immunohistochemistry

Question 1

what is IHC

Answer 1

technique used to identify cellular or tissue constituents (antigens) using antigen-antibody binding

Question 2

What is avidity and what does it depend on

Answer 2

Avidity is functional affinity which represents the overall strength of the antigen antibody ineteraction and is infulenced by:
- binding affinity
- valency (no. of binding sites on antibody or antigen
- structural arrangement of the antibody and antigen in question

Question 3

What are polyclonal antibodies

Answer 3

a heterogenous mix of antibodies where each antibody rexognizes different epitopes of the same antigne

Question 4

How are polyclonal antibodies produced?

Answer 4

Commonly carried out in rabbits:
- immunization with taregt antigen
- humoral response leads to activation of plasma cell clones each made for different epitopes on the antigen
- repeated immunization for larger number and affinity of antibodies
- bleed animal to obtain serum
-purification by precipitation or chromatography

Question 5

disadvantages of polyclonal a

Answer 5

batch to batch variation, lack of reproducability, cross reactivity and lack of specificty because of a higher risk of binding to other proteins with similar sequences

Question 6

What are monoclonal antibodies

Answer 6

Obtained from mice mainly. anitbodies obtained from antibody producing hybridoma clones, made specific for 1 epitope of the antigen (highly specific)

Question 7

advantges and disadvantges of monoclonal antibodies

Answer 7

reproducible, inexhaustabile supply. dis of monospecifity. Keep in mind that they are prone to experience genetic drift over time - antibodies have slight variation from the original antibodies.

Question 8

Monoclonal antibodies production process using hybridomas

Answer 8

-immunization of mice with the antigen for humoral response trigger, repeated immunization.
- plasma cells are harvested from the spleen and fused with immortal tumour cells grown in tissue culture. This forms a hybridoma.
- Hybridomas are screened for antibody production
- These antibody producing hybridomas are cloned and cultivated using cell culturing techniques.
- antibodies are secreted into the culture media and harvested.

Question 9

Recombinant monoclonal antibody production

Answer 9

produced by cloning the antibody coding genes into a high yield mammalian expression vector. the resutling vectors are introduced into expression hosts to manufacture functional antibodies.

Question 10

What can be used a label , conjugated with the antiboud

Answer 10

• fluorochromes (fluorescin, rhodamine)
• enzymes (HRP, Alkaline phosphatase) +chromogen
  metals(Colloidal gold)

Question 11

Why is HRP commonly used as a label

Answer 11

• very small size therefore removes any issues of stearic hinderance
• easily obtained and highly purified
• stable enzyme and remains unchanged during manufacturing and storage
• endogenous activity can be easily quencehd

Question 12

What are the 3 types of antigens when it comes to retrieval

Answer 12

• highly formalin sensitive epitopes (vimentin)
• moderately formalin sensitive epitopes (PCNA)
• formalin resistant epitopes(CAM5.2)

Question 13

methods of antigen retrievel

Answer 13

physical using detergents (tween 20 and saponin)
enzymatic digestion (trypsin, pepsin, proteinase K)
head mediated antigen retrievel (most used)

Question 14

antigen types when it comes to pH values

Answer 14

• stable type: Acceptable reaction through the whole scale of pHs (CD20)
• v form type :antigens with best results at extremes pH (MIB-1
• ascending type: antigens with best results at alkaline pH (CK20).

Question 15

direct vs indirect IHC

Answer 15

direct - labelled primary antibody
indirect - more sensitive, labelled secondary antibody

Question 16

What has to be blocked for IHC

Answer 16

• endogenous enzyme
• endogenous biotin
• endogenous proteins

Question 17

what is used to block endogenous peroxidase and alkaline phosphatase

Answer 17

peroxidase - 0.3% hydorgen peroxide
AP -1mM concentrated levamisole

Question 18

how is endogenous biotin blocked

Answer 18

avidin is added - binds to biotin. biotin is added to saturate all avidin added, and found in tissue, further binding of biotinylated or avidinylated reagents is blocked as they are all saturated.

Question 19

How is protein blocking carried out and why

Answer 19

It preevents the non specific binding of antibodies to tissues or to endogenous Fc receptors. They are blocked using serum or prtoein blocking reagent. The serum used is commonly rabbit - it must be a different species than the primary antibodies.

Question 20

Interanl quality controls

Answer 20

positive - using known ppositive tissue
negative - omitting primary antibody

Question 21

applications of IHC

Answer 21

• confirmation of tumour histiogenesis
• prognosis
• classifcication of tumour
• prediction fo response to therapy
• muscel diseases
• detect infectious agents