Immunology techniques

Question 1

Define antigen

Answer 1

Anything recognised by the immune system as non-self

Question 2

Define antibody

Answer 2

Proteins produced in response to an antigen. Can only bind wth antigen that induced its formation.

Question 3

Define epitope

Answer 3

Specific part of antigen that binds to the antibody

Question 4

Define affinity

Answer 4

Measure of binding strength between an epitope and antibody binding site. Higher affinity = stronger the interaction

Question 5

How is polyclonal antiserum formed?

Answer 5

1. Based on vaccination principle of Ab production to foreign Ag
2. Injec Ag into antimal, leave for 4 weeks for primary Ab response
3. Give booster injec of same Ag
4. Leave 4 weeks for secondary Ab response which is bigger due to memory
5. Collect blood and centrifuge to isolate serum
6. Collect serum and check for specificity - ELISA

Question 6

Production of monoclonal antibodies?

Answer 6

1. Mouse immunised with antigen
2. Mouse produce Ab to Ag
3. Spleen removed to get plasma cells
4. Plasma cells fused with immortal B cells using polyethylene glycol to produce immortal hybridomas
5. Cells are placed into 96-well plates containing HAT - kills off non-fused cells so only hybridoma cells aliva
6. Dilute so have only 1 hybridoma per well = produces single mAB with 1 specificity
7. Hybridomas secreting high affinity mAB selected using ELISA against original Ag
8. = Limited supply of high affinity mAB

Question 7

How are antibodies labelled (conjugated)?

Answer 7

Unlabelled, Stick enzyme, fluorescence or gold on FC region

Question 8

Serological diagnosis - what is it?

Answer 8

Use of Ab specificity to detect Ag

Question 9

What is a titre of an Ab?

Answer 9

The lowest dilution of the sample that retains a detectable activity

Question 10

What can serological tests be used to do?

Answer 10

Diagnose infecs, identify microorganisms, quantify proteins in the serum, type blood - for blood banks and tissue transplants
ONLY SHOW THAT YOU HAVE HAD AN INFECTION

Question 11

What do ppt and immunodiffusion techniques rely on?

Answer 11

Ability of Ab to form complexes with Ag and ppt

Question 12

Immunoprecipitation - Ouchterlony diffusion test?

Answer 12

Ab and Ag diffuse through agar gel and form a ppt at the equivalence point
Used to detect diphtheria toxin in serum - now PCR used to detect bac

Question 13

Immunodouble diffusion steps?

Answer 13

1. Preciptinin band formed with single antigen
2. Two independent Ag react with their specific Ab
3. Ab are specific for their Ag

Question 14

Single radial immunodiffusion?

Answer 14

Involves diffusion of Ag into Ab-containing gel. Precipitin rings indicate an immune rxn and the area of the ring is proportional to the conc of antigen (larger ring = more ag)

Question 15

Immunoelectroporesis?

No longer used

Answer 15

1. Ag placed in a well and separated by electrophoresis

2. Ab is laced in trough and precipitin lines form as Ag and Ab diffuse toward each other

Question 16

Agglutinin tests?

Answer 16

Used in serology for infecs, relies on polyclonal nature of serum, relies on polyclonal serum to cross-link Ab but involves cells or beads

Question 17

How to detect influenza?

Answer 17

Haemagglutination inhibition test;

1. Influenza has haemagglutinin molecules on outer surface
2. Haemagglutinin binds the virus to red blood cells
3. When virus particles are mixed with red cells they cause haemagglutination = forms aggregate
4. In presence of specific Ab, binding of haemagglutinin to RBC is inhibited. RBC settle to bottom of tube.

Question 18

Haemophilus influenzae detection?

Answer 18

1. CSF sample taken from pt with meningitis
2. Sample mixed with suspension of latex beads coated with specific anti-H. influenzae Ab
3. Interaction between Ag and Ab causes immediate agglutination of beads which can be seen by eye - positive diagnosis of H influenzae

Question 19

Detection of infection by streptococcus bacteria?

Answer 19

Anti-streptolysin O test;

1. Serum taken from pt and diluted in tubes containing standard amount of sheep RBC and O toxin
2. If pt has Ab to O toxin = it will neutralise O toxin and stop it from lysin RBC (tube clear)
3. At low Ab conc there is not enough Ab to stop RBC lysis
4. = Gives the Ab titre

Question 20

What do streptococcus bac secrete?

Answer 20

Streptolysin O toxin = lyses RBC by punching holes into them

Question 21

When can blood be transfused?

Answer 21

When no agglutination occurs

Question 22

Enzyme linked immunosorbent assay uses?

MOST IMPORTANT

Answer 22

• Detects level of Ag, Ab or proteins in sample
• Non-agglutination and non-ppt test
• Accurately quantifies levels of test molecules in a sample using a standard curve
• Uses Ag or Ab bound to a solid phase

Question 23

Enzyme linked immunosorbent assay - ELISA types?

Answer 23

1. Sandwich ELISA (most important)

2. Antigen ELISA

Question 24

What is sandwich ELISA?

