Setting up Microneutralization Assay for Coxsackie B1 and B3 viruses
Korhonen, Ulla (2025)
2025
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2025-12-11
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-2025121111503
https://urn.fi/URN:NBN:fi:tuni-2025121111503
Tiivistelmä
Coxsackie B viruses (CVB) belong to Enterovirus (EV) family and represent important human pathogens causing acute and chronic diseases. CVBs can cause mild illness and serious life-threatening conditions such as myocarditis, pancreatitis, and possibly the etiology of type 1 diabetes (T1D). Improved methods for neutralizing antibody detection are needed to support CVB vaccine development. This study aimed to develop a microneutralization assay (MNA) for CVB1 and CVB3 as a faster, automated alternative to the traditional plaque neutralization assay.
CVB1 and CVB3 were produced in GMK cells at a high multiplicity of infection ~10 and purified using sucrose cushion ultracentrifugation. Virus purity and identity were confirmed by SDS-PAGE, densitometry, and BCA protein assay, while particle size and morphology were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Infectivity was quantified using the end-point dilution (EPD) method. Optimal assay conditions including cell line, cell density, cell viability readout, and incubation parameters were determined to establish the microneutralization assay using mouse sera from CVB1 vaccine studies.
CVB1 and CVB3 were successfully produced and purified with ≥95% purity. DLS analysis and TEM imaging confirmed intact particles with expected diameters (~26 nm). Infectivity titration showed >1 × 108 tissue-culture infectious dose / ml for both viruses. GMK cells at 16 000 cells/well provided the most reproducible results in the virus infectivity measurement and Alamar Blue was selected as the viability readout due to its simplicity and compatibility with automation; a 2-hour incubation with the dye was optimal. The MNA detected neutralizing antibodies in CVB1 mouse sera, but reproducibility was affected by cell condition and virus titer variability. CVB3 MNA assay development was initiated but requires further optimization.
The developed MNA for CVB1 offers a faster, less labor-intensive approach for measuring neutralizing antibodies compared to plaque assays. While promising for high-through-put applications, additional optimization is needed to improve reproducibility and extend the method to CVB3 and other enteroviruses.
CVB1 and CVB3 were produced in GMK cells at a high multiplicity of infection ~10 and purified using sucrose cushion ultracentrifugation. Virus purity and identity were confirmed by SDS-PAGE, densitometry, and BCA protein assay, while particle size and morphology were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Infectivity was quantified using the end-point dilution (EPD) method. Optimal assay conditions including cell line, cell density, cell viability readout, and incubation parameters were determined to establish the microneutralization assay using mouse sera from CVB1 vaccine studies.
CVB1 and CVB3 were successfully produced and purified with ≥95% purity. DLS analysis and TEM imaging confirmed intact particles with expected diameters (~26 nm). Infectivity titration showed >1 × 108 tissue-culture infectious dose / ml for both viruses. GMK cells at 16 000 cells/well provided the most reproducible results in the virus infectivity measurement and Alamar Blue was selected as the viability readout due to its simplicity and compatibility with automation; a 2-hour incubation with the dye was optimal. The MNA detected neutralizing antibodies in CVB1 mouse sera, but reproducibility was affected by cell condition and virus titer variability. CVB3 MNA assay development was initiated but requires further optimization.
The developed MNA for CVB1 offers a faster, less labor-intensive approach for measuring neutralizing antibodies compared to plaque assays. While promising for high-through-put applications, additional optimization is needed to improve reproducibility and extend the method to CVB3 and other enteroviruses.