immunohistochemistry Flashcards
what is IHC
technique used to identify cellular or tissue constituents (antigens) using antigen-antibody binding
What is avidity and what does it depend on
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
What are polyclonal antibodies
a heterogenous mix of antibodies where each antibody rexognizes different epitopes of the same antigne
How are polyclonal antibodies produced?
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
disadvantages of polyclonal a
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
What are monoclonal antibodies
Obtained from mice mainly. anitbodies obtained from antibody producing hybridoma clones, made specific for 1 epitope of the antigen (highly specific)
advantges and disadvantges of monoclonal antibodies
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.
Monoclonal antibodies production process using hybridomas
-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.
Recombinant monoclonal antibody production
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.
What can be used a label , conjugated with the antiboud
- fluorochromes (fluorescin, rhodamine)
- enzymes (HRP, Alkaline phosphatase) +chromogen
metals(Colloidal gold)
Why is HRP commonly used as a label
- 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
What are the 3 types of antigens when it comes to retrieval
- highly formalin sensitive epitopes (vimentin)
- moderately formalin sensitive epitopes (PCNA)
- formalin resistant epitopes(CAM5.2)
methods of antigen retrievel
physical using detergents (tween 20 and saponin)
enzymatic digestion (trypsin, pepsin, proteinase K)
head mediated antigen retrievel (most used)
antigen types when it comes to pH values
- 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).
direct vs indirect IHC
direct - labelled primary antibody
indirect - more sensitive, labelled secondary antibody
What has to be blocked for IHC
- endogenous enzyme
- endogenous biotin
- endogenous proteins
what is used to block endogenous peroxidase and alkaline phosphatase
peroxidase - 0.3% hydorgen peroxide
AP -1mM concentrated levamisole
how is endogenous biotin blocked
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.
How is protein blocking carried out and why
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.
Interanl quality controls
positive - using known ppositive tissue
negative - omitting primary antibody
applications of IHC
- confirmation of tumour histiogenesis
- prognosis
- classifcication of tumour
- prediction fo response to therapy
- muscel diseases
- detect infectious agents
