Immunohistochemistry

General ImmunologyThe immune system exerts its control

through humoral ( antibody) and cellular components.

Spring 2014

Antibody

• What is an antibody? An antibody is a host protein host protein ( immunoglobulin) produced in response to the presence of foreign molecules, organisms, or other agents in the body.

• Antibodies commonly known as immunoglobulins are proteins that are produced by B- lymphocytes in response to antigenic stimulation.

• An immunoglobulin is a Y- shaped protein a Y- shaped protein molecule that is composed of both heavy and light chains.

Antibody classification

• Classes of antibody differ in structure and function with each immunoglobulin antigenically distinct.

• An immunoglobulin is composed of 2 identical heavy chains α, γ,ε,μ, or delta, which determine the immunoglobulin subclass ( Ig A, IgG, Ig E, Ig M or Ig D)

• The light chain is either κ or λ. These proteins are expressed on the cell surface or membrane, and secreted into the blood and other fluids by plasma cells.

• The upper arm of the Y are the regions of the antibody that bind to the short arms of their specific antigen.

Antibody classification Contd.

• Epitope- the site at which the antibody attaches to the tissue.

• An antibody may target more than 1 antigen but is specific for for only 1 epitope.

• Proteins are very good antigens because of their large size. The fact that anibodies ( immunoglobulins ) are proteins makes them very potent antigens.

Antigen

• What is an antigen? An antigen is a molecule made up of proteins, carbohydrate or other polymers, and is capable of producing an immune response in animals or cell cultures for the production of antibodies.

• Antigens in tissues bind antibodies at different cell sites,: the membrane, cytoplasm, nucleus, organelles or a combination of more than 1.

• Many compounds are antigenic. • Because of their large size proteins are ideal for immunization and

for binding but polysaccharides, nucleic acids, and other polymers can also be antigenic.

• The most common antigen that induce antibody production are bacteria and viruses.

Polyclonal antisera

• Pooling of antibodies to make polyclonal antisera• Blood drawn from human arm, isolate Ig M antibody Ig M antibody

from it which is injected into a rabbit. The human Ig M antibody would act as an antigen and stimulate the rabbit to make Anti-human Ig M antibody. Serum from the immunized rabbit could be used as a polyclonal anti-human Ig M antibody reagent.

• Polyclonal antiserum is highly sensitive because it binds to more than 1 epitope, but is not as selective and gives non specific staining background.

• Examples of hosts that are used to produce polyclonal antisera include rabbit, goat, horse, sheep and humans.

Monoclonal antibodies

• Monoclonal antibodies (MABs) How are they prepared?

• MABs- prepared by injecting mice with an antigen.• B-lymphocytes are fused with nonsecreting myeloma

cells ( non secreting plasma cell tumor) MABs can be characterized, standardized, and produced in unlimited quantities. Mouse MABs are purer than polyclonal antibodies. MABs have high affinity and selectivity.

Immunofluorescence

• What is immunofluorescence ?• Imf- the oldest pathology immunohistochemical technique.• Fluorochrome- dye that absorbs light and then emits its own light emits its own light

at a longer wavelength. This phenomenon of absorption and emission of light is called fluorescence.

• Fluorochrome attached or conjugated to antibody, the sites of reaction between antigen and labeled antibody can be visualized easily.

• Commonly used fluorochrome in immunofluofluorescence technique are fluoescein isothiocyanate ( FITC) and rhodamine.

• Both of these dyes absorb light that is not visible to the human eye. (UV) and emit light that is visible.

Invasive Carcinoma of the Breast

Chromogen used akloline phosphatase

Breast Carcinoma

Her 2 Dab Chromogen

Define the following

1. Antigen

2. Antibody

3. Chromogen

4. Epitope

5. For what purpose is immunoistochemistry used?

6. What type of compound is antigenic?

7. What common antigens induce antibody production?

8. Define immunoglobin

9. Define Polyclonal Antisera

10.Define Monoclonal

11.Which antibodies are purer?

12.How long should breast tissue be fixed for IHC?

13.Define Fluorochrom

14.What 2 frozen section tissue are routinely examined with immunoflourescence?

15.Define Direct Method

16.Define Indirect Method

Immunohistochemistry Questions

Enzyme Immunohistochemistry Questions

15. What enzmes are used in enzyme immmunohistochemistry?

16. What are the most common enzymes chosen for antibody visualization?

17. List property of AEC

18. List property of DAB