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Principles of Science > Immunological And Molecular Diagnostics > Flashcards

Immunological And Molecular Diagnostics Flashcards

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1
Q

What can serology be used for?

A

Evaluation of immune status/function

  • exposure to infection
  • response to vax
  • diagnosing immune-mediated disease
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2
Q

What does immunoassay involve?

A

Using labelled ABs as detection reagents

  • ID presence of a pathogen
  • measurement of biomarkers
  • Immunophenotyping
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3
Q

WHat two types of sample may be submitted for immunodiagnostics?

A 
> Blood
- clotted sample for serum 
- citrate/heparin anticoagulant for cells 
> Tissue biopsy 
- Lymphoid tissue 
- lesion
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4
Q

What are serological markers of the innate immune system?

A

Acute phase proteins

  • C reactive proteins
  • Serum amyloid A
  • Fibrinogen
  • Haptoglobin
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5
Q

What are the serological markers of the adaptive immune system?

A
  • Antibodies

- Cytokines

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6
Q

What are the 2 uses of measuring serum antibody? In what situations are these used?

A 
> 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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7
Q

What age is FPT blood test carried out in foals?

A

15-18hours

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8
Q

What is the smallest serum protein?

A

Albumen

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9
Q

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

A

Gamma globulins = Immunoglobulins (mainly)

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10
Q

Give 3 common ways of measuring Ag specific AB

A
  • ELISA
  • IFAT (immunoflouroescent AB test)
  • VN assay
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11
Q

Give 4 less common ways of measuring Ag specific AB

A
  • CFT (Complement fixation test)
  • HI (Haemaglutination inhibition)
  • MAT (Microscopic agglutination test)
  • AGID (Agar gel immunodiffusion)
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12
Q

How soon after vax should rabies serology be carried out? When else may a similar protocol be used?

A

> 3-4 weeks

> WSAVA strongly recommend serology in dogs 3-4 weeks post 1* course of normal vax

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13
Q

How long may the plateaux phase of AB production last?

A

Variable - weeks/months/yeasr

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14
Q

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

A

Ag 5d post infection

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15
Q

What may be carried out following a positive elisa?

A

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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16
Q

How does IFA differ from ELISA?

A

Fluorescent marker rather than enzymes

17
Q

Outline how VNAs are carried out

A
  • Infect cells with virus -> cytopathic effects seen

- Add serum to cells prior to virus infection -> look for cytopathic effect

18
Q

How do the results from a VN differ in information cf. ELISA?

A
  • VN indicates biologically active, functional AB

- Presence of Ab on ELISA does not mean it is protective

19
Q

How can T-cell responses be evaluated? Give an example.

A

> 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

20
Q

How does the bovine IFNg test compare to the tuberculin skin test?

A

IFNg more sensitive but less specific than skin test

21
Q

How are immunodiagnostics used for allergy?

A 
> Serology based 
- measurement of allergen specific IgE
> Intra-dermal 
- Immediate IgE mediated *usually used for this*
- Delayed T cell mediated
22
Q

What trademarked immunoassay test is specific for IgE?

A

Allercept

23
Q

What other Igs may be produced in an allergic reaction? Are these of clinical significance?

A

IgG can be produced with NO clinical signs

24
Q

How long should intradermal type 4 sensitivity tests be left before checking result?

A

72 hours -> 1 week

25
Q

Outline the tuberculin intra dermal test. Why are 2 types of PPD injected?

A
  • 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
26
Q

In which species is autoimmune disease most common? Which tests are available for autoimmune disease?

A

Dogs

  • Coombs test for IMHA
  • ANA test for SLE
  • Specific Ab serology eg. AChR Abs in myasthenia gravis; TGAA in hypothyroidism
27
Q

What tests can be used to identify pathogens or biomarkers in a sample?

A 
> biological fluid 
- Sandwich ELISA
- Immunofluorescence 
> tissue 
- Immunofluorescence 
- immunohistochemistry
28
Q

What is immunohistochemistry?

A

ELISA on a slide instead of in a liquid well

- tissue section on slide, enzyme labelled Ab against cell surface marker

29
Q

What tissue sample can BVDV be detected in?

A

Immunohistochemistry of ear notch biopsy (automatically biopsied when ear pierced)

30
Q

How are PI BVDV calves treated?

A

CULL

31
Q

What tissue sample is tested for parvo? Leptospira?

A 
Parvo = diluted faecal sample 
Leptospira = urine
32
Q

Give 3 methods of using labelled ABs for measurement of a biomarker

A
  1. ELISA
  2. Chemiluminescence eg. immulite
  3. Radioimmunoassat (RIA)
33
Q

How does immunophenotyping work? Give egs.

A
  • 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
34
Q

How an molecular diagnositcs be used to ID a pathogen?

A
  • ID pathogen nucleic adic (DNA/RNA)
  • PCR or qPCR for pathogen specific primers/probes
  • sequence analysis (look up online database to ID bacterial spp)
35
Q

Give 2 uses of molecular diagnositcs of the pathogen

A
  1. Genotyping pathogen
    - influenza virus H and N gnees
    - virulence factors of E. Coli
  2. Epidemiological studies
    - FMDV genotype
    - M. Bovis spoligotype
36
Q

Give 2 uses of molecular diagnostics of the host

A
  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
37
Q

What molecular diagnostic technique may be used to identify mutations in the host?

A

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
38
Q

What may DNA genotyping be used for?

A
  • 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)
39
Q

What is KIT? What disease is it associated with?

A
  • Stem cell factor receptor
  • Mast cell tumours
    > presence of KIT -> more aggressive tumours but also more responsive to tyrosine kinase inhibition treatment

Principles of Science (27 decks)

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