffer solution in the following examples is prepared as follows:

[0072] 1. 0.05M glycine-hydrochloric acid buffer (pH 2.8)

[0073] 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.

[0074] 2. 0.05M citric acid-sodium citrate buffer (pH 3.0-5.0)

[0075] 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

[0088] Example 1. Establishment of an enzyme-linked immunoassay kit for detecting poultry interleukin 2 (IL-2) and a double-antibody sandwich ELISA detection method and optimization of conditions

[0089] One, the preparation of the enzyme-linked immunoassay kit for detecting poultry interleukin 2 (IL-2)

[0090] The enzyme-linked immunoassay kit for detecting poultry interleukin 2 (IL-2) of the present invention comprises polystyrene enzyme-labeled reaction plate, chIL-2 standard product (recombinant protein His-chIL-2, sandwich protein), Anti-chIL-2 monoclonal antibody FM-1 (coating antibody, the present invention selects the antibody secreted by hybridoma cell line FM-1 as coating antibody), anti-chIL-2 monoclonal antibody FM-2 (detection antibody, the present invention Select the antibody secreted by the hybridoma cell line FM-2 as the detection antibody), 0.05M phosphate buffer (pH8.0, coating solution), PBST (pH7.4, washing buffer), 5% skimmed milk (blocking solution ),...

Embodiment 2

[0164] Example 2. Sensitivity detection of the enzyme-linked immunoassay kit for detecting poultry IL-2

[0165] 1. Sensitivity detection of the enzyme-linked immunoassay kit for detecting poultry IL-2 of the present invention

[0166] According to the optimal conditions of the double-antibody sandwich ELISA detection method determined in Implementation 1, use FM-1 antibody as the coating antibody and biotinylated antibody FM-2 as the detection antibody, and perform ELISA after doubling dilution of the sandwich protein to different concentrations Test, and take the sandwich protein concentration as the abscissa, and take the OD 450 Create a standard curve for the ordinate. Specific steps are as follows:

[0167] 1. Dilute the FM-1 antibody to 5 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 inside of the well residual liquid;

[0168] 2. 5% skimmed milk, 300 μL / well, after blocking for ...

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Abstract

The invention discloses a method for detecting poultry interleukin-2 content and a special kit thereof. The present invention uses chIL-2 monoclonal antibody to establish a double-sandwich ELISA detection method for chIL-2 at the protein level. It is proved by experiments that the anti-chIL-2 monoclonal antibody of the present invention has good specificity and affinity, can accurately reflect the content of chIL-2 in serum or cell supernatant, and the detection method of the present invention is fast, efficient and accurate , which solves the problems of 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‑2 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 2 (IL-2) content and a special kit thereof. Background technique [0002] Interleukin-2 (IL-2) is an essential cytokine for immune response, and plays an important role in the growth and differentiation of T cells, the development of B cells and the activation of NK cells. However, due to the low content of cytokines, there have been difficulties in detection. With the popularization of monoclonal antibody technology, the immunoassay technology established by using monoclonal antibody technology can detect cytokines very quickly, which greatly accelerates the research process of cytokines. [0003] Monoclonal antibody technology utilizes the characteristics that myeloma cells can proliferate in large quantities but cannot secrete specific antibodies in vitro, while B lymphocytes immunized with antigens can produce specific antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6869
Inventor 郑世军付梦娇王永强李晓齐曹红
Owner CHINA AGRI UNIV
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