Immunological Techniques In Diagnostics And Research

Question 1

What is a hybridoma?

Answer 1

B cells and tumour cells fused together

Question 2

What do the hybridomas produce?

Answer 2

Antibodies against the target protein

Question 3

What is a monoclonal antibody?

Answer 3

Only has one binding site for one specific shape on one specific antigen

Question 4

What is the blood sample mixed with when they’re trying to work out a blood type?

Answer 4

Antibodies raised against A,B or RhD

Question 5

What happens to the RBCs when they’re mixed with antibodies in blood typing?

Answer 5

Agglutination

Question 6

What happens in blood typing if the samples are all positive or negative?

Answer 6

You’ve done it wrong

Question 7

What is flow cytometry?

Answer 7

Technology used to analyse the proteins on cells that are in suspension

Question 8

What can flow cytometry tell you (4)?

Answer 8

• cell size and density
• if a cell expresses a target protein
• The amount of expression of a target protein
• The cells identity

Question 9

What does flow cytometry often involve the use of?

Answer 9

Commercially produced antibodies that are then conjugated to fluorochromes

Question 10

What is a fluorochrome?

Answer 10

A fluorescent molecule that absorbs light of a certain wavelength and in turn emits light of a certain wavelength

Question 11

What is detected in flow cytometry?

Answer 11

The amount of light emitted

Question 12

How is flow cytometry used in research(2)?

Answer 12

Identification and analysis of immune cells

Cell sorting

Question 13

How is flow cytometry used in diagnostics(2)?

Answer 13

CD4 T cell counts in HIV

Diagnostics of haematological malignancies

Question 14

What happens to the immune cells in B cell lymphoma?

Answer 14

Reduced T cell and granulocytes and increased B cell clonality

Question 15

Why do you need to use flow cytometry in HIV?

Answer 15

HIV destroys white cells so its important to know how many you have to know the progression of the disease

Question 16

What are 3 applications of confocal microscopy?

Answer 16

Mainly research
Identification and analysis of cells within tissues
Co-localisation of different antigens

Question 17

Name the four differences between confocal microscopy and flow cytometry

Answer 17

In confocal microscopy:

• 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 (not plotted graphically)
• has the advantage of visualising where the protein is in a cell

Question 18

What does IHC stand for?

Answer 18

Immunohistochemistry

Question 19

What is IHC used for?

Answer 19

To show the distribution and localisation of antigens in tissue sections using antibody-antigen interactions

Question 20

What does a brown precipitate mean in IHC?

Answer 20

The protein is present

Question 21

Why is IHC helpful?

Answer 21

You can see where, in organs, certain things can be targeted to

Question 22

How is IHC used in cancer diagnosis?

Answer 22

Used to stain B-Raf protein in tissue sections selecting patients eligible for B-Raf inhibitor therapy

Question 23

What is visualised using chromogenic detection in IHC?

Answer 23

The antibody-antigen interaction

Question 24

What is the secondary antibody conjugated to in IHC?

Answer 24

Horseradish peroxidase (HRP)

Question 25

What does the horseradish peroxidase do in IHC?

Answer 25

Catalyses the conversion of the chromagen 3,3-diaminobenzidine substrate to produce a brown precipitate

Question 26

What does ELISA stand for?

Answer 26

Enzyme-linked immunoabsorbant assay

Question 27

What does the ELISA test do?

Answer 27

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

Question 28

What are the 4 applications of ELISA tests?

Answer 28

Antibody titres sera (HIV and HepB) Bacterial toxins in food Home pregnancy testing Research quantification of cytokines/ chemokines/ growth factors in tissue culture supernatants

Question 29

What are the four types of ELISA?

Answer 29

Direct Indirect Sandwich Competitive

Question 30

Give the 6 steps in the sandwich ELISA

Answer 30

``` Add capture antibody Add sample Wash Add detection antibody Add substrate Determine optical density ```

Question 31

What does western blotting do?

Answer 31

Detects proteins

Question 32

What are the 2 applications of western blotting?

Answer 32

Antibody detection of specific sized proteins from viral infections or parasites Cell signalling proteins

Question 33

What are the four steps of a western blot?

Answer 33

Sample prep Electrophoresis Transfer to membrane Stain for protein of interest

Question 34

What happens in the sample prep step of a western blot?

Answer 34

Cells are lysed and proteins denatured

Question 35

What happens in the electrophoresis step of a western blot?

Answer 35

Lysates are loaded onto a gel and proteins are separated based on size

Question 36

What happens in the transfer to membrane step of a western blot?

Answer 36

Fractionated proteins are transferred onto a membrane

Question 37

How are the proteins transferred onto a membrane in a western blot?

Answer 37

Put filter paper on top and pass electricity through it onto the paper

Question 38

What happens in the staining step of a western blot (4 steps)?

Answer 38

- the membrane is incubated with a primary antibody specific for the target protein - the membrane is incubated with a HRP conjugated secondary antibody for the primary antibody - a chemiluminescent HRP substrate is added to the membrane - the membrane is expose to an X-ray film that bleaches when exposed to light