Immunohistochemistry
Principles
Immunohistochemistry = tissues
Immunocytometry = cells
Three types of IHC patterns
o Nuclear
o Membranous
o Cytoplasmic
Advantages of IHC include
o Possible to use fresh or frozen tissue
o Well established and readily available
o Cost is relatively low
o Fast turn around time
o No live infectious agents, so no risk to health
Disadvantages of IHC
o IHC stains are not standardised worldwide
o Equipment is costly
o Quantifying results is difficult
o Subject to human error
Antibodies
Five classes of antibodies
o IgA
o IgG
o IgM
o IgD
Light chain consists of two monovalent antigen binding fragments
Heavy chain consists of a constant structure backbone
, Affinity and Avidity
Affinity Avidity
The total binding strength of all of its binding sites
3D fit of the antibody to its specific antigen
together. How strong the bond is
Measure of the binding strength between
Influenced by the affinity of the antibody for its antigen
antigenic epitope and its specific binding
and the valences of both the antibody and the antigen
site
Titre and Dilution
Titre Dilution
The highest dilution of an antibody that Determined by the antibody titre evaluation. This may
results in the maximum intensity and be pre-determined by the manufacturer and may be
specificity of the final reaction product with variable depending on the type of antigen retrieval
minimal background staining used and sensitivity of method.
IHC Target
Antigenic epitopes
o A small sequence of amino acids or monosaccharides that interact with an antibody
complex
o The epitope indicates the binding components that may be involved in the antibody
antigen interactions
Monoclonal antibodies are targeted
o More precise for antigen specificity
o Different antigens may share similar epitopes
Polyclonal antiserum
o Provide many more specificities for antigenic determinants
o Many more variation giving more background staining
Preserving Antigenicity
Fixation - 10% buffered formalin
o Preserving morphological appearance
Principles
Immunohistochemistry = tissues
Immunocytometry = cells
Three types of IHC patterns
o Nuclear
o Membranous
o Cytoplasmic
Advantages of IHC include
o Possible to use fresh or frozen tissue
o Well established and readily available
o Cost is relatively low
o Fast turn around time
o No live infectious agents, so no risk to health
Disadvantages of IHC
o IHC stains are not standardised worldwide
o Equipment is costly
o Quantifying results is difficult
o Subject to human error
Antibodies
Five classes of antibodies
o IgA
o IgG
o IgM
o IgD
Light chain consists of two monovalent antigen binding fragments
Heavy chain consists of a constant structure backbone
, Affinity and Avidity
Affinity Avidity
The total binding strength of all of its binding sites
3D fit of the antibody to its specific antigen
together. How strong the bond is
Measure of the binding strength between
Influenced by the affinity of the antibody for its antigen
antigenic epitope and its specific binding
and the valences of both the antibody and the antigen
site
Titre and Dilution
Titre Dilution
The highest dilution of an antibody that Determined by the antibody titre evaluation. This may
results in the maximum intensity and be pre-determined by the manufacturer and may be
specificity of the final reaction product with variable depending on the type of antigen retrieval
minimal background staining used and sensitivity of method.
IHC Target
Antigenic epitopes
o A small sequence of amino acids or monosaccharides that interact with an antibody
complex
o The epitope indicates the binding components that may be involved in the antibody
antigen interactions
Monoclonal antibodies are targeted
o More precise for antigen specificity
o Different antigens may share similar epitopes
Polyclonal antiserum
o Provide many more specificities for antigenic determinants
o Many more variation giving more background staining
Preserving Antigenicity
Fixation - 10% buffered formalin
o Preserving morphological appearance