V IMPORTANT

Answer 24

1. Capture Ab bound to plastic surface that is specific for desired Ag.
2. Add patient serum, CSF or supernatant (and standards to other wells)
3. Ab will bind specifically to Ag it is raised to. All other Ag are washed away.
4. Add Detection Ab – this has an Enzyme (usually HRP) conjugated to it.
5. Wash off excess detection Ab.
6. Add substrate for enzyme – this is
   colourless and turns blue in the presence of enzyme.
7. The more Ag the more colour is produced
8. Measure absorbance @450nm
9. Calculate concentration of Ag in the sample

Used to measure cytokines, virus, bac products in serum

Question 25

What is antigen ELISA?

Answer 25

Used to measure conc of human Ab in serum to bacteria – ie anti-Strep. Pneumoniae Ab 1. Bind Ag/bac/cell to solid phase - wash off excess Ag 2. Add primary Ab specific for Ag. Wash excess primary Ab. 3. Add 2ndry Ab specific to primary Ab and is conjugated with an enzyme 4. Wash excess 2ndry Ab 5. Add substrate = turns from colourless to blue 6. Measure absorbance on spectrophometer 7. Calculate Ag conc

Question 26

Measuring cell-associated antigen?

Answer 26

By cell based ELISA

Question 27

Flow cytometry?

Answer 27

- Analyses cells and cell surface receptors in clinic and research - Uses Ab conjugated with fluorescent tags - Laser excites FL tags - Emission intensity from the FL tags is recorded - More emission intensity = more receptors on cell

Question 28

How does fluorescence work?

Answer 28

1. Fluorescent molecule is excited by a laser = provides electromagnetic energy that excites an electron in the fluorescent molecule . 2. The electron moves to an excitation state at the next energy level. 3. Energy is released in the form of a photon of light (fluorescence) and the electron moves back down to the lower energy level.

Question 29

What does HIV infect?

Answer 29

CD4 T cells (T helper cells)

Question 30

How does flow cytometry work in HIV?

Answer 30

Flow cytometry determines no. of CD4 T cells compared to CD8 T cells In HIV = low CD4 can progress to AIDS

Question 31

Uses of Ab in microscopy?

Answer 31

Used to localise Ag in tissue | Used in cancer and auto-immune disease diagnosis

Question 32

Types of Ab in microscopy?

Answer 32

Immunohistochemistry - staining of sections of tissues | Immunocytochemistry - staining of cells

Question 33

How does immunohistochemistry of wax occur?

Answer 33

1. Tissue biopsy from patient/animal 2. Tissue fixed in 10% Formalin 3. Tissue processed then paraffin embedded 4. 5µM sections cut using a microtome and placed onto glass slides 5. Tissue de-waxed with xylene, processed though alcohol gradient to water 6. Antigen retrieval 7. Primary Ab added 8. Secondary Ab added (with enzyme – HRP or Alkaline Phosphatase) 9. Use substrate (colourless to brown, red or p

Question 34

How does immunohistochemistry of frozen samples occur?

Answer 34

1. Tissue biopsy taken from patient/animal 2. Tissue frozen in liquid nitrogen 3. 5µM sections cut using a microtome and placed onto glass slides 4. Primary Ab added (with Fluorescent tag – for direct) 5. Secondary Ab added (with Fluorescent tag – for indirect)

Question 35

Pros and cons of immunohistochemistry - frozen?

Answer 35

``` Pros = wax preserves tissue architecture better Cons = not as may Ab can be used due to antigen retrieval ```

Question 36

In immunohistochemistry -frozen, what are green and white cells? What is blue?

Answer 36

``` Green = tumour associated macrophages stained with anti-CD68 White = endothelial cells (BV) stained with anti-CD31 Blue = cell nuclei ```

Question 37

What is immunoblotting?

Answer 37

1. Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 2. Electroblotted onto nitrocellulose (NC) paper 3. Incubated with antigen-specific 1°Ab and then rabbit anti-mouse HRP conjugated antiserum (2°Ab). 4. Add substrate and this is converted to light (not colour) 5. Develop on light-sensitive paper to identify if protein is there.

Question 38

Use of therapeutic Ab?

Answer 38

- Monoclonal Ab specifically bind to Ag it is raised against - Monoclonal Ab can block the activity of proteins if they bind to its active site - They can be used to block proteins binding to receptors on cells or viruses binding to and entering cells

Question 39

Problems with therapeutic Ab?

Answer 39

- Most monoclonal Ab are raised in mice - These are NOT human (not-self) - The human immune system will raise Ab to the therapeutic Ab and will inactivate it

Question 40

Solutions for therapeutic Ab?

Answer 40

- Make humanised antibodies = Ab where only the Ab binding site is mouse, the rest has been engineered to be human. Enough of the therapeutic Ab is human for it not to be recognised as nonself

Question 41

What do all drugs that are monoclonal Ab end in?

Answer 41

Mab

Question 42

Example of therapeutic Ab?

Answer 42

Trastuzumab (trade name = Herceptin)

Question 43

Actions of therapeutic Ab?

Answer 43

``` Therapeutic Ab taken into cell via endocytosis = enters the lysosome = proteolytic enzymes and pH release the drug inside the cell Drug gets transported to the nucleus = affects cell division. Other cytotoxic molecules can be used to kill cells directly ```