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Quantitation of adenovirus type 5 empty capsids
Eddie Takahashi1, Steven L Cohen, P K Tsai
1Bioprocess and Bioanalytical Research, Bioprocess R&D, Merck Research Laboratories, West Point, PA 19486, USA.
Analytical Biochemistry
|December 20, 2005
Summary
This study introduces a novel method using reverse-phased HPLC to accurately quantify empty adenovirus capsids. This technique offers a faster and more precise alternative to traditional methods for analyzing viral preparations.
Area of Science:
- Virology
- Biochemistry
- Proteomics
Background:
- Adenovirus empty capsids are immature viral intermediates lacking DNA and core proteins.
- Purified preparations of empty and full capsids are essential for studying viral maturation and assembly.
Purpose of the Study:
- To develop and validate a novel, rapid, and precise method for quantifying empty adenovirus capsids.
- To identify and characterize proteins specific to empty capsids.
Main Methods:
- Purification of adenovirus empty and full capsids using cesium chloride gradient separation.
- Analysis of capsid proteins by polyacrylamide gel electrophoresis (PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
- Quantification of empty capsids using reverse-phased HPLC (RP-HPLC) with precursor protein VIII (pVIII) as a marker.
Main Results:
- Empty capsids contain unique proteins, including varying forms of the L1 52/55kDa protein involved in DNA encapsidation.
- A 31kDa band was identified as precursors to proteins VI and VIII, which are uncleaved and absent in mature capsids.
- The novel RP-HPLC method accurately quantifies the percentage of empty capsids with high precision.
Conclusions:
- The developed RP-HPLC method provides an accurate, precise, and rapid alternative for quantifying empty adenovirus capsids.
- This method eliminates the need for laborious cesium chloride gradient separations and extractions.
- The findings contribute to a better understanding of adenovirus assembly and provide a valuable tool for viral characterization.