Immunological and Molecular Diagnostics

Question 1

How do you evaluate immune function?

Answer 1

serology

Question 2

what is an immnoassay? 3 examples

Answer 2

using Abs as detection reagents (presence of a specific pathogen, measurement of a biomarker, immunophenotyping)

Question 3

which anticoagulant do you use for immune function?

Answer 3

citrate (best), heparin (ok)

Question 4

What are serological markers of innate immunity?

Answer 4

acute phase proteins (APPs)

Question 5

Examples of APPs

Answer 5

c-reactive protein, serum amyloid A, fibrinogen, haptoglobulin

Question 6

Serological markers of adaptive immunity - 2

Answer 6

Antibodies (usually) or cytokines (new to veterinary medicine)

Question 7

How to measure total immunoglobulin level?

Answer 7

radial immunodiffusion or serum protein electrophoresis

Question 8

When would you want to measure total immunoglobulin?

Answer 8

FPT (foals especially)
specific Ig deficiency syndromes
monoclonal/polyclonal gammopathies (e.g. myeloma)

Question 9

When do you look at antigen-specific immunoglobulins? 3

Answer 9

• exposure of an individual or herd to a specific pathogen
• response to vaccination
• diagnosis of antibody-mediated hypersensitivity (allery/autoimmunity)

Question 10

Methods ot detect antigen-specific immunoglobulin - 7

Answer 10

ELISA, IFAT, VNA, CFT, HI, MAT, AGID

Question 11

What does an indirect ELISA detect?

Answer 11

Ab in serum

Question 12

What does a direct ELISA detect?

Answer 12

Antigen

Question 13

How is IFA different to ELISA?

Answer 13

The detection Ab is labelled with a fluorescent marker rather than an enzyme so the presence of fluorescence is a positive result

Question 14

What are the two results of cells infected in a VNA?

Answer 14

Cytopathic viruses –> kill cells

NCP viruses –> infection of the target cells needs to be visualised by direct IFA.

Question 15

What is the virus neutralising titre?

Answer 15

The greatest dilution of serum that precents the cells from becoming infected

Question 16

Why is VNA better than ELISA or IFA?

Answer 16

It indicates the presence of biologically active antibody (i.e. VN in vitro = VN in vivo)

Question 17

Define MAT. Use?

Answer 17

Microscopic agglutination test (e.g. leptospirossis)

Question 18

What is AGID and what is it used for?

Answer 18

Agar gel immunodiffusion test (equine infectious anaemia)

Question 19

How can CD4+ TC responses be evaluated in vitro?

Answer 19

Measuring cytokine release following stimulation with specific antigen (e.g. bovine IFN-gamma ELISA)

Question 20

What is a DTH?

Answer 20

Delayed-type hypersensitivity test

Question 21

What is SICCT? What is it the basis of?

Answer 21

Single intradermal comparitive cervical tuberculin - the basis for bovine TB testing in the UK

Question 22

When is adaptive immunodeficiency usually seen? What is it usually associated with? 2

Answer 22

Seen after MDA wanes. Usually associated with lymphopaenia and/or agammaglobulinaemia

Question 23

What is the Allercept assay?

Answer 23

Used for small animal allergic skin disease. Uses a labelled recombinant Fc-epsilon receptor detection system (shows a colour change).

Question 24

What can intradermal skin testing be used for?

Answer 24

To identify immediate-type (IgE-mediated) and delayed-type (TC-mediated) hypersensitivities

Question 25

What do you use the Coomb’s test for?

Answer 25

IMHA

Question 26

What do you use ANA for?

Answer 26

SLE

Question 27

Example of where you would use specific autoantibodies?

Answer 27

• AChR in myasthenia gravis,

- Thyroglobulin in hypothyroidism (TGAA)

Question 28

What is the PPD that you inject cows with for the TB test?

Answer 28

Purified protein derivative - contains avian and bovine types of mycobacteria (since cattle are routinely exposed to the avian type anyway).

Question 29

How would you detect the presence of pathogen antigen in biological fluid or tissue biopsy (i.e. a clinical sample)?

Answer 29

• sandwich ELISA (fluids/faeces)
• immunofluorescence (smears, tissue sections)
• immunohistochemistry (tissue sections)

Question 30

What type of molecules are biomarkers?

Answer 30

usually proteins (e.g. cytokine, hormone)

Question 31

Define HRP

Answer 31

horseradish peroxidase (used in ELISAs)

Question 32

What is molecular diagnostics?

Answer 32

determining the presence of nucleic acid

Question 33

What does molecular diagnostics involve? 4

Answer 33

• PCR or quantitative PCR (usually)
• sequencing for identification (for less obvious cases)
• characterise the pathogen (e.g. H and N influenza genes)
• epidemiological studies (spread of specific subtypes/genotypes)

Question 34

What can you use molecular diagnostics to look at?

Answer 34

Pathogen AND/OR host

Question 35

What should be considered when doing molecular diagnostic genetic testing of animals? 5

Answer 35

• Mutation type (insertion, deletion or substitution)
• simple or complex genetic disorder?
• autosomal or sex-linked?
• dominant or recessive?
• complete or incomplete penetrance?

Question 36

How can the target region be analysed after PCR primer flanking and amplification? 4

Answer 36

• amplicon size
• direct sequence analysis
• use of WT/mutant-specific probes
• RFLP where the mutation alters the digestion pattern of the amplicon with a specific restriction enzyme

Question 37

Uses of DNA genotyping -4

Answer 37

• establishing parentage
• diagnosis of a disease (SOD1 - degenerative myelopathy in GSDs)
• screening for carrier animals (CLAD in irish setters)
• establishing potential risk of disease later in life

Question 38

What is KIT?

Answer 38

A gene that codes for stem cell factor receptor, mast cells use this to survive and proliferate.

Question 39

What happens when KIT mutates?

Answer 39

Canine mast cell tumours: mutation of KIT gene found in 30-50% of these and seems to be associated with a more aggressive disease. There is a large insertion mutation in exon 11 (detect by larger sample on gel electrophoresis versus normal). Makes spread of the neoplasia much more likely. Susceptibility to certain tyrosine kinase inhibitors is actually enhanced so although the mutation carries a worse prognosis, the animals affected actually respond better to TKI drugs.