Week 1 Fundamentals of IHC

What are the most frequently utilised Ab for IHC?

IgG and IgM

Why are monoclonal Ab immunochemically identical?

They are produced by the same plasma cell

What is the product of combining plasma cells and tumour cells called?

Hybridoma

Compare between mAb and pAb.

mAb:

• unique specificity

• lower sensitivity

• unlimited supply

• lower titre

• lower batch to batch variability

• lower robustness to staining conditions variability

pAb:

• lower specificity

• higher sensitivity

• limited supply

• higher titre

• higher batch to batch variability

• higher robustness to staining conditions variability

What is the most widely used enzyme? Why?

• HRP

• small size→not hinder the Ab binding

• easily obtainable in a highly purified form→min contamination

• stable

List the visualization systems from least sensitive to the most sensitive.

direct IHC, indirect IHC, PAP, ABC, LSAB, polymer technology, tyramide

What label is more commonly used in direct IHC?

fluorochrome

When will we use direct IHC?

frozen sections of skin and renal biopsies

What does PAP reagent consist of?

HRP and Ab against HRP

What is the principle behind ABC?

avidin and biotin has high affinity

What is the difference between ABC and LSAB? Why is LSAB better than ABC?

• streptavidin replaces avidin

• streptavidin has fewer bg staining, more stable, higher sensitivity

What is the advantage of polymer method?

avoid biotin→no need to block endogenous biotin→less false +ve and less errors due to less steps

How to reduce non specific Ab binding?

Preincubation with normal serum that is the same species as the 2nd Ab→proteins within can occupy the charged tissue components

We have to block endogenous peroxidase after 1st Ab application. Why?

H2O2 may damage some epitopes→less signals

What is optimal dilution?

The finest conc that gives the best contrast between specific Ag and non specific bg staining

How do we know the specific Ag is really what it is?

• use isotype Ab

• add 2nd Ab directly

What is the difference between specific -ve control and non specific -ve control?

• specific uses tissues without the Ag and normal Ab to detect Ab cross reactivity to cells

• non specific uses pt tissue that doesn’t contain tumour and diluent to replace Ab to detect bg staining

What is the most widely used Ag retrieval method?

HIER

What are some possible problems that IHC can encounter?

• wrong Ab selection

• false +ve

• false -ve

What are some possible reasons for false +ve?

• bg staining

• endogenous enzymes

• too high Ab conc

• pigment mistaken for +ve signals

• drying artefact

• pseudospecific signal

What are some possible reasons for false -ve?

• poor fixation

• too low Ab conc

• incorrect Ag retrieval