Immunological Techniques in Diagnostics & Research

Question 1

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

Answer 1

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

Question 2

What is determined by flow cytometry?

Answer 2

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

Question 3

What technique is used to determine blood types?

Answer 3

Blood typing done by agglutination reaction

Question 4

Describe the key features of ABO blood types

Answer 4

• 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

Question 5

How is western blotting used in research ?

Answer 5

Cell signalling proteins

Mechanism of action for cancer drug

Question 6

Outline the process of flow cytometry

Answer 6

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

Question 7

How is IHC used to diagnose cancer?

Answer 7

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

Question 8

How is flow cytometry used in research?

Answer 8

Identification and analysis of immune cells in infections and cancer immunotherapy

Question 9

Outline how raising antibodies is carried out in a lab

Answer 9

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

Question 10

What are the diagnostic uses of western blotting?

Answer 10

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

Question 11

Outline the process of IHC

Answer 11

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

Question 12

Describe the structure of Antibodies

Answer 12

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

Question 13

What are the diagnostic applications of Flow cytometry?

Answer 13

Diagnosis of haematological malignancies

CD4 T cell counts in HIV

Question 14

What is the significance of antibodies?

Answer 14

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

Question 15

Explain the role of B-Raf in cancer

Answer 15

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

Question 16

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

Answer 16

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

Question 17

What is ELISA?

Answer 17

Enzyme linked immunosorbant assays

Question 18

What are the applications of confocal microscopy?

Answer 18

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

Question 19

What is IHC?

Answer 19

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

Question 20

What is the role of ELISA?

Answer 20

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

Question 21

What are antibodies?

Answer 21

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

Question 22

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

Answer 22

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

Question 23

What are the 4 different types of ELISA?

Answer 23

Direct
Indirect
Sandwich
Competitive

Question 24

What are the applications of ELISA?

Answer 24

• 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

Question 25

Outline the process of blood typing

Answer 25

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

Question 26

What is flow cytometry?

Answer 26

Used to analyse proteins on cells, in suspension | Often involves use of antibodies (commercially produced) that are conjugated to fluorochromes

Question 27

What are immunological techniques?

Answer 27

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

Question 28

Outline how sandwich ELISA is carried out?

Answer 28

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

Question 29

What are the most common immunological technique?

Answer 29

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

Question 30

Describe the process of western blotting

Answer 30

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

Question 31

What is western blotting?

Answer 31

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