Immunofluorescence and Viral Disease Diagnosis

Question 1

What is a method for efficient and definitive detection of viral antigens in virus-infected cells?

Answer 1

Immunofluorescence (FA) Staining in Virology

Question 2

3 applications of FA

Answer 2

1. Transport medium sediment from samples collected on swabs
2. Cells from infected cell culture monolayers
3. Shell vial monolayers

Question 3

Procedure of Immunofluorescence staining (4 steps)

Answer 3

1. Cells are fixed to a slide
2. Monoclonal antibodies applied in direct or indirect staining protocols
3. One of the antibody preparation is labeled with a fluorescent dye
4. Results are red with a fluorescence microscope

Question 4

FA stain: fluorescein isothiocyanate (FITC) is used most. What color does it fluoresce? What does it stain?

Answer 4

bright green…stains the target

Question 5

FA stain: Evans Blue is used as a counterstain. What color does it fluoresce? What does it stain?

Answer 5

Red…stains the background

Question 6

Steps in direct FA staining?

Answer 6

1. Fluorescein-labled antibodies are added to a microscope slide with smear of virus-infected cells
2. Incubate and rinse
3. If Ag/Ab binding then green fluorescence
4. If no binding, no fluorescence

Question 7

Steps in indirect FA staining?

Answer 7

1. Antibodies added to a microscope slide with smear of virus-infected cells
2. incubate and rinse
3. Stage 1: There will either be Ag/Ab binding or not
4. Stage 2: Fluorescein-labled anti-species globulin added
5. incubate and rinse
6. If Ag/Ab binding then fluorescence

Question 8

In FA testing what are smears evaluated for?

Answer 8

Intensity and distribution of fluorescence

Question 9

How do we stain cells from a cell culture monolayer?

Answer 9

• use pipette/scraper to scrape cells off tube wall
• spin to sediment the cells
• make smear of sediment
• stain, read

Question 10

How do we stain cells from cells collected on swabs submitted in transport medium?

Answer 10

• 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)

Question 11

Enzyme-linked Virus Inducible System (ELVIS) is specific for what virus?

Answer 11

Herpes Simplex Virus

Question 12

How does ELVIS work?

Answer 12

• 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)

Question 13

Advantages of ELVIS

Answer 13

• Very little skill required
• Expensive monoclonal antibodies not needed
• Sensitive and specific compared to cell culture

Question 14

What are two ways that antivirals work?

Answer 14

1. nucleoside analogs (allow virus to add it in and then destroy it)
2. enzyme inhibitors

Question 15

Are antivirals “virus specific” or “broad spectrum”

Answer 15

virus specific

Question 16

What is CMV’s antiviral

Answer 16

Gangciclovir

Question 17

What is HSV’s antiviral

Answer 17

Acyclovir

Question 18

What is HIV-1’s antiviral

Answer 18

Didanosine

Question 19

What is RSV’s antiviral

Answer 19

Ribavirin

Question 20

What test involves using components labeled with active enzymes?

Answer 20

Enzyme Immunoassay EIA Antigen Detection

Question 21

What is EIA used for?

Answer 21

to detect unknown Antigen or antibody (widely used for testing fecal samples for rotavirus antigen)

Question 22

Non competitive Solid-Phase Enzyme immunoassay for Antigen Detection

Answer 22

• viral antibody coats the tube
• viral antigen and enzyme is added
• incubate and rinse
• add substrate and if Ag/Ab binding then color change

Question 23

Strains of influenza A vary in regards to what?

Answer 23

hemagglutinin (H) and neuraminidase (N) types

Question 24

Non-competitive EIA for antibody detection

- reagents and equipment

Answer 24

• antigen coated tube
• Antibodies in patients serum
• Enzyme-labled antihuman igG
• Substrate solution

Question 25

Non-competitive EIA for antibody detection | - list the steps of preforming

Answer 25

- 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

Question 26

Non-competitive EIA for antibody detection | - appearance of pos and neg

Answer 26

Color change pos | No color change neg

Question 27

Competitive EIA for antibody detection | - List the steps

Answer 27

- 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

Question 28

Non-competitive EIA for ANTIGEN

Answer 28

- 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

Question 29

Advantages of Enzyme Immunoassay

Answer 29

1. Well standardized 2. Good for large volume testing 3. Can be automated or semi-automated 4. Cost effective 5. Requires less technical skill than many methods

Question 30

disadvantages of Enzyme Immunoassay

Answer 30

1. Unable to distinguish specific from nonspecific binding | 2. May require repeat and/or confirmatory testing

Question 31

BioMerieux Vidas | - general format

Answer 31

- Automated EIA = walk-away function | - pipette dips into reagent wells both antigen and antibody detection methods are available

Question 32

BioMerieux Vidas | - solid phase

Answer 32

pipette tip (coated on the inside)

Question 33

microwell-based EIA instruments | - general format

Answer 33

Automated EIA = completely or largely walk away | - used for detection unknown antibodies

Question 34

microwell-based EIA instruments | - solid phase

Answer 34

wall of microwell

Question 35

Multiplex Micro-bead systems

Answer 35

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