Immunocytochemistry Flashcards
(32 cards)
What is immunocyto(histo)chemistry?
Identification of tissue constituent (antigen) in situ by means of a specific antigen-antibody reaction tagged by a visible label (e.g., fluorophore)
What does in situ mean?
Directly at site. Intact cells and tissue.
What are the advantages of immunocytochemistry?
Can visualise exact location of antigen which is not possible with other biochemical techniques. Visualise antigens in low concentrations or in limited samples. Not detectable through western blots for example.
What are the disadvantages or immunocytochemistry?
Doesn’t show if protein is active or not. E.g., when visualising an enzyme. Doesn’t clarify whether the antigen is being produced at site of the location (need mRNA technology).
What do we need for immunostaining?
Antigen (substance capable of immune response). Epitope/antigenic determinant, antibody.
What is an epitope?
Part of an antigen that combines with antigen binding site of antibody. Proteins that differ by a single ammino acid or 2 optical isomers can still be distinguished.
What is the basic structure of antibodies?
4 polypeptide chains. 2 light chains 220 aa. 2 heavy chains 440 aa.
What is the tail or FC of heavy chain for?
Flexible hinge regions to allow the distance between binding sites to be varied. Increases binding efficiency.
What determines an antigen being multivalent?
If two or more determinants. Polymer with repeat structures.
What is affinity?
Strength of binding to a single antigenic determinant. Independent of number of binding sites.
What is avidity?
Total binding strength of all binding sites together.
What does equilibrium point depend on?
Concentrations of Ab and antigen strength of interaction.
Why split antibody molecules with proteolytic enzymes?
Enhance performance of certain procedures.
What are 3 advantages of antibody fragments?
Reduced nonspecific binding from Fc interactions (many cells have receptors that bind Fc region) Ability to control binding in experiments with immunoprecipitation or western blot. More efficient penetration of tissue sections.
What are 4 more advantages of antibody fragments?
Higher sensitivity of antigen detection in solid phase as steric hindrance is reduced. Elimination of Fc-associated effector functions. Simpler system for studying structural basis for immune recognition using X-ray crystallography or NMR. Lower immunogenicity than intact antibody.
Why cut out the IgG chain?
Reduce site of antibody make it lighter so antibody can travel further.
What is papain?
Thiol-type protease. Cuts 2 separate but identical Fab fragments each with 1 binding site and 1 Fc fragment.
What is pepsin?
Acid type protease. Cuts 1 Fab fragment with 2 covalently linked Fab fragments which are bivalent and can still cross link.
Compare monyclonal and polyclonal antibodies.
Monyclonal reactive to single epitome. Polyclonal more likely to detect epitome so less antibody needed however is less specific.
What are the 5 classes of antibodies?
IgA, IgD, IgE, IgG and IgM. Major class in blood is IgG. 4 subclasses of this.
What are polyclonal antibodies?
Whole sera of immunised animal antibodies are purified off other circulating antibodies and contain a mixture of monoclonal antibodies.
What are monoclonal antibodies?
An antibody secreting cell isolated from an immunised animal and fused with B-cell tumour. Hybridoma maintained in vitro and secrete antibodies within a defined specificity. Each mAb is immunoreactive to single epitope.
What is peroxidase anti-peroxidase PAP method?
Enzyme linked horseradish peroxidase chromogenic substrate that changes colour if reacted with DAB alkaline phosphatase. Light microscopy.
Why block endogenous peroxidase activity?
Some cells or tissues contain endogenous peroxidase. Using HRP conjugated antibody may result in high non specific background staining. Can be reduced by pre treatment of cells with hydrogen peroxide.
