Immunohistochemistry Flashcards
Define ‘immunohistochemistry’.
Any method using labelled antibodies to detect a specific antigen
_________ antibodies, which are sera, containing multiple antibodies to the antigenic epitopes.
Polyclonal
List four characteristics of polyclonal antibodies.
Many different species are involved
Tend to have more non-specific reactivity
Can have wildly different affinity or avidity from batch to batch
More likely to have success in uncertain applications
What is a monoclonal antibody?
Antibody-producing cells that are harvested from the spleen of the animal, fused with tumour cells, and grown in cell culture (hybridoma technology)
True or false: monoclonal antibodies are less likely to get false negative results.
False
Antibody attaches to the cell/tissue antigen, and is then visualised with a __________ through a label.
Chromagen
________ can be intra- or extracellular, membranous, nuclear components, etc..
Antigens
List three features of IHC which must be considered when choosing reagents.
Controls
Direct vs. indirect methods
Fluorescence
Multiple labelling characteristics
What are primary antibodies?
Usually monoclonal antibodies which detect and bind to specific epitope(s) of antigen which is usually a cell surface antigen, e.g., mouse antihuman macrophage
What are secondary antibodies?
Antibodies that bind to primary antibodies or antibody fragments
True or false: secondary antibodies are usually labelled.
True
Secondary antibodies bind to the primary antibody only, and not to the ______ ________ _______.
Cell surface antigen
___________ methods employ primary antibodies only.
Direct
What do indirect antibodies employ?
Primary and secondary antibodies
True or false: enzymatic methods are direct.
False
Name a popular enzymatic method.
PAP method
Applying layers of antibodies (primary, secondary and maybe tertiary) and labels is more _____________ for antigen detection.
Sensitive
What is one disadvantage of direct methods?
They are expensive
Describe the avidin-biotin-enzyme complex.
An indirect, three-step method, whereby the primary antibody unlabelled, and secondary antibody is biotin-labelled. The third layer is an avidin-enzyme complex. Avidin and biotin have very high affinity for one another. The enzyme conjugate catalyses a reaction with a substrate (chromogen), which changes colour
What method of detection does the EnVision FLEX exploit?
Polymer
Describe the polymer method of detection.
The primary antibody is unlabelled, and the second layer is a dextran polymer of multiple antibodies and labels
Localising the antigen-antibody complex in IHC relies on _______, which are very readily attached to antibodies.
Labels
Labels may be radioactive isotopes, enzymes, ________, metals, and fluorescent dyes.
Biotin
In cells and _________, the label is used to create a colour at the site of staining.
Tissues
List two fluorescent dyes.
Rhodamine and fluorescein IsoThioCyanate FITC
____________ dyes include peroxidase and glucose oxidase.
Enzymatic
________ ____________ uses an Azo dye reaction to visualise a red colour.
Alkaline phosphatase
With _________________, oestrogen receptor-positive breast tumour cells can be identified by brown stain.
Immunoperoxidase
What is the principle of the DAB reaction?
The DAB reaction uses peroxidase, which is attached at site of antigen during IHC reactions. Peroxidase then acts on a hydrogen peroxide substrate to release oxygen, which causes a brown polymer of diaminobenzidene to form
True or false: IHC is an adjunct to tissue morphology and used in addition to the H&E.
True
List four reasons why IHC testing would be requested.
Confirmation of tumour type suspected on H&E
Identification of adjacent tumour spread
Identification of distant metastases
Identification of tumours of unknow etiology
A panel of antibodies is used in IHC to different _____ _______ _________, which are present on tumour cell.
Cell type antigens
____________ is present on epithelial tumours; antibody names include MNF116, AE1/AE3, and PanCytokeratin.
Cytokeratin
Expression of antigens/proteins in the _______ ______ is predictive of response to therapy, and/or more aggressive tumours.
Tumour cells
For breast cancer, oestrogen and progesterone receptors are responsive to oestrogen blocking with ____________.
Tamoxifen
