Virus Assays

Question 1

Plaque assay method

Answer 1

10fold serial dilutions of birus stock prepped in culture medium. 0.2ml of each added to small tissue culture plate growing monolayers of susceptible cells and virus left to absorb at 37degree c for 1hr. Inoc removed and agar growth medium replaces it to prev virus spreading throughout the medium so amount of infectious units in a sample measured as areas of infection only arise from viruses that bound to cells within the hr they can’t diffuse or spread to other cells
After 4d fixed with formalin and stained with giemsa, agar removed and monolayer examined for CPE unser microscope. Holes in monolayer indicate where virus infected monolayer
By counting plaque no and allowing for dilution no of plaque forming units per ml can be calc

Question 2

Which viruses is tissue culture infectious dose 50% used for

Answer 2

Those that can’t spread by cell to cell contact so don’t form plaques. It determines amount of virus req to infect 50% of cultures, not individ cells

Question 3

TCID50% method

Answer 3

10fold serial dilutions of virus stock icubated with cell monolayers on 96 well tissue culture plate after 1hr inoc removed and replaced with media
After normally 4-7d media removed, cells fixed and stained
End point is dilution of virus that causes CPE in 50% of cultures
Most cases it doesn’t fall on an actual dilution used and statistical methods developed to calc end point of titration

Question 4

Neutralisation test principle

Answer 4

Some virus spec Ab can block virus infection
Termed neutralisation amd these Abs are called neutralising Abs
Simplest neut test carried out as a type of plaque assay
Similar assay can be done to determine efficacy of antiviral drugs in red infection

Question 5

Neutralisation test method

Answer 5

Serial dilutions of test serum or experimentally raised monoclonal Ab are incubated with a known no of PFU for 1hr
Virus-Ab mix used as standard plaque assay
Percent neut plotted against Ab dilution and half max titre calc
Serum % can then be read off the standard graph to work out Ab dilution in serum

Question 6

Percentage neutralisation calculation

Answer 6

((No of plaques in absence of Ab - no of plaques in Ab presence) divided by no of plaques in Ab absence) x100

Question 7

Haemagglutination assay premise

Answer 7

Many viruses cont pros thag can bind RBCs and can link many RBCs forming a lattice, called haemagglutination and can be used to detect virus presence in a sample

Question 8

Haemagglutination assay method

Answer 8

2fold serial dilutions of virus prepped and mixed with a defined no of RBC
Each sample added to well of v-bottomed 96 well plate
If no virus present RBC fall to bottom and form a sharp dot, agglutinatd ones form lattice that remains dispersed in well
Rapid (30min) assay provide quick indicator of relative viral quantity

Question 9

Haemagglutination inhib assay premise

Answer 9

Ab to virus surf pros can block agglutination ability so can determine virus Ab presence

Question 10

Haemagglut inhib assay method

Answer 10

Dilutions of serum incub with known amount of virus and then RBCs added after incubation HAI titre is read as highest serum dilution that inhibits heamagglutination
Assay is quick inexpensive and sensitive