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Method for detecting ChIFN-gamma double-monoclonal antibody sandwich ELISA

A monoclonal antibody and antibody technology, applied in the field of immunological detection, can solve the problems of lack of detection tools and detection systems, and achieve the effect of good detection tools

Inactive Publication Date: 2009-05-27
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Chicken is an important animal model for a large number of poultry intracellular infectious diseases. So far, the choice of chicken as an animal model for poultry diseases has been limited by many limitations, mainly due to the lack of appropriate detection tools and detection systems.

Method used

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  • Method for detecting ChIFN-gamma double-monoclonal antibody sandwich ELISA
  • Method for detecting ChIFN-gamma double-monoclonal antibody sandwich ELISA
  • Method for detecting ChIFN-gamma double-monoclonal antibody sandwich ELISA

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Embodiment 1

[0026] Example 1: Establishment of the double monoclonal antibody sandwich ELISA method for detecting ChIFN-γ of the present invention

[0027] 1. Purify and label anti-ChIFN-γ specific monoclonal antibodies: 3E3, 3E5, 1G10, 2C3 (mAbs), use Proein G to purify the mAb, and use biotin to label the mAb. The specific operation is carried out according to the standard method.

[0028]2. Preliminary establishment of ChIFN-γ antigen capture ELISA: using prokaryotic expressed rChIFN-γ as capture antigen (development and preliminary identification of anti-chicken γ-interferon monoclonal antibody, Journal of Cellular and Molecular Immunology, 2006, 22 (4 ): 510-512), the pairing experiment was carried out between the above-mentioned labeled 3E3, 3E5, 1G10, 2C3 mAbs and commercially available mAbs (such as Abs: Serotec: Rabbit anti chicken interferon gamma (polyclonal antibody, AHP945Z), and the checkerboard method was used to find out Establish the optimal antibody for ChIFN-γ antigen c...

Embodiment 2

[0030] Embodiment 2: Application of the method of the present invention to detect natural ChIFN-γ

[0031] Preparation of natural ChIFN-γ: Spleen of 4-week-old SPF chicken was aseptically removed, ground and filtered through 200-mesh copper mesh; the filtrate was collected, centrifuged at 4°C, 1000rpm, 5min; lymphocyte separation medium (Sigma Company) was used to separate lymphocytes, 21 ℃, 2000rpm, 30min, centrifuge; collect the middle cloud layer, which is the lymphocytes, wash the collected cells twice with PBS, and then use complete RPMI 1640 (10% fetal bovine serum, 100U / ml penicillin, 100U / ml Streptomycin) was washed once, and finally the cells were suspended with complete RPMI 1640, and the cell concentration was adjusted to 1×10 7 pcs / ml, 5×10 6 pcs / ml, 2×10 6 cells / ml; take a 96-well cell culture plate, add 100ul cells, and then add 100 μl of culture medium containing stimulator ConA (Sigma Company), so that the final concentration of ConA is 12 μg / ml, and set up a...

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Abstract

The invention discloses a double monoclonal-antibodies sandwich ELISA method for detecting ChIFN-gamma, wherein the specific monoclonal antibodies (mAbs) against ChIFN-gamma are used to build the double monoclonal-antibodies sandwich ELISA method for detecting ChIFN-gamma. The double monoclonal-antibodies sandwich ELISA method for detecting ChIFN-gamma, with mAb3E5 as capture antibody and biotin-labeled mAb 3E3 as detection antibody, can quickly detect the trace ChIFN-gamma (125-500pg / ml). The method is rapid, useful, sensitive, and effective, can be used to detect the ChIFN-gamma and recombinant ChIFN-gamma in biological sample, and provides a sensitive detection tool for detecting and evaluating the cell immune response of poultry, and is useful for the research on the function of the ChIFN-gamma in various immune response mechanisms of poultry, and efficiently makes up for the prior ChIFN-gamma detection.

Description

technical field [0001] The invention belongs to the field of immunological detection methods, in particular to a double monoclonal antibody sandwich ELISA method for detecting poultry cytokines. Background technique [0002] γ-Interferon (IFN-γ, also known as immune interferon or type II interferon) is an important mediator of cellular immune response and plays an important role in the regulation of the body's immune response, especially in inducing and maintaining Th1 cell immune response effect. Th1 response is very important to resist the infection of intracellular pathogens, and the appearance of IFN-γ-secreting T lymphocytes is the hallmark of Th1 response. The level of IFN-γ in biological samples and the appearance of IFN-γ-secreting cells are usually used as markers for evaluating the occurrence of Th1 immune response. [0003] At present, there are many methods for detecting cytokines, mainly biological activity assays, immunological assays, and the use of nucleic ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543
Inventor 焦新安戴华郑佳玉陈俊华潘志明陈祥孙林
Owner YANGZHOU UNIV
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