Immunohistochemistry Flashcards
What is IHC?
Locating (known) antigens (proteins) in tissue, using antibodies specific for that antigen.
Antibody is tagged with an enzyme or fluorophore
What is ICC?
Locating antigen/protein in a cell or cell compartment using antigen-antibody complex and a biomarker
What is a polyclonal antibody?
Multiple different antibodies that bind to multiple epitopes on antigen
What is a monoclonal antibody?
1 specific type of antibody binds to 1 specific epitope on antigen
How are monoclonal antibodies made?
Mouse injected with antigen.
Lymphocytes isolated (spleen cells)
Spleen cells hybridised with myeloma cells = hybridoma, and rapidly multiply
They are immortalised
Positively selected
Antibody clones selected
How are polyclonal antibodies formed?
Multiple antigens injected into mouse(s)
Produces different lymphocytes (Spleen cells) which are isolated and hybridised with myeloma cells
Different types of cells are immortalised and different antibodies are isolated from each cell
Alternative name for antibodies
Immunoglobulin
What is the bottom of antibody called?
FC fragment - constant domain
What do FC fragments bind to?
Cell receptors
What is the top (V) of antibody called
FAB fragment - variable domain
What does FAB fragment bind to?
Antigen
If indirect IHC is used, what do the primary and secondary antibodies bind to?
Primary - Fab fragment binds to antigen
Secondary - Fab fragment binds to primary FC fragment (1st AB = antigen for 2nd AB)
Why is tissue prepared in IHC?
Improve antibody penetration
Retrieve antigen (ihc version of dewaxing)
How is tissue prepared for IHC?
Heat
Proteolysis
What does heat/proteolysis do to tissue?
Reduce cross linking
Makes membrane more permeable
Improves antigen presentation
What is blocking?
Blocking unwanted protein binding to the antibody
What is used for blocking?
Serum of antibody (usually secondary)
What is the most common conjugate used for antibodies
Horseradish Peroxidase (HRP)
Why HRP?
Increases detectability of target molecules
Produces weak signal
What is the signal produced by HRP in IHC?
What is the other type of signal and it’s substrate name?
Insoluble Redish/brown colour
Chemiluminescence with luminol substrate
5 steps of indirect detection
- Blocking reagent - unoccupied membrane sites
- Primary antibody specific to antigen - Fab region binds to form complex
- Conjugated secondary antibody Fab fragment binds to FC fragment of primary antibody
- Substrate addition
- ESC = product = signal (colour or chemiluminescence
Why do multiple secondary antibody bind to primary antibody FC fragment?
FC fragment has repeated amino acid sequences
Or
Secondary antibody in polyclonal
What is an alternative conjugate to enzyme/fluorophore?
Biotin
Which enzyme does biotin interact with?
Streptavidin enzyme
Where is biotin located in the assay?
Secondary antibody in bionylated
Why is HRP used in sABC?
It cleaves the strepavidin-biotin complex to form the products = signal = light/colour
What does streptavidin bind to in IHC?
Biotin on secondary antibody
When is direct immunoassay performed?
Immunocytochemistry
Outline direct immunoassay
- Primary Antibody conjugated with enzyme or fluorophore
- Antibody Fab fragment binds to antigen
- Substrate added for enzyme or UV light to detect fluorescence
- Level of signal proportional to antigen concentration
What is direct immunofluorescence?
A direct assay - primary antibody conjugated to fluorophore
Why does increased signal in IHC/ICC mean?
Increased antigen concentration meaning more binding sites for antibody
