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Serology > Immunofluorescence and Viral Disease Diagnosis > Flashcards

Immunofluorescence and Viral Disease Diagnosis Flashcards

1
Q

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

A

Immunofluorescence (FA) Staining in Virology

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2
Q

3 applications of FA

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

Procedure of Immunofluorescence staining (4 steps)

A
  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
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4
Q

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

A

bright green…stains the target

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5
Q

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

A

Red…stains the background

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6
Q

Steps in direct FA staining?

A
  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
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7
Q

Steps in indirect FA staining?

A
  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
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8
Q

In FA testing what are smears evaluated for?

A

Intensity and distribution of fluorescence

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9
Q

How do we stain cells from a cell culture monolayer?

A
  • use pipette/scraper to scrape cells off tube wall
  • spin to sediment the cells
  • make smear of sediment
  • stain, read
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10
Q

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

A
  • 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)
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11
Q

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

A

Herpes Simplex Virus

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12
Q

How does ELVIS work?

A
  • 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)
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13
Q

Advantages of ELVIS

A
  • Very little skill required
  • Expensive monoclonal antibodies not needed
  • Sensitive and specific compared to cell culture
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14
Q

What are two ways that antivirals work?

A
  1. nucleoside analogs (allow virus to add it in and then destroy it)
  2. enzyme inhibitors
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15
Q

Are antivirals “virus specific” or “broad spectrum”

A

virus specific

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16
Q

What is CMV’s antiviral

A

Gangciclovir

17
Q

What is HSV’s antiviral

A

Acyclovir

18
Q

What is HIV-1’s antiviral

A

Didanosine

19
Q

What is RSV’s antiviral

A

Ribavirin

20
Q

What test involves using components labeled with active enzymes?

A

Enzyme Immunoassay EIA Antigen Detection

21
Q

What is EIA used for?

A

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

22
Q

Non competitive Solid-Phase Enzyme immunoassay for Antigen Detection

A
  • viral antibody coats the tube
  • viral antigen and enzyme is added
  • incubate and rinse
  • add substrate and if Ag/Ab binding then color change
23
Q

Strains of influenza A vary in regards to what?

A

hemagglutinin (H) and neuraminidase (N) types

24
Q

Non-competitive EIA for antibody detection

- reagents and equipment

A
  • antigen coated tube
  • Antibodies in patients serum
  • Enzyme-labled antihuman igG
  • Substrate solution
25
Q

Non-competitive EIA for antibody detection

- list the steps of preforming

A
  • 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
26
Q

Non-competitive EIA for antibody detection

- appearance of pos and neg

A

Color change pos

No color change neg

27
Q

Competitive EIA for antibody detection

- List the steps

A
  • 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
28
Q

Non-competitive EIA for ANTIGEN

A
  • 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
29
Q

Advantages of Enzyme Immunoassay

A
  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
30
Q

disadvantages of Enzyme Immunoassay

A
  1. Unable to distinguish specific from nonspecific binding

2. May require repeat and/or confirmatory testing

31
Q

BioMerieux Vidas

- general format

A
  • Automated EIA = walk-away function

- pipette dips into reagent wells both antigen and antibody detection methods are available

32
Q

BioMerieux Vidas

- solid phase

A

pipette tip (coated on the inside)

33
Q

microwell-based EIA instruments

- general format

A

Automated EIA = completely or largely walk away

- used for detection unknown antibodies

34
Q

microwell-based EIA instruments

- solid phase

A

wall of microwell

35
Q

Multiplex Micro-bead systems

A

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

Serology (17 decks)

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