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Updated: May 22, 2026
Detection of Abnormal Prion Protein by Immunohistochemistry
Published on: May 5, 2023
Detection of prion protein oligomers by single molecule fluorescence imaging
Satoko Shibano1, Kensuke Sasaki, Satoru Kidoaki
1Department of Neuropathology, Graduate School of Medical Sciences Division of Biomolecular Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan.
Abstract:
The degree of polymerization of PrP has a close relationship with the pathological mechanisms of prion diseases. We examined, at the molecular level, the polymerization state of PrP in lysates of prion-infected cells using total internal reflection fluorescence microscopy (TIRFM). The crude lysates were fractionated by gel-filtration spin columns according to their molecular size. Both the oligomer-rich and the monomer-rich fractions were probed with fluorescein-labeled anti-PrP antibodies (mAb SAF70 and mAb 8G8). Fluorescent spots of varying intensity were detected, with the ratio of intense fluorescent spots being greater in the oligomer fraction samples with mAb SAF70 than those with 8G8, the specific epitope of which is thought to be buried in abnormal PrP molecules. The results indicated that PrP oligomers could be specifically detected and conformational changes of abnormal PrP molecules observed. Imaging by TIRFM may aid in determining the polymerization state and properties of PrP oligomers in pathological processes.
Insights
Researchers used advanced microscopy to visualize prion protein (PrP) polymerization in infected cells. This technique helps detect abnormal PrP oligomers and their conformational changes, crucial for understanding prion diseases.
Area of Science:
- Neuroscience
- Biochemistry
- Microscopy
Background:
- Prion diseases are linked to the polymerization state of prion protein (PrP).
- Understanding PrP polymerization is key to elucidating prion disease pathogenesis.
- Current methods may not fully capture the molecular details of PrP aggregation.
Purpose of the Study:
- To investigate the polymerization state of PrP in prion-infected cells at the molecular level.
- To develop and apply a method for specifically detecting PrP oligomers.
- To observe conformational changes in abnormal PrP molecules.
Main Methods:
- Utilized Total Internal Reflection Fluorescence Microscopy (TIRFM) for high-resolution imaging.
- Fractionated cell lysates using gel-filtration spin columns to separate PrP by molecular size.
- Employed fluorescein-labeled anti-PrP antibodies (mAb SAF70 and mAb 8G8) for detection.
Main Results:
- TIRFM successfully detected fluorescent PrP species in different molecular size fractions.
- A higher ratio of intense fluorescent spots was observed in oligomer-rich fractions using mAb SAF70 compared to mAb 8G8.
- The differential antibody binding suggests specific detection of PrP oligomers and potential conformational changes.
Conclusions:
- TIRFM enables specific detection and characterization of PrP oligomers.
- The study provides insights into the polymerization state and conformational properties of abnormal PrP.
- This imaging approach holds promise for advancing prion disease research.
