IF, IHC and ICC Flashcards
What does IF stand for?
Immunofluorescence
What does IHC stand for?
Immunohistochemistry
What does ICC stand for?
Immunocytochemistry
Where is the variable region of an antibody? Top or bottom?
Top
What does the antibody bind to?
Epitope on the antigen
How many types of mammalian immunoglobulin chains are there?
5
IgA, IgD, IgE, IgG and IgM
What are the three sections an antibody can be divided into?
Two Fab regions
One Fc region
Which region of the antibody is the variable domain in?
Fab regions
What binds to the Fc fragment?
- Fc receptors on lymphocytes
- Secondary antibodies
- Dye labels and enzymes
What enzyme do we use if we want to produce a fragment both Fab regions and no Fc?
Pepsin
What enzyme do we use if we want to produce all three parts?
Papain
Which antibody is widely produced, found in mucosal areas and prevents colonisation of pathogens?
IgA
Which antibody is found with IgM on the surface of B cells? And is involved in B cell development?
IgD
Which antibody is important in allergies and binds to allergens causing histamine release?
IgE
Which is the major antibody and produces the most antibody-based immunity against invading pathogens?
IgG
Which is the first response antibody, has high avidity (strength) and eliminates pathogens in early B cell immunity? (before sufficient IgG?)
IgM
Which antibody type is a mixture of many types of antibodies?
Polyclonal antibody
Why are multiple antibodies produced in response to an antigen?
Because antigens have multiple epitopes.
From where in the animal’s body are antibodies collected?
Antiserum (whole serum following exposure to antigen).
Why are lab animals injected twice with the same antigen?
2nd infection activates memory cells to make IgG antibodies.
What is affinity maturation?
Affinity maturation is the process by which B cells undergo selection to produce antibodies with increased binding affinity for a specific antigen.
Leads to a more effective immune response upon re-exposure.
What is an adjuvant?
Adjuvant is a substance that is added to a vaccine to enhance the body’s immune response to the vaccine’s antigen.
Stimulates greater antibody production
Monoclonal antibody production:
What is collected from the lab animal after infecting it? And where in the body it collected from?
B cells
Collected from spleen of lab animal
What are collected B cells fused with in monoclonal antibody production?
B cells fused with immortal cancerous B cells (myeloma cells).
Why are B cells fused with immortal cancerous B cells?
To yield hybridoma cells that can proliferate and produce antibodies continuously.
What are hybridoma cells screened for?
Desired monoclonal antibody (recognising single epitope).
How do we develop a secondary antibody?
Immunise a second animal with IgG from mouse.
Animal produces IgG in response to constant region of mouse IgG.
What is immunofluorescence?
Visualises presence and location of specific proteins/ antigens in cells/tissues using antibodies labeled with fluorescent dyes
Can Immunofluorescence also be used to detect protein modifications?
Yes
What are the two types of Immunofluorescence (IF)?
- Direct
- Indirect
What is the purpose of the fixation step in Immunofluorescence (IF)?
To preserve original cell architecture and ensuring antigens are immobilised.
What is the purpose of organic solvents such as methanol and acetone in the fixation step of IF?
Organic solvents remove lipids from the cell membrane, facilitating antibody binding.
Acetone is more harsh.
What types of aldehyde are used in aldehyde based fixing?
What is the purpose of aldehyde-based fixing?
4% formaldehyde or 10% formalin
To cross-link proteins.
Why is permeabilisation step in IF needed?
Allows antibodies to access intracellular antigens within cells.
Creates openings in membrane.
If we used organic solvent in fixation step, do we need to permeate?
No, as cell membrane holes are already punched.
If aldehyde fixative used, do we permeate?
If so, what do we use to do so?
Yes.
Non-ionic detergent such as Triton X-100 or saponin.
How do we reduce non-specific antibody binding during IF?
Blocking
What are two commonly used blocking reagents in IF?
1% milk powder in PBS
Serum from the 2nd animal
Both bind unoccupied sites
What is the function of the microtone during IF and IHC?
To create uniform sections of tissue samples of a specified thickness.
How does ImmunoHistoChemistry (IHC) differ from Immunofluorescence (IF)?
secondary antibody is enzyme-linked in IHC
How does ImmunoCytoChemsitry (ICC) differ from ImmunoHistoChemsitry (IHC)?
ICC involves staining cells (with secondary antibodies), not tissue.
