Immunological Techniques in Diagnostics & Research

Question 1

What are immunological techniques and what is the most common kind?

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

Describe the process of raising antibodies

Answer 2

• The process of producing an antibody specific for a target protein
• These antibodies have a wide range of applications in immunological techniques
• How do we raise antibodies?
Mice are immunised with the target protein
B cells are harvested & fused with tumour cells to form hybridoma
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

Question 3

Describe blood typing

Answer 3

• Blood typing is an example of an “agglutination reaction” used to determine blood type
• ABO system of blood typing
A & B glycoproteins on RBCs
Four types of blood A, B, AB & O
Rhesus factor (D protein or RhD) is also a protein on the surface of RBCs (positive or negative)
• A, B & 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 4

What is flow cytometry?

Answer 4

• Flow cytometry is a technology used to analyse the proteins on cells that are in suspension
• Often involves the use of commercially produced antibodies that are conjugated to fluorochromes

Question 5

What does flow cytometry determine about the cell?

Answer 5

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 involves the use of commercially produced antibodies that are conjugated to fluorochromes

Question 6

How does a flow cytometer work?

Answer 6

• 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 7

What are the diagnostic and research applications of a flow cytometer?

Answer 7

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

Question 8

What are the differences between confocal microscopy and the flow cytometer

Answer 8

• 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

Question 9

What are the applications of confocal microscopy?

Answer 9

• Applications
Mainly research
Identification & analyse cells within tissues

Question 10

What does IHC stand for and why is it carried out?

Answer 10

• IHC stands for ImmunoHistoChemistry

* Used to show the distribution & localization of antigens in tissue sections using antibody-antigen interactions

Question 11

What is the process for IHC?

Answer 11

• 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 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

Question 12

What does ELISA stand for and what does this technique do?

Answer 12

• Enzyme-Linked ImmunoSorbant Assay

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

Question 13

What are the applications of ELISA and what are the 4 different types?

Answer 13

• Applications
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
• Four different types
Direct
Indirect
Sandwich
Competitive

Question 14

What is western blotting and what are the other types of blotting?

Answer 14

• Western blotting is a technique used to detect proteins
A Southern Blot (Edwin Southern) detects DNA
A Northern blot detects RNA
Western blot involves four steps

Question 15

What is the procedure for western blotting?

Answer 15

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

Question 16

What is the application of western blotting?

Answer 16

•	Diagnostic use
o	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. eg. Viral infections (HIV) parasites (spirochete Borrelia burgdorferi that causes Lyme disease)
o	Research
 	Cell signalling proteins
 	Mechanism of action for cancer drugs