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Method for detecting chicken interleukin 12 content and special kit thereof

A content and auxiliary detection technology, applied in the biological field, can solve the problem of not being able to directly and truly reflect the protein level, etc., and achieve the effect of fast detection method, low instrument requirements, and cumbersome operation.

Inactive Publication Date: 2019-04-19
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because there are still many regulations in the cell during the process of mRNA translation into protein, it cannot directly and truly reflect the protein level of chIL-12 in chickens

Method used

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  • Method for detecting chicken interleukin 12 content and special kit thereof
  • Method for detecting chicken interleukin 12 content and special kit thereof
  • Method for detecting chicken interleukin 12 content and special kit thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0069] The buffer solution in the following examples is prepared as follows:

[0070] 1. 0.05M citric acid-sodium citrate buffer (pH 3.0-5.0)

[0071] 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 citrate buffer solution (pH 3.0).

[0072] Mix 65.5mL of solution A and 34.5mL of solution B to obtain a 0.05M citric acid-sodium citrate buffer solution (pH 4.0).

[0073] Mix 41mL of solution A and 59mL of solution B to obtain a 0.05M citric acid-sodium citrate buffer solution (pH 5.0).

[0074] 2. 0.05M phosphate buffer (pH 6.0-8.0)

[0075] Liquid A: Weigh 3.9 g of sodium dihydrogen phosphate dihydrate and dissolve it in 500 mL of distilled water to ...

Embodiment 1

[0082] Embodiment 1, the enzyme-linked immunoassay kit for detecting chicken interleukin 12 (chIL-12) and its application method

[0083] One, the preparation of the enzyme-linked immunoassay kit for detecting chicken interleukin 12 (chIL-12)

[0084] The ELISA kit for detecting chicken interleukin 12 (chIL-12) of the present invention comprises chIL-12 standard product (recombinant protein GST-chIL-12, sandwich protein), monoclonal antibody LHK-1 (including Antibody), monoclonal antibody LHK-2 (detection antibody), polystyrene enzyme-labeled reaction plate, 0.05M carbonate buffer (pH 6.0, 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).

[0085] 1. Preparation of immunogen

[0086] (1) Construction of chicken IL-12 prokaryotic expression vector

[0087] (1-1) Obtain the published nucleotide sequence (NM_213571.1) of chi...

Embodiment 2

[0156] Example 2. Sensitivity detection of the enzyme-linked immunoassay kit for detecting chicken IL-12

[0157] According to the optimal conditions of the double-antibody sandwich ELISA detection method determined in Example 1, the LHK-1 antibody was used as the coating antibody, and the biotinylated antibody LHK-2 was used as the detection antibody, and the sandwich protein was diluted to different concentrations after multiple dilutions. ELISA 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:

[0158] 1. Dilute LHK-1 antibody to 5 μg / mL with 0.05M phosphate buffer (pH6.0), 100 μL / well, coat at 4°C for 12 hours, wash with washing buffer 6 times, 300 μL / well, and spin dry the well residual liquid;

[0159] 2. 5% skimmed milk, 300 μL / well, after blocking for 2 hours at 37°C, wash as above;

[0160] 3. Dilute the serum sample to be tested with 1% BSA at a ratio of ...

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Abstract

The invention discloses a method for detecting chicken interleukin 12 content and a special kit thereof. A chIL-12 monoclonal antibody is utilized to establish a double-sandwich ELISA detection methodfor chIL-12 on the protein level. Experiments prove that the chIL-12 monoclonal antibody has good specificity and affinity and can accurately reflect the content of the chIL-12 in serum or cell supernatant. The detection method has the advantages that the method is fast, efficient and accurate, the problems of cumbersome operation, time and labor consumption, susceptibility to operation and otherexternal condition influences which are caused by a fluorescence quantitative PCR detection method in the prior art are solved, not only is the method conductive to the evaluation of the dynamic change level of the chIL-12 in the body, a good reference for the prevention and treatment of diseases is provided, but also the method is conductive to knowing the occurrence, development, prognosis anddynamics of protective immune responses in chickens.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting the content of chicken interleukin 12 (chIL-12) and a special kit thereof. Background technique [0002] Interleukin-12 (IL-12) is a cytokine released by B cells, macrophages, and neutrophils when they receive various stimuli. IL-12 is a cytokine with the strongest induction and regulation effect on immune active cells in vivo and the widest range among the cytokines found so far. IL-12 has a variety of biological functions and plays an important role in the immunotherapy of tumors, viral diseases and parasitic diseases. [0003] Monoclonal antibody technology is the use of myeloma cells that can proliferate in large quantities but cannot secrete specific antibodies in vitro and B lymphocytes that can produce specific antibodies but cannot proliferate in vitro for cell fusion to obtain both infinite proliferation and Antibody-secreting hybridoma c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558G01N33/535
Inventor 郑世军刘海坤王永强李晓齐曹红
Owner CHINA AGRI UNIV
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