Assays For Viruses Flashcards
How are viruses grown
Grown in tissue culture
Then infect cells and incubated within cells
The cells are then harvested if infected
What does CPE mean which some viruses do to cells they infect
Cytopathic effects
What is MOI
Multiplicity of infection
What does multiplicity of infection measure
Number of infectious only particles which infect a cell
What would an MOI of 10 mean
10 infectious particles found within a cell
Why isn’t Multiplicity of infection same as particle per cell
Not all viruses have cytopathic effects
What type of distribution has MOI have
Poisson
What is a plaque assay
Way of quantifying viruses using serial dilutions and agar
Why is agar important in plaque assays
Stops new viral progeny infecting cells
What does PFU mean found on plaque assays in agar
Plaque forming units (from a single viral particle)
Why wouldn’t all viruses show on the plaque assay
Not all have CPE cytopathic effects
Does plaque asssays take a long time
Yes
What is the difference in plaque assay and focus forming assays
Focus forming don’t use agar and are faster
What is used to detect viruses in focus forming assays
Immunofluorescence which attach to viruses
How are cells dna detected in focus forming assays
Via counter stain
Why wouldn’t a FFU be the same as a PFU
Some particles are FFU (show up in immunofluorescence)
They don’t necessarily cause CPE which is what needs to show in PFU
What does end point dilution assays use to test how many particles present
They use dilutions until no CPE is detected
What is TCID50 which also uses end point dilution assays
It’s the conc of virus needed to kill 50% of the cells
How many cells killed if there’s 100 TCID50
50/100
What is TCID50 used for to counteract plaque issues
Allows the detection of non plaquing/CPE viruses
What is an issue with TCID50
Takes a long time
Which microscopes are used for protein assays for detection of viruses and what does it allow to find
Electron microscopy
Allows finding of the MOI (infectious particles in cell)
What does haemogglutination measure
Number or viruses relatively (viable and non viable)
Via the clumping of RBC causing cross linking
How can viruses non specifically mostly cause rbc clumping
Via their GP eg influenza HA
What would need to be counted to get absolute number or viruses in haemogluttination
How many particles via EM
Why doesn’t haemogluttination measure infectivity
Because even if cells are infectious they can be reactive and cause rbc cross linking
Name the 3 types of Elisa
Direct
Indirect
Sandwich
How does direct Elisa work
Sample added with possible antigen
The primary antibody with an enzyme attached would bind if antigen present
When substrate added it reacts and causes positive for the viral antigen
How is indirect Elisa different
It uses a secondary antibody conjugate which binds to the antibody already present bound to antigen if the person is positive
What do sandwich Elisa detect in sanple
CAPTURE Antibodies for the virus
What is added to test capture antibody in sandwich
The antigen
Which then if present and attached will cause another antibody to bind
This antibody detected by a tertiary antibody with enzyme present and substrate added
