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Published on: July 11, 2015
Development and applications of a monoclonal antibody against caprine interferon-gamma
Wen-Tao Ma1, Qi Liu1, Meng-Xia Ning1
1College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi Province, China.
Insights
Researchers developed a specific monoclonal antibody (mAb) for caprine interferon-gamma (IFN-γ). This new antibody can detect IFN-γ expression in goats, aiding in the diagnosis of contagious ecthyma.
Area of Science:
- Immunology
- Veterinary Medicine
- Molecular Biology
Background:
- Interferon-gamma (IFN-γ) is crucial for type I immune responses, exhibiting antiviral, immunoregulatory, and anti-tumor properties.
- IFN-γ plays significant roles in inflammation and autoimmune diseases, making it a key target for research.
- Understanding caprine IFN-γ is vital for veterinary immunology and disease management in goats.
Purpose of the Study:
- To develop a specific monoclonal antibody (mAb) targeting caprine interferon-gamma (IFN-γ).
- To validate the specificity and utility of the generated mAb for detecting caprine IFN-γ.
- To assess the potential application of the mAb in diagnosing caprine diseases.
Main Methods:
- Production of recombinant caprine IFN-γ in Escherichia coli.
- Immunization of BALB/c mice with purified recombinant caprine IFN-γ to generate hybridomas.
- Screening and characterization of monoclonal antibodies using ELISA, Western blot, and immunofluorescence assays.
- Evaluation of mAb application in goat lip tissues infected with Orf virus.
Main Results:
- A hybridoma secreting a specific mAb (designated 2C) against caprine IFN-γ was successfully established.
- ELISA and Western blot confirmed that mAb 2C specifically recognizes caprine IFN-γ and not the fusion tag protein.
- Immunofluorescence analysis demonstrated the mAb's ability to detect IFN-γ in histopathological sites of Orf virus-infected goats.
Conclusions:
- A caprine IFN-γ-specific monoclonal antibody (mAb) was successfully developed.
- The developed mAb is a valuable tool for detecting IFN-γ expression levels.
- This mAb can be utilized for diagnosing contagious ecthyma in goats.
Background:
Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ.
Results:
Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus.
Conclusions:
A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.
