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Development of an enzyme-linked immunosorbent assay based on the murine leukemia virus p30 capsid protein
Dai-Tze Wu1, Sriram Aiyer, Rodrigo A Villanueva
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854 USA.
Abstract:
Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was developed using several goat-polyclonal serum against MuLV p30 generated by the NCI as primary antibody and a rat-monoclonal antibody to CA available from ATCC. The MuLV p30 CA antigen was standardized against recombinant MuLV p30 CA expressed from bacteria. The assay is sensitive, accurate and linear within a defined concentration range of CA. Comparison with different MuLV quantitative methods including reporter gene transfer, reverse transcriptase activity assay, and viral RNA quantitative PCR, showed this ELISA protocol to be highly quantifiable within defined ranges, which can be correlated with infectious viral titer.
Insights
A new enzyme-linked immunosorbent assay (ELISA) accurately quantifies murine leukemia virus (MuLV) infectious particles by measuring the p30 capsid (CA) core antigen. This sensitive method correlates well with infectious viral titers, aiding gene therapy vector development.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Murine leukemia virus (MuLV) vectors are crucial for gene therapy and viral pathogenesis research.
- The p30 capsid (CA) protein is a major internal core antigen in MuLV particles.
Purpose of the Study:
- To develop a sensitive and accurate enzyme-linked immunosorbent assay (ELISA) for quantifying MuLV infectious particles.
- To measure MuLV particles using the p30 CA core antigen.
Main Methods:
- Developed an ELISA using goat-polyclonal anti-MuLV p30 serum and rat-monoclonal anti-CA antibody.
- Standardized the assay against recombinant MuLV p30 CA expressed in bacteria.
- Validated the ELISA against reporter gene transfer, reverse transcriptase activity, and viral RNA qPCR.
Main Results:
- The developed ELISA is sensitive, accurate, and linear within a specific concentration range.
- The assay demonstrated high quantifiability and correlated with infectious viral titers.
- Compared favorably to existing MuLV quantification methods.
Conclusions:
- The novel ELISA provides a reliable method for quantifying MuLV infectious particles.
- This assay is valuable for gene therapy vector development and viral pathogenesis studies.
- The p30 CA antigen serves as an effective target for MuLV quantitation.
