Immunohistochemistry

Question 1

Define ‘immunohistochemistry’.

Answer 1

Any method using labelled antibodies to detect a specific antigen

Question 2

_________ antibodies, which are sera, containing multiple antibodies to the antigenic epitopes.

Answer 2

Polyclonal

Question 3

List four characteristics of polyclonal antibodies.

Answer 3

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

Question 4

What is a monoclonal antibody?

Answer 4

Antibody-producing cells that are harvested from the spleen of the animal, fused with tumour cells, and grown in cell culture (hybridoma technology)

Question 5

True or false: monoclonal antibodies are less likely to get false negative results.

Answer 5

False

Question 6

Antibody attaches to the cell/tissue antigen, and is then visualised with a __________ through a label.

Answer 6

Chromagen

Question 7

________ can be intra- or extracellular, membranous, nuclear components, etc..

Answer 7

Antigens

Question 8

List three features of IHC which must be considered when choosing reagents.

Answer 8

Controls
Direct vs. indirect methods
Fluorescence
Multiple labelling characteristics

Question 9

What are primary antibodies?

Answer 9

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

Question 10

What are secondary antibodies?

Answer 10

Antibodies that bind to primary antibodies or antibody fragments

Question 11

True or false: secondary antibodies are usually labelled.

Answer 11

True

Question 12

Secondary antibodies bind to the primary antibody only, and not to the ______ ________ _______.

Answer 12

Cell surface antigen

Question 13

___________ methods employ primary antibodies only.

Answer 13

Direct

Question 14

What do indirect antibodies employ?

Answer 14

Primary and secondary antibodies

Question 15

True or false: enzymatic methods are direct.

Answer 15

False

Question 16

Name a popular enzymatic method.

Answer 16

PAP method

Question 17

Applying layers of antibodies (primary, secondary and maybe tertiary) and labels is more _____________ for antigen detection.

Answer 17

Sensitive

Question 18

What is one disadvantage of direct methods?

Answer 18

They are expensive

Question 19

Describe the avidin-biotin-enzyme complex.

Answer 19

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

Question 20

What method of detection does the EnVision FLEX exploit?

Answer 20

Polymer

Question 21

Describe the polymer method of detection.

Answer 21

The primary antibody is unlabelled, and the second layer is a dextran polymer of multiple antibodies and labels

Question 22

Localising the antigen-antibody complex in IHC relies on _______, which are very readily attached to antibodies.

Answer 22

Labels

Question 23

Labels may be radioactive isotopes, enzymes, ________, metals, and fluorescent dyes.

Answer 23

Biotin

Question 24

In cells and _________, the label is used to create a colour at the site of staining.

Answer 24

Tissues

Question 25

List two fluorescent dyes.

Answer 25

Rhodamine and fluorescein IsoThioCyanate FITC

Question 26

____________ dyes include peroxidase and glucose oxidase.

Answer 26

Enzymatic

Question 27

________ ____________ uses an Azo dye reaction to visualise a red colour.

Answer 27

Alkaline phosphatase

Question 28

With _________________, oestrogen receptor-positive breast tumour cells can be identified by brown stain.

Answer 28

Immunoperoxidase

Question 29

What is the principle of the DAB reaction?

Answer 29

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

Question 30

True or false: IHC is an adjunct to tissue morphology and used in addition to the H&E.

Answer 30

True

Question 31

List four reasons why IHC testing would be requested.

Answer 31

Confirmation of tumour type suspected on H&E Identification of adjacent tumour spread Identification of distant metastases Identification of tumours of unknow etiology

Question 32

A panel of antibodies is used in IHC to different _____ _______ _________, which are present on tumour cell.

Answer 32

Cell type antigens

Question 33

____________ is present on epithelial tumours; antibody names include MNF116, AE1/AE3, and PanCytokeratin.

Answer 33

Cytokeratin

Question 34

Expression of antigens/proteins in the _______ ______ is predictive of response to therapy, and/or more aggressive tumours.

Answer 34

Tumour cells

Question 35

For breast cancer, oestrogen and progesterone receptors are responsive to oestrogen blocking with ____________.

Answer 35

Tamoxifen