Immunological And Molecular Diagnostics Flashcards
What can serology be used for?
Evaluation of immune status/function
- exposure to infection
- response to vax
- diagnosing immune-mediated disease
What does immunoassay involve?
Using labelled ABs as detection reagents
- ID presence of a pathogen
- measurement of biomarkers
- Immunophenotyping
WHat two types of sample may be submitted for immunodiagnostics?
> Blood - clotted sample for serum - citrate/heparin anticoagulant for cells > Tissue biopsy - Lymphoid tissue - lesion
What are serological markers of the innate immune system?
Acute phase proteins
- C reactive proteins
- Serum amyloid A
- Fibrinogen
- Haptoglobin
What are the serological markers of the adaptive immune system?
- Antibodies
- Cytokines
What are the 2 uses of measuring serum antibody? In what situations are these used?
> Total Ig - FPT foals - Specific Ig deficiency sybdromes -Mono/polyclonal gammopathy > Ag specific Ig - exposure to pathogens (eg. EIA, BVDV) - Vax response (eg. rabies for PETS) - Dx Ab-mediated hypersensitivity (allergy and autoimmunity)
What age is FPT blood test carried out in foals?
15-18hours
What is the smallest serum protein?
Albumen
What makes up the majority of the rest of the spectrum of serum proteins?
Gamma globulins = Immunoglobulins (mainly)
Give 3 common ways of measuring Ag specific AB
- ELISA
- IFAT (immunoflouroescent AB test)
- VN assay
Give 4 less common ways of measuring Ag specific AB
- CFT (Complement fixation test)
- HI (Haemaglutination inhibition)
- MAT (Microscopic agglutination test)
- AGID (Agar gel immunodiffusion)
How soon after vax should rabies serology be carried out? When else may a similar protocol be used?
> 3-4 weeks
> WSAVA strongly recommend serology in dogs 3-4 weeks post 1* course of normal vax
How long may the plateaux phase of AB production last?
Variable - weeks/months/yeasr
When should ELISA testing for Ag be carried out? Ab?
Ag 5d post infection
What may be carried out following a positive elisa?
Antibody titre - dilute solution and repeat ELISA until result is positive. Smaller the number (ie. ^ dilution) the more Abs are present in original sample
How does IFA differ from ELISA?
Fluorescent marker rather than enzymes
Outline how VNAs are carried out
- Infect cells with virus -> cytopathic effects seen
- Add serum to cells prior to virus infection -> look for cytopathic effect
How do the results from a VN differ in information cf. ELISA?
- VN indicates biologically active, functional AB
- Presence of Ab on ELISA does not mean it is protective
How can T-cell responses be evaluated? Give an example.
> Measure cytokine production released by T cells to encourage proliferation of fellow T cells (can only be carried out on live cells immediately after sample collection)
eg. bovine TB gamma IFN test
- heparinised blood sample sent to AHVLA
- culture cells with mycobacterial Ag (PPD purified protein derivative)
- measure IFN-g production by ELISA
How does the bovine IFNg test compare to the tuberculin skin test?
IFNg more sensitive but less specific than skin test
How are immunodiagnostics used for allergy?
> Serology based - measurement of allergen specific IgE > Intra-dermal - Immediate IgE mediated *usually used for this* - Delayed T cell mediated
What trademarked immunoassay test is specific for IgE?
Allercept
What other Igs may be produced in an allergic reaction? Are these of clinical significance?
IgG can be produced with NO clinical signs
How long should intradermal type 4 sensitivity tests be left before checking result?
72 hours -> 1 week
Outline the tuberculin intra dermal test. Why are 2 types of PPD injected?
- Measure skin thickness, inject AVIAN and BOVINE PPD, measure skin thickness 72hrs later
- Avian strain of TB doesn’t cause clinical problems but can cross react in test -> look to check the bovine bump is ^ than avian to confirm immunity
In which species is autoimmune disease most common? Which tests are available for autoimmune disease?
Dogs
- Coombs test for IMHA
- ANA test for SLE
- Specific Ab serology eg. AChR Abs in myasthenia gravis; TGAA in hypothyroidism
What tests can be used to identify pathogens or biomarkers in a sample?
> biological fluid - Sandwich ELISA - Immunofluorescence > tissue - Immunofluorescence - immunohistochemistry
What is immunohistochemistry?
ELISA on a slide instead of in a liquid well
- tissue section on slide, enzyme labelled Ab against cell surface marker
What tissue sample can BVDV be detected in?
Immunohistochemistry of ear notch biopsy (automatically biopsied when ear pierced)
How are PI BVDV calves treated?
CULL
What tissue sample is tested for parvo? Leptospira?
Parvo = diluted faecal sample Leptospira = urine
Give 3 methods of using labelled ABs for measurement of a biomarker
- ELISA
- Chemiluminescence eg. immulite
- Radioimmunoassat (RIA)
How does immunophenotyping work? Give egs.
- ABs against cell surface markers used to phenotype different cells within a fluid (flow cytometry) or tissue biopsy (IHC/IF)
> cell phenotypes within a lesion eg. canine lymphoma sub-types (T v B cell) prognosis differs
How an molecular diagnositcs be used to ID a pathogen?
- ID pathogen nucleic adic (DNA/RNA)
- PCR or qPCR for pathogen specific primers/probes
- sequence analysis (look up online database to ID bacterial spp)
Give 2 uses of molecular diagnositcs of the pathogen
- Genotyping pathogen
- influenza virus H and N gnees
- virulence factors of E. Coli - Epidemiological studies
- FMDV genotype
- M. Bovis spoligotype
Give 2 uses of molecular diagnostics of the host
- Gene polymorphisms and mutations
- insertions, deletions, substitutions - Disease susceptibility genes NB consider:
- monogenic v complex genetic disorders
- autosomal v sex linked
- dominant v recessive
- complete v incomplete penetrance
What molecular diagnostic technique may be used to identify mutations in the host?
PCR- based
- gene specific primers flank mutation
- amplicon size analysis for larger insertions and deletions
- dequence analysis for point mutations
- wild-type/mutant specific probes
- RFLPs
What may DNA genotyping be used for?
- parentage
- disease (eg. SOD1 mutation in degenerative myelopathy of GSDs; KIT mutations in mast cell tumours)
- potential disease risk/susceptibility genes [becoming more common use]
- screening for carrier animals in breeding population (eg. CLAD in Irish Setters, PRA several breeds)
What is KIT? What disease is it associated with?
- Stem cell factor receptor
- Mast cell tumours
> presence of KIT -> more aggressive tumours but also more responsive to tyrosine kinase inhibition treatment
