Immunological and Molecular Diagnostics Flashcards
How do you evaluate immune function?
serology
what is an immnoassay? 3 examples
using Abs as detection reagents (presence of a specific pathogen, measurement of a biomarker, immunophenotyping)
which anticoagulant do you use for immune function?
citrate (best), heparin (ok)
What are serological markers of innate immunity?
acute phase proteins (APPs)
Examples of APPs
c-reactive protein, serum amyloid A, fibrinogen, haptoglobulin
Serological markers of adaptive immunity - 2
Antibodies (usually) or cytokines (new to veterinary medicine)
How to measure total immunoglobulin level?
radial immunodiffusion or serum protein electrophoresis
When would you want to measure total immunoglobulin?
FPT (foals especially)
specific Ig deficiency syndromes
monoclonal/polyclonal gammopathies (e.g. myeloma)
When do you look at antigen-specific immunoglobulins? 3
- exposure of an individual or herd to a specific pathogen
- response to vaccination
- diagnosis of antibody-mediated hypersensitivity (allery/autoimmunity)
Methods ot detect antigen-specific immunoglobulin - 7
ELISA, IFAT, VNA, CFT, HI, MAT, AGID
What does an indirect ELISA detect?
Ab in serum
What does a direct ELISA detect?
Antigen
How is IFA different to ELISA?
The detection Ab is labelled with a fluorescent marker rather than an enzyme so the presence of fluorescence is a positive result
What are the two results of cells infected in a VNA?
Cytopathic viruses –> kill cells
NCP viruses –> infection of the target cells needs to be visualised by direct IFA.
What is the virus neutralising titre?
The greatest dilution of serum that precents the cells from becoming infected
Why is VNA better than ELISA or IFA?
It indicates the presence of biologically active antibody (i.e. VN in vitro = VN in vivo)
Define MAT. Use?
Microscopic agglutination test (e.g. leptospirossis)
What is AGID and what is it used for?
Agar gel immunodiffusion test (equine infectious anaemia)
How can CD4+ TC responses be evaluated in vitro?
Measuring cytokine release following stimulation with specific antigen (e.g. bovine IFN-gamma ELISA)
What is a DTH?
Delayed-type hypersensitivity test
What is SICCT? What is it the basis of?
Single intradermal comparitive cervical tuberculin - the basis for bovine TB testing in the UK
When is adaptive immunodeficiency usually seen? What is it usually associated with? 2
Seen after MDA wanes. Usually associated with lymphopaenia and/or agammaglobulinaemia
What is the Allercept assay?
Used for small animal allergic skin disease. Uses a labelled recombinant Fc-epsilon receptor detection system (shows a colour change).
What can intradermal skin testing be used for?
To identify immediate-type (IgE-mediated) and delayed-type (TC-mediated) hypersensitivities
What do you use the Coomb’s test for?
IMHA
What do you use ANA for?
SLE
Example of where you would use specific autoantibodies?
- AChR in myasthenia gravis,
- Thyroglobulin in hypothyroidism (TGAA)
What is the PPD that you inject cows with for the TB test?
Purified protein derivative - contains avian and bovine types of mycobacteria (since cattle are routinely exposed to the avian type anyway).
How would you detect the presence of pathogen antigen in biological fluid or tissue biopsy (i.e. a clinical sample)?
- sandwich ELISA (fluids/faeces)
- immunofluorescence (smears, tissue sections)
- immunohistochemistry (tissue sections)
What type of molecules are biomarkers?
usually proteins (e.g. cytokine, hormone)
Define HRP
horseradish peroxidase (used in ELISAs)
What is molecular diagnostics?
determining the presence of nucleic acid
What does molecular diagnostics involve? 4
- 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)
What can you use molecular diagnostics to look at?
Pathogen AND/OR host
What should be considered when doing molecular diagnostic genetic testing of animals? 5
- Mutation type (insertion, deletion or substitution)
- simple or complex genetic disorder?
- autosomal or sex-linked?
- dominant or recessive?
- complete or incomplete penetrance?
How can the target region be analysed after PCR primer flanking and amplification? 4
- 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
Uses of DNA genotyping -4
- 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
What is KIT?
A gene that codes for stem cell factor receptor, mast cells use this to survive and proliferate.
What happens when KIT mutates?
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.
