Immunological Techniques

Question 1

What are immunlogical techniques?

Answer 1

Immunological techniques include both experimental methods to study the immune system AND methods to generate and use immunological reagents as experimental tools.
The most common immunological techniques relate to the production and use of antibodies to detect specific proteins in biological samples.

Question 2

What is raising antibodies?

Answer 2

It is the process of producing an antibody specific for a target protein. These antibodies have a wide range of applications in immunological techniques.

Question 3

How do we raise antibodies?

Answer 3

1) Mice are immunised with the target protein
2) B-cells are harvested and fused with tumour cells to form a hybridoma
3) A hybridoma that produces antibodies against the target protein is selected and cloned
4) The antibodies secreted by the clones hybridoma are harvested and used in immunological techniques

Question 4

What is blood typing?

Answer 4

It is an example of an ‘agglutination reaction’ used to determine blood type.
A, B and RhD are antigens that elicit immune responses in mismatched donor/recipient blood transfusions.

Question 5

How does blood typing work?

Answer 5

1) A blood sample is mixed with antibodies raised against A, B or RhD antigens
2) The sample is visually checked for agglutination (blood cells sticking together)

Agglutination indicates the presence of antigens in the blood sample.

Question 6

What is flow cytometry?

Answer 6

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

It can determine:

• whether or not a cell expresses a target protein
• the amount of expression of a target protein

It often involves the use of commercially produced antibodies that are conjugated to a fluorochrome.

Fluorochromes absorb light of a certain wavelength and emit light of a certain wavelength.

Question 7

How does a flow cytometer work?

Answer 7

1) Fluorochrome-conjugated antibodies that are specific for the target protein are added to the cells.
2) The cells are passed through lasers that excite the fluorochrome (eg. blue laser excites FITC, which then emits green light)
3) The light emitted from the excited fluorochrome is detected and plotted on a graph.

The amount of light emitted is proportional to the amount of antibody bound to the protein, which is proportional to the amount of protein expressed by the cell.

Question 8

What are some applications of flow cytometry?

Answer 8

DIAGNOSTICS:

• diagnosis of haematological malignancies
• CD4 T-cell counts in HIV

RESEARCH:
- identification and analysis of immune cells in the context of infection and cancer immunotherapy

Question 9

What is confocal microscopy?

Answer 9

It is similar to flow cytometry, although there are some key differences:

• the cells to be analysed are not in suspension
• it’s 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 being plotted graphically
• confocal microscopy has the advantage of visualising where the protein is on the cell

Its applications are:

• mainly research
• indentification and analysis of cells within tissues
• co-localisation of different antigens

Question 10

What is IHC?

Answer 10

IHC stands for ImmunoHistoChemistry. It’s used to show the distribution and localisation of antigens in tissue sections using antibody-antigen interactions.

Question 11

How does IHC work?

Answer 11

1) Thin sections of the tissue are cut.
2) Primary antibodies that recognise the target protein are added to the tissue.
3) The antibody-antigen interaction is visualised using chromogenic detection.
4) A secondary antibody specific for the primary antibody and conjugated to horseradish peroxidase (HRP) is added.
5) HRP catalyses the conversion of the chromogen 3,3-diaminobenzidine (DAB) substrate to produce a brown precipitate at the location of the protein.
6) The brown precipitate is then visualised by a light microscope.

Question 12

Give an example of a use of IHC.

Answer 12

IHC is used routinely in the diagnosis of cancer.
Cancer can be caused by mutations in tumour supressor or tumour promoter genes (eg. BRAF is a gene that encodes the B-Raf protein that promotes cell division).

IHC is used to stain B-Raf protein in tissue sections of cancer patients in order to check for eligibility for treatment with B-Raf inhibitors.

Question 13

What is ELISA?

Answer 13

It stands for Enzyme-Linked ImmunoSorbant Assay. It quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants.

There are four different types:

• direct
• indirect
• sandwich
• competitive

Question 14

What are some applications of ELISA?

Answer 14

• antibody titres in patient serum (eg. viral infections such as HIV and Hepatitis B, as well as bacterial infections such as Mycobacterium tubercolosis (TB))
• the home pregnancy test uses the principles of ELISA to measure human chorionic supernatants hormone (HCG) in urine
• in research, it is used to quantify cytokines/chemokines/growth factors in tissue culture supernatants

Question 15

What are the steps to perform a sandwich ELISA?

Answer 15

1) add capture antibody
2) add the sample
3) add the secondary with detection antibody (HRP)
4) add the substrate
5) determine the optical density using a spectrophotometer

Question 16

What is Western Blotting?

Answer 16

It is a technique used to detect proteins in a tissue homogenate.

It involves four steps:

1) sample preparation
2) electrophoresis
3) transfer to membrane
4) stain for protein of interest

Question 17

What is the procedure for Western Blotting?

Answer 17

1) The sample is prepared by denaturing and lysing.
2) It is then loaded onto a gel for electrophoresis, where the proteins are separated based on size.
3) Then, it is transferred to a membrane.
4) We then stain for our protein of interest by incubating the membrane with a primary antibody specific for our target protein.
5) We then incubate the membrane with an HRP-conjugated secondary antibody specific for the primary antibody.
6) A chemiluminescent HRP substrate is added to the membrane.
7) The membrane is exposed to X-ray film that ‘bleaches’ when exposed to light.

Question 18

List some applications of Western Blotting.

Answer 18

DIAGNISTIC USE:

• viral infections (eg. HIV)
• bacterial infections (eg. spirochete Borrelia burgdorferi that causes Lyme disease)
• used to detect the presence of antibodies against a pathogen in patient serum
• The pathogen is lysed and its proteins are seperated on a gel. The proteins are transferred to a membrane. the membrane is mixed with petient serum which may or may not contain anitbodies. Secondary antibodies are added, etc.

RESEARCH:

• cell signalling proteins
• mechanism of action for cancer drugs