Diagnostic Tests

Question 1

Diagnostic tests used to determine:

Answer 1

• exposure to specific pathogen
• status of infection
• antibody levels
• cell-mediated immune response
• determine vaccine efficacy
• allergies or hypersensitivity reactions
• autoimmune reactions

Question 2

Conjugates/labelling of Fc fragment of antibodies for testing

Answer 2

• enzymes
• Fluorochromes
• radioactivity

Question 3

Two methods for labelling certain cell surfaces

Answer 3

1. using a directly conjugated antibody
2. using a primary and secondary antibody (second has conjugate)

**usually depends on what is commercially available

Question 4

Polyclonal antibody

Answer 4

• cheap to produce
• mixed population of antibodies
• may bind to different areas of the target molecule
• tolerant or small changes in protein structure

Question 5

Monoclonal antibody

Answer 5

• expensive to produce
• single antibody species
• will only bind single specific sites
• may recognise a particular protein form

Question 6

Production of monoclonal antibodies

Answer 6

1. Immunize rodent with antigen
2. form antibody-forming cells
3. Fuse with tumor cell
4. Produce antibody-producing hybridomas, clone them
5. Produce monoclonal antibodies

Question 7

Separate molecules by size

Answer 7

• Matrix that makes it hard for material to migrate through. Smaller molecules will travel faster than larger molecules and highly charged molecules travel faste.
• migrate with electric field or centrifugation

Question 8

Two possibilities for immune diagnostics

Answer 8

1. detect antigen
2. detect immune response (humoral, cell mediated, cytokines)

Question 9

Ways to detect pathogens

Answer 9

DNA or RNA
- PCR
- Southern Blot
- Northern Blot
- in situ hybridization

PROTEIN
- immunohistochemistry
- microscopy

LIVE PROTEIN
- isolation and amplification in culture
- microscopy

Question 10

PCR

Answer 10

• amplify DNA or RNA
• detect presence of pathogens

Question 11

Southern Blot

Answer 11

• method to detect specific DNA fragments (size/strain) and visualize them using electrophoresis and blot
  *Can find specific gene within genome
• gel, blot onto membrane, stain, displays size of fragments based on where they end up

Question 12

Northern Blot

Answer 12

• method to detect RNA fragments (size/strain of pathogen)
• gel, blot onto membrane, stain, detect size

Question 13

in situ hybridization

Answer 13

• labelling tissue samples but no gel
• not really used much anymore

Question 14

Immunohistochemistry

Answer 14

• using either direct or indirect assays (enzyme= form of label)
• pathogen on tissue slide, bind antibody (either primary with enzyme or primary without and then secondary with enzyme)
• label or enzyme will result in colour change if pathogen present (under microscope)

Question 15

Immunofluorescence

Answer 15

• Same principle as immunohistochemistry but with flurochromes as the “label”
• will fluoresce different colours

Question 16

Pathogen isolation and culture

Answer 16

1. Take tissue sample
2. Homogenize
3. Grow on plates (dilutions)
4. Stain plates
5. Perform a plaque assay (More plaques= more pathogen and more dead cells)

Question 17

What do plaque assays measure?

Answer 17

Measure the virus titer (quantity of infectious virus)

Question 18

Detect immune response (RNA)

Answer 18

• PCR
• Real-time PCR
• Multiplex assays

Used for cytokines and other specific proteins of interest

Question 19

Real-time PCR

Answer 19

• Monitors the amplification of DNA or RNA in real time, not only at the end
• As more copies are made, there is a greater signal. There is a threshold that has to be met to be seen/detected.
  **The fewer cycles it takes, the more you had to start with.

Question 20

Detect immune response (antibodies)

Answer 20

• ELISA
• Agglutination

**used for antibodies, subclasses of antibodies, pathogens, cytokines, other specific proteins of interest

Question 21

ELISA (enzyme-linked immunosorbent assays)

Answer 21

Used:
1. detect and quantify specific-antibody titer (direct/indirect assays or sandwich)
2, Quantify antigen (capture: cytokines, viral antgens)

Question 22

Capture Assay of ELISA (Sandwich)

Answer 22

Sandwich method

• antibody has to capture antigen first (because the antigen is not on the tissues)
• then use primary and secondary antibodies to do the rest

Question 23

ELISA dilutions

Answer 23

• Highest presence of antibodies will be present prior to dilution

Question 24

Titre

Answer 24

A titre is the last dilution to give a positive test response

**If testing serum for antibodies, the more antibodies present the greater the dilution factor that will still yield a positive test

Question 25

Signal amplification

Answer 25

Used when need to enhance the signal reaction

• Secondary antibodies are linked to another signal (streptavidin) which link more molecules and increase the amount of reactions that will occur therefore increasing the signal produced

Question 26

Agglutination

Answer 26

The immune complexes are formed between antigen and antibody. If the antibodies recognize their antigen, they can bind to more than one antigen which results in visible clumping and agglutination

Question 27

Agglutination based assays

Answer 27

• make a series of dilutions of patient serum with saline
• RBCs are coated with antigen (acting like a sensitized particulate)
• RBCs that are not agglutinated will fall down into the well. If agglutinated, will sit on surface

**Titer- last dilution of the test serum that agglutinates the antigen-coated particles/RBCs

Question 28

Immunoprecipitation

Answer 28

• in both antigen and antibody excess, small and soluble immune complexes are produced
• at optimal proportions, due to cross linking of the molecules, large insoluble complexes are generated
• Antibody and antigen will move towards each other and forms a lattice or visible line or band in the gel

Question 29

Coggins Test

Answer 29

• detects antibodies in horse infectious anemia
• Positive test- will see line between antigen and antibodies

Question 30

Radial immunodiffusion

Answer 30

• Tests the IgG levels in serum
• Gel has anti-sera to the antibody
• Antibodies and anti-sera interact. Diameter of ring of diffusion is proportional to the amount of antibody present in the sample

Question 31

Neutralization Assay

Answer 31

• neutralizing antibodies will destroy the pathogen before it destroys the cells
• Less plaques, no cytopathic effect therefore not killing of cells and lots of neutralizing antibodies
  *Dilutions will increase Cytopathic effect (more plaques) and less neutralizing antibodies