Immunological Techniques in Diagnostics & Research

Question 1

Describe the structure of antibodies

Answer 1

 Membrane-bound (B cell receptor)
 Secreted
 Antibodies have a Y-shaped structure
 2 × heavy chains & 2× light chains
 2 parts: Fc & Fab fragments
 Fab region is specific for a protein (“antigen”)
 5 different types of heavy chain in mammals generates 5 “isotypes” IgA, IgG, IgD, IgE & IgM

Question 2

How do we raise antibodies?(6)

Answer 2

 Mice are immunised with the target protein
 B cells are harvested & fused with tumour cells to form hybridoma
 Tumour cells are drug sensitive but not b-cells
 A hybridoma that produces antibody against the target protein is selected & cloned
 The antibodies secreted by the cloned hybridoma are harvested & used in immunological techniques
 Monoclonal antibodies have one binding site for one antigen.

Question 3

How does agglutination test work?

Answer 3

• A blood sample is mixed with antibodies raised against A, B or RhD antigens
• The sample is visually checked for agglutination (blood cells sticking together)
• Agglutination indicates the presence of antigens in the blood sample

Question 4

What are the 4 blood types?

Answer 4

A, B, AB & O

Question 5

What is Rhesus factor?

Answer 5

(D protein or RhD) is also a protein on the surface of RBCs (positive or negative)

Question 6

What is Flow cytometry?

Answer 6

a technology used to analyse the proteins on cells that are in suspension

Question 7

What can flow cytometry determine about a cell?

Answer 7

 The cell size and density
 Whether or not a cell expresses a target protein
 The amount of expression of a target protein
 The cells identity

Question 8

Often involves the use of commercially produced antibodies that are conjugated to…

Answer 8

fluorochromes on the Fc

Question 9

How a flow cytometer works?(4)

Answer 9

 Fluorochrome-conjugated antibodies specific for the target protein are added to the cells
 Cells are channelled past lasers that excite the fluorochrome (e.g. blue laser excites FITC which then emits green light and PE emits Red light)
 The light emitted from the excited fluorochromes is detected & plotted on a graph
 Amount of light emitted = amount of antibody bound to protein = amount of protein expressed by the cell

Question 10

Name some gated filters in flow cytometry

Answer 10

CD3+CD4+ and CD3+CD8+ T cells

Question 11

What are the applications of flow cytometry?(2)

Answer 11

 Diagnostics: CD4 T cell counts in HIV
Diagnosis of haematological malignancies
 Research: Identification & analysis of immune cells

Question 12

What is the role of confocal microscopy?

Answer 12

 Used to analyse tissue sections or cells attached to a microscope slide
 The light emitted by the fluorochrome-conjugated antibodies is observed under a microscope instead of plotted graphically

Question 13

What is a difference between confocal and flow cytometry?

Answer 13

 The cells to be analysed are not in suspension

Question 14

What is an advantage of confocal microscopy?

Answer 14

visualising where the protein is on the cell

Question 15

What are the applications of confocal microscopy?

Answer 15

 Mainly research eg. Zebrafish
 Identification & analyse cells within tissues
 Co-localisation of different antigens

Question 16

What is IHC? (3)

Answer 16

 IHC stands for ImmunoHistoChemistry
 Used to show the distribution & localization of antigens in tissue sections using antibody-antigen interactions.
 Very reliable and can be used to localise antibodies

Question 17

How does IHC work?(7)

Answer 17

 Thin sections of tissue are cut
 Primary antibodies that recognise the target protein are added to the tissue
 The antibody-antigen interaction is visualised using chromogenic detection
 A secondary antibody specific for the primary antibody conjugated to horseradish peroxidase (HRP) is added
 HRP catalyses the conversion of the chromogen 3,3-diaminobenzidine (DAB) substrate to produce a brown precipitate at the location of the protein
 The brown precipitate is then visualised using a light microscope
 You can see cells that are most likely there eg cancer cells and where they are made

Question 18

Examples of IHC…

Answer 18

 IHC is routinely used in the diagnosis of cancer

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

Question 19

What is a BRAF gene?

Answer 19

a gene that encodes B-Raf protein that promotes cell division

Question 20

What are Vemurafenib and dabrafenib?

Answer 20

are inhibitors used to treat B-Raf-positive tumours

Question 21

What is ELISA?

Answer 21

 Enzyme-Linked ImmunoSorbant Assay

 Quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants

Question 22

Applications of ELISA…

Answer 22

 Antibody titres in patient serum e.g viral infections such as HIV & Hepatitis B
 Detection of bacterial toxins in food such as Escherichia coli O157:H7
 Home pregnancy testing detection of human chorionic gonadotropin hormone (HCG) in urine
 Research quantification of cytokines/chemokines/growth factors in tissue culture supernatants

Question 23

Name the 4 types of ELISA

Answer 23

 Direct
 Indirect
 Sandwich
 Competitive

Question 24

What does density of blue solution indicate in sandwich ELISA?

Answer 24

Amount of protein in a sample

Question 25

What is the process Western blotting?

Answer 25

1. Sample preparation
    Cells are lysed & proteins denatured
2. Electrophoresis
    Lysates are loaded onto a gel & proteins separated based on size
3. Transfer to membrane
    Fractionated proteins are transferred onto a membrane
4. Stain for protein of interest
    The membrane is incubated with a primary antibody specific for the target protein
    The membrane is then incubated with a HRP conjugated secondary antibody specific for the primary antibody
    A chemiluminescent HRP substrate is added to the membrane
    The membrane is exposed to x ray film that “bleaches” when exposed to light

Question 26

What are the applications of Western blotting?

Answer 26

 Diagnostic use

 Research: Cell signalling proteins. Mechanism of action for cancer drugs

Question 27

How is Western blotting used as a diagnostic tool for pathogens?

Answer 27

 Diagnostic use
The pathogen is lysed & its proteins are separated on a gel. The proteins are transferred to a membrane.

The membrane is mixed with patient serum to capture antibodies.

Excess is washed off then secondary antibodies are added to visualise.

spirochete Borrelia burgdorferi that
causes Lyme disease