Immunological And Molecular Diagnostics Flashcards by laura knight

What can serology be used for?

Evaluation of immune status/function

exposure to infection
response to vax
diagnosing immune-mediated disease

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What does immunoassay involve?

Using labelled ABs as detection reagents

ID presence of a pathogen
measurement of biomarkers
Immunophenotyping

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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

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What are serological markers of the innate immune system?

Acute phase proteins

C reactive proteins
Serum amyloid A
Fibrinogen
Haptoglobin

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What are the serological markers of the adaptive immune system?

Antibodies

- Cytokines

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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)

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What age is FPT blood test carried out in foals?

15-18hours

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What is the smallest serum protein?

Albumen

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What makes up the majority of the rest of the spectrum of serum proteins?

Gamma globulins = Immunoglobulins (mainly)

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Give 3 common ways of measuring Ag specific AB

ELISA
IFAT (immunoflouroescent AB test)
VN assay

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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)

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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

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How long may the plateaux phase of AB production last?

Variable - weeks/months/yeasr

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When should ELISA testing for Ag be carried out? Ab?

Ag 5d post infection

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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

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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

1. ELISA 2. Chemiluminescence eg. immulite 3. 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

1. Genotyping pathogen - influenza virus H and N gnees - virulence factors of E. Coli 2. Epidemiological studies - FMDV genotype - M. Bovis spoligotype

Give 2 uses of molecular diagnostics of the host

1. Gene polymorphisms and mutations - insertions, deletions, substitutions 2. 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