Immunological techniques

Question 1

How do we raise antibodies?

Answer 1

Mice are immunised with the target protein
B cells are harvested and fused with tumour cells to form hybridoma
A hybridoma that produces antibody against the target protein is selected and cloned
The antibodies secreted by the cloned hybridoma are harvested and used in immunological techniques

Question 2

How does blood typing work?

Answer 2

Blood typing is an example of an agglutination reaction used to determine blood type
ABO system of blood typing:
A and B glycoproteins on RBCs
Four types of blood- A, B, AB and O
Rhesus factor (D protein or RhD) is also a protein on the surface of RBCs (positive or negative)
A, B and RhD are antigens that elicit immune responses in mismatched donor/recipient blood transfusions
How does the test work?
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 3

What is flow cytometry?

Answer 3

Flow cytometry is a technology used to analyse the proteins on cells that are in suspension
What can it determine about a cell?
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
Often involved the use of commercially produced antibodies that are conjugated to fluorochromes

Question 4

How does a flow cytometer work?

Answer 4

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 form the excited fluorochromes is detected and plotted on a graph
Amount of light emitted= amount of antibody bound to protein= amount of protein expressed by the cell

Question 5

What are the applications of flow cytometry?

Answer 5

Diagnostics:
CD4 T cell counts in HIV
Diagnosis of haematological malignancies
Research:
Cell sorting
Identification and analysis of immune cells

Question 6

What is confocal microscopy and what are its applications?

Answer 6

Similar to flow cytometry although there are key differences
The cells to be analysed are not in suspension
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
Confocal microscopy has the advantage of visualising where the protein is on the cell
Applications:
Mainly research
Identification and analysis of cells within tissues
Co-localisation of different antigens

Question 7

What is IHC?

Answer 7

IHC stands for Immunohistochemistry
Used to show the distribution and localisation of antigens in tissue sections using antibody-antigen interactions
How does it work?
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 peroxide (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

Question 8

Give an example of IHC?

Answer 8

IHC is routinely used in the diagnosis of cancer
Cancer can be caused by mutations in tumour suppressor or tumour promoter genes
These genes encode proteins that are involved in suppressing or promoting cell division
Mutations in these genes can cause uncontrollable cell division
E.g. BRAF is a gene that encodes B-Raf protein that promotes cell division
Many cancers have BRAF mutations that result in over-production of B-Raf and thus uncontrolled cell division
IHC is used to stain B-Raf protein in tissue sections from cancer patients and direct eligible for treatment with B-Raf inhibitors

Question 9

What is ELISA?

Answer 9

Enzyme-linked Immunosorbent Assay
Quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants
Applications:
Antibody titres in patient serum e.g. viral infections such as HIV and hepatitis B
Detection of bacterial toxins in food such as Escherichia coli
Home pregnancy testing detection of human chorionic gonadotropin hormone (HCG) in urine
Research quantification of cytokines/chemokines/growth factors in tissue culture supernatants
Four different types:
Direct
Indirect
Sandwich
Competitive

Question 10

How is Western blotting carried out? What is it used for?

Answer 10

Sample preparation:
Cells are lysed and proteins denatured
Electrophoresis:
Lysates are loaded onto a gel and proteins separated based on size
Transfer to membrane:
Fractionated proteins are transferred onto a membrane
Stain for protein of interest:
The membrane is incubated with a primary antibody specific for the target protein
The membrane is then incubated with an HRP conjugated secondary antibody specific for the primary antibody
The chemiluminescent HRP substrate is added to the membrane
The membrane is exposed to X-ray film that “bleaches” when exposed to light
Applications:
Diagnostic use:
The pathogen is lysed and 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 e.g. viral infections (HIV)
Research:
Cell signalling protein
Mechanism of action for cancer drugs

Question 11

What is Western blotting?

Answer 11

Western blotting is a technique used to detect proteins
A southern blot detects DNA
A northern blot detects RNA
Western blot involves four steps:
	1. Sample preparation
	2. Electrophoresis
	3. Transfer to membrane
	4. Stain for protein of interest