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Pathology 1 > Immunological Techniques in Diagnostics & Research > Flashcards

Immunological Techniques in Diagnostics & Research Flashcards

1
Q

What is the significance of A, B and RhD on RBCs?

A

Act as antigens eliciting an immune response in donor mismatches / blood transfusions

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2
Q

What is determined by flow cytometry?

A
  • Whether a cell expresses a target protein
  • The amount of expression of target protein
  • The cells identity and analysis by protein expression
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3
Q

What technique is used to determine blood types?

A

Blood typing done by agglutination reaction

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4
Q

Describe the key features of ABO blood types

A
  • A and B are glycoproteins on RBCs
  • There are 4 blood types - A, B, AB, O
  • Rhesus factors (D protein or RhD) proteins on RBC
    surface determine charge -ve/+ve
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5
Q

How is western blotting used in research ?

A

Cell signalling proteins

Mechanism of action for cancer drug

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6
Q

Outline the process of flow cytometry

A
  1. Fluorochrome-conjugated antibodies specific for target
    protein added to cells
  2. Cells passed through lasers exciting fluorochrome
    (blue laser excites HTC emitting green light)
  3. Emitted light detected and plotted on graph
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7
Q

How is IHC used to diagnose cancer?

A

IHC used to stain B-Raf cancer protein in tissue sections from cancer patients and direct eligible for treatment with B-Raf inhibitors

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8
Q

How is flow cytometry used in research?

A

Identification and analysis of immune cells in infections and cancer immunotherapy

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9
Q

Outline how raising antibodies is carried out in a lab

A
  1. Mice immunised with target protein
  2. B cells harvested & fused with tumour cells forming
    hybridoma
  3. Hybridoma produces antibodies against target protein
    which is selected and cloned
  4. Antibodies are secreted by cloned hybridoma which
    are collected & harvested to be used in immunological
    techniques
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10
Q

What are the diagnostic uses of western blotting?

A

Pathogen used, proteins are separated on a gel and transferred to a membrane which is mixed with patient serum to catch ab
Excess is washed off then secondary ab are added to visualise
e.g. HIV, Parasites

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11
Q

Outline the process of IHC

A
  1. Thin sections of tissue are cut
  2. Primary antibodies recognising target proteins are
    added to tissue
  3. Ab-Ag complex visualised using chromogenic detection
  4. Secondary ab specific to primary ab conjugated to HRP
    added
  5. HRP catalyses chromogen3,3-DAB into a brown
    precipitate at the protein’s location
  6. Visualised using light microscope
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12
Q

Describe the structure of Antibodies

A

Antibodies have a Y shaped structure

  • 2 Heavy chains and 2 light chains
  • Fab and Fc compartments
  • Fab specific for antigens
  • 5 different types of heavy chains producing 5 isotopes
  • IgA, IgG, IgM, IgD, IgE
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13
Q

What are the diagnostic applications of Flow cytometry?

A

Diagnosis of haematological malignancies

CD4 T cell counts in HIV

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14
Q

What is the significance of antibodies?

A

Important part of our immune response causing neutralisation and opsonisation in phagocytosis

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15
Q

Explain the role of B-Raf in cancer

A

Cancer caused by mutations in tumour suppressor /promoter genes causing uncontrolled cell division
B-Raf encodes B-Raf protein promoting cell division
many cancers have B-Raf mutation resulting in overproduction of B-Raf leading to uncontrolled cell division

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16
Q

How does flow cytometry determine amount of target protein in cell?

A

The amount of light emitted = amount of ab bound to protein = amount of protein expressed in cell

17
Q

What is ELISA?

A

Enzyme linked immunosorbant assays

18
Q

What are the applications of confocal microscopy?

A
  • mainly research
  • Identification and analysis of cells within tissues
  • Co localisation of different antigens
19
Q

What is IHC?

A

Immunohistochemistry
- shows the distribution and localisation of antigens in
tissue sections using antibody-antigen interactions

20
Q

What is the role of ELISA?

A

Quantifies amount of protein / antibody in liquid samples such as sera / tissue culture supernatants

21
Q

What are antibodies?

A

Specialised proteins produced by B cells that act as B cell receptors when membrane bound and can also be secreted

22
Q

What are the key differences in confocal microscopy compared to flow cytometry?

A

The cells analysed aren’t in suspension
Tissue sections / cells analysed are attached to microscope slides
Light emitted isn’t plotted, its examined under microscope
Can visualise protein location on cell

23
Q

What are the 4 different types of ELISA?

A

Direct
Indirect
Sandwich
Competitive

24
Q

What are the applications of ELISA?

A
  • ab titres in HIV & HepB patients serum
  • Bacterial toxin detection in food (E.Coli)
  • Home pregnancy testing detection of chorionic
    gonadotropin hormone (HCG) in urine
  • Research quantification of cytokines / chemokines /
    growth factors in tissue culture supernatants
25
Q

Outline the process of blood typing

A
  1. Blood sample mixed with antibody raised against A, B
    or RhD antigens
  2. Sample checked for agglutination by eye
  3. Agglutination indicates presence of antigen in blood
    sample
26
Q

What is flow cytometry?

A

Used to analyse proteins on cells, in suspension

Often involves use of antibodies (commercially produced) that are conjugated to fluorochromes

27
Q

What are immunological techniques?

A

Include both experimental methods to study immune response and methods to generate and use immunological agents as experimental tools

28
Q

Outline how sandwich ELISA is carried out?

A
  1. Add capture ab
  2. Add sample serum (Serum/Tc)
  3. Add detection ab (HRP)
  4. Add substrate
  5. Determine optical density using spectrophotometer

Density of blue solution = amount of protein in sample

29
Q

What are the most common immunological technique?

A

The most common techniques relate antibody production to detect specific proteins in biological samples

30
Q

Describe the process of western blotting

A
  1. Sample prepped - cells lysed, protein denatured
  2. Electrophoresis - lysates laoded on gel, proteins
    separated based on size
  3. Transfer to membrane - fractionated proteins
    transferred to membrane
  4. Stain for protein of interest
    • membrane incubated with primary ab specific for
      target protein
    • then incubated with HRP conjugated secondary ab
      specific for primary ab
    • Chemiluminescent HRP substrate added to membrane
      exposed to Xray
    • bleaches when exposed to light
31
Q

What is western blotting?

A

A technique used to detect proteins
Southern blot detects DNA
Northern blot detects RNA

Pathology 1 (20 decks)

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