Immunological and Molecular Diagnostics Flashcards
function of serology
Evaluation of immune status / function
Exposure of animal to infection
Response to vaccination
Diagnosis of immune-mediated disease
function of immunoassay
Using labelled antibodies as detection reagents
Presence of pathogen in a biological sample
Measurement of a biomarker
Immunophenotyping
blood sample for serum
clotted sample
blood sample for cells
citrate/heparin
acute phase proteins
C-reactive protein, Serum amyloid A, fibrinogen,
haptoglobin
provide evidence of infl in horses
Serological markers of adaptive immunity
antibodies
cytokines
measuring total immunoglobulin can be useful in
Failure of passive transfer in foals
Specific Ig deficiency syndromes
Monoclonal / polyclonal gammopathy
antigen-specific immunoglobulin useful for
Exposure to pathogen
Response to vaccination
diagnosis of antibody-mediated hypersensitivity - Allergy, Autoimmunity
Diagnosis of FPT in foals
blood test 15 – 18 hours of age
ELISA can be used to detect either ___ or ____
antigen or antibodies
ELISA test early on in an infection vs later on
early test for antigen as antibodies in lag phase
later on test for antibodies as the body is responding to the disease
immunofluorescence assay (IFA)
same principle as ELISA exept detection antibody marked with fluorescent marker not enzyme
virus neutralization (VN) assay
cultured cells infected with the virus +/- serum from patient
if antibody absent - cells infected
VN assay advantage over ELISA or IFA
indicates presence of biologically active antibody
presence of antibody in ELISA does not necessarily mean that it is protective
virus neutralising titre
greatest dilation of serum that prevents cell from becoming infected
Evaluation of T cell responses
CD4+ T cell
measure cytokine release following stimulation with specific antigen
Bovine TB gamma-IFN test
Heparinised blood sample to AHVLA
Culture cells in vitro with mycobacterial antigens (PPD)
Measure IFN-gamma production by ELISA
More sensitive but less specific than tuberculin “skin test”
Intradermal skin tesing
Used a lot for diagnosis of small animal allergic skin disease
Immediate-type (IgE-mediated) hypersensitivity
Tuberculin test for TB in cattle
Clip hair
Measure skin thickness
Inject PPD (avian & bovine)
Measure skin thickness 72 hrs later
Immunodiagnostics for Autoimmunity
coombs test for IMHA
ANA for SLE
measurement of specific autoantibodies
detection of pathogen in a sample
sandwich ELISA
IFA
immunohistochemistry
immunophenotyping
antibodies against cell-surface markers can tell which cell types are in a sample
can be done on cells in suspension by flow cytometry
Identification of pathogen’s nucleic acid in a biological sample
Usually using PCR or qPCR (quantitative PCR)
Pathogen-specific primers / probes tell if sample is +/-ve
16S rRNA sequencing can also be used
Genotyping pathogen
Influenza virus H + N genes
Virulence factors
disease susceptibility genes
Monogenic vs complex genetic disorder Autosomal vs sex-linked Dominant vs recessive Complete vs incomplete penetrance PCR based test
use of DNA genotyping
parentage
Diagnosis of disease – SOD1 mutation in degenerative myelopathy of GSD, KIT mutations in mast cell tumours
Establishing potential risk of disease in later life
Screening for carrier animals in a breeding population
Mast cells and KIT
Tissue mast cell viability is dependent upon the presence of stem cell factor (SCF)
SCF acts through the KIT (c-kit) receptor, which has tyrosine kinase activity.
KIT gene mutations are found in 30-50% of mast cell tumours
The presence of a KIT mutation seems to be associated with more aggressive disease.
acute phase proteins serological markers of innate or adaptive immunity?
innate
