Immunofluorescence and Viral Disease Diagnosis Flashcards
What is a method for efficient and definitive detection of viral antigens in virus-infected cells?
Immunofluorescence (FA) Staining in Virology
3 applications of FA
- Transport medium sediment from samples collected on swabs
- Cells from infected cell culture monolayers
- Shell vial monolayers
Procedure of Immunofluorescence staining (4 steps)
- Cells are fixed to a slide
- Monoclonal antibodies applied in direct or indirect staining protocols
- One of the antibody preparation is labeled with a fluorescent dye
- Results are red with a fluorescence microscope
FA stain: fluorescein isothiocyanate (FITC) is used most. What color does it fluoresce? What does it stain?
bright green…stains the target
FA stain: Evans Blue is used as a counterstain. What color does it fluoresce? What does it stain?
Red…stains the background
Steps in direct FA staining?
- Fluorescein-labled antibodies are added to a microscope slide with smear of virus-infected cells
- Incubate and rinse
- If Ag/Ab binding then green fluorescence
- If no binding, no fluorescence
Steps in indirect FA staining?
- Antibodies added to a microscope slide with smear of virus-infected cells
- incubate and rinse
- Stage 1: There will either be Ag/Ab binding or not
- Stage 2: Fluorescein-labled anti-species globulin added
- incubate and rinse
- If Ag/Ab binding then fluorescence
In FA testing what are smears evaluated for?
Intensity and distribution of fluorescence
How do we stain cells from a cell culture monolayer?
- use pipette/scraper to scrape cells off tube wall
- spin to sediment the cells
- make smear of sediment
- stain, read
How do we stain cells from cells collected on swabs submitted in transport medium?
- use sediment found in tp medium after centrifugation
- make a smear on a microscope slide
- stain and read (at least 20-25 cells must be present to be valid test)
Enzyme-linked Virus Inducible System (ELVIS) is specific for what virus?
Herpes Simplex Virus
How does ELVIS work?
- E. coli LacZ gene is cloned into cells behind an HSV promoter
- A substrate is added to infected cells. If beta galactosidase enzyme is present (only produced by HSV infected cells), it acts on the substrate to produce a color change (DARK BLUE)
Advantages of ELVIS
- Very little skill required
- Expensive monoclonal antibodies not needed
- Sensitive and specific compared to cell culture
What are two ways that antivirals work?
- nucleoside analogs (allow virus to add it in and then destroy it)
- enzyme inhibitors
Are antivirals “virus specific” or “broad spectrum”
virus specific
What is CMV’s antiviral
Gangciclovir
What is HSV’s antiviral
Acyclovir
What is HIV-1’s antiviral
Didanosine
What is RSV’s antiviral
Ribavirin
What test involves using components labeled with active enzymes?
Enzyme Immunoassay EIA Antigen Detection
What is EIA used for?
to detect unknown Antigen or antibody (widely used for testing fecal samples for rotavirus antigen)
Non competitive Solid-Phase Enzyme immunoassay for Antigen Detection
- viral antibody coats the tube
- viral antigen and enzyme is added
- incubate and rinse
- add substrate and if Ag/Ab binding then color change
Strains of influenza A vary in regards to what?
hemagglutinin (H) and neuraminidase (N) types
Non-competitive EIA for antibody detection
- reagents and equipment
- antigen coated tube
- Antibodies in patients serum
- Enzyme-labled antihuman igG
- Substrate solution
Non-competitive EIA for antibody detection
- list the steps of preforming
- Antibodies in patients serum is added to an antigen coated tube
- incubate and rinse
- Add enzyme labeled antihuman IgG and incubate and rinse
- AddSubstrate solution and will change color if positive
Non-competitive EIA for antibody detection
- appearance of pos and neg
Color change pos
No color change neg
Competitive EIA for antibody detection
- List the steps
- Put both Antibodies in patients serum and enzyme labeled antibodies into the Antigen coated tube
- incubate and rinse
- If Ab was present it sill bind and substrate solution is added and no color change
- If Ab was absent enzymes will bind, substrate will be added and there will be a color change
Non-competitive EIA for ANTIGEN
- Add viral antigen to a antibody coated tube
- incubate and rinse
- Add enzyme labeled antiviral antibody and incubate and rinse
- add substrate solution, if color change then antigen was bound so POS
Advantages of Enzyme Immunoassay
- Well standardized
- Good for large volume testing
- Can be automated or semi-automated
- Cost effective
- Requires less technical skill than many methods
disadvantages of Enzyme Immunoassay
- Unable to distinguish specific from nonspecific binding
2. May require repeat and/or confirmatory testing
BioMerieux Vidas
- general format
- Automated EIA = walk-away function
- pipette dips into reagent wells both antigen and antibody detection methods are available
BioMerieux Vidas
- solid phase
pipette tip (coated on the inside)
microwell-based EIA instruments
- general format
Automated EIA = completely or largely walk away
- used for detection unknown antibodies
microwell-based EIA instruments
- solid phase
wall of microwell
Multiplex Micro-bead systems
designed to detect and distinguish many different antibodies simultaneously in a single test well, microbes dyed various shades of colors, each color is coated with particular antigen, laser interrogation
One Laser identifies among of patients antibody bound and the other identifies the color
