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Characterization of the mouse monoclonal antibody TJ4C4 directed at human p68 kinase
J A Radosevich1, G D Ghadge, G K Haines
1Department of Otolaryngology-Head and Neck Surgery, University/VA Lakeside Medical Center, Chicago, Ill.
Abstract:
This report describes the production and characterization of a mouse monoclonal antibody (mAb) TJ4C4, which is directed against the interferon-induced double-stranded RNA-activated protein kinase p68 (PKR). The mAb TJ4C4 was produced against p68 which was isolated using a partially purified p68 preparation from human cells. The specificity of this mAb is demonstrated in competitive inhibition assays using recombinantly produced p68 protein and by immunoprecipitation. This mAb is of particular value in that it detects an epitope on p68 which is not destroyed in routinely prepared, formalin-fixed paraffin-embedded tissues, or in a variety of other commonly used clinical fixatives. The inhibition of mAb TJ4C4 by recombinantly produced p68, on human tissues sections, validates the specificity of this mAb in histological studies. Therefore, mAb TJ4C4 serves as a specific probe to study p68 expression in clinically derived tissue specimens.
Insights
A new mouse monoclonal antibody, TJ4C4, has been developed to detect the protein kinase p68 (PKR). This antibody is valuable for studying p68 expression in clinical tissue samples, even after fixation.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Interferon-induced double-stranded RNA-activated protein kinase p68 (PKR) plays a role in cellular responses to viral infection and stress.
- Understanding PKR expression is crucial for investigating various biological processes and disease states.
- Existing methods for detecting PKR in clinical samples can be limited by tissue fixation methods.
Purpose of the Study:
- To produce and characterize a novel mouse monoclonal antibody (mAb) TJ4C4 specific for the p68 (PKR) protein.
- To validate the utility of mAb TJ4C4 for detecting p68 in various tissue preparation and fixation conditions relevant to clinical diagnostics.
Main Methods:
- Production of a mouse monoclonal antibody (mAb) TJ4C4 against human p68 (PKR).
- Characterization of mAb TJ4C4 specificity using competitive inhibition assays with recombinant p68 and immunoprecipitation.
- Assessment of mAb TJ4C4 reactivity with formalin-fixed paraffin-embedded (FFPE) human tissues and other clinical fixatives.
Main Results:
- Monoclonal antibody TJ4C4 was successfully produced and directed against the interferon-induced double-stranded RNA-activated protein kinase p68 (PKR).
- Specificity of mAb TJ4C4 was confirmed through competitive inhibition assays and immunoprecipitation using recombinant p68.
- Crucially, mAb TJ4C4 detects an epitope on p68 that remains intact in routinely prepared FFPE tissues and other common clinical fixatives.
Conclusions:
- mAb TJ4C4 is a specific and valuable tool for detecting p68 (PKR) expression.
- This antibody is suitable for histological studies using clinically derived tissue specimens, including those processed with standard fixation methods.
- mAb TJ4C4 facilitates the study of p68 expression in a wide range of clinical research applications.