Analyzing the Antigenic Relationship between Viruses using an Image-Based Microneutralization Assay
Transcrição
Add media and serially diluted receptor-destroying enzyme-treated antisera onto mammalian cell monolayers, preventing nonspecific virus binding.
Add an influenza virus suspension. Antisera antibodies, specific to viral proteins, bind, neutralizing viruses and preventing cellular entry.
Low neutralizing antibody concentration or non-neutralizing antibodies enable ineffective viral antigen binding, allowing virus attachment to cell receptors.
Discard media. Apply cellulose-containing media, forming a semi-solid overlay.
The virus uses host cell machinery to form viral particles and cause cell lysis.
The overlay restricts virus movement, facilitating localized infection and creating holes in cell monolayers.
Remove the overlay. Fix and permeabilize cells.
Wash with buffer. Add antibodies binding viral antigens within permeabilized cells.
Pipette horseradish peroxidase-conjugated secondary antibodies binding to primary antibodies.
Introduce peroxidase substrates and hydrogen peroxide, resulting in a blue product.
Image wells to quantify virus-infected cell populations against antiserum dilutions.
Identify antisera dilutions indicating a specified reduction in an infected cell population to determine neutralization titers.
