L2 Introduction to antibody-based assays Flashcards
Understand the antibody-antigen interaction. Understand the importance of antibodies in biomedical research. Understand and explain various uses and limitations of antibodies. Describe general principles of antibody based assays.
What is a linear determinant epitope?
Linear epitope is only recognised in denaturing conditions, perhaps due to epitope being sequestered within protein. Hidden epitope.
What is a conformational determinant epitope?
Conformational determinant epitope is where epitope may only be recognised by antibody in the native conformation of protein. Requires tertiary protein structure for recognition.
What is a neoantigenic determinant epitope?
Where epitope is only recognised by proteolysis. Breaking of peptide bonds to expose epitope.
What is antibody affinity?
Measures the strength of the interaction between epitope and paratope.
What is antibody avidity? What are the three major parameters it depends upon?
This gives a measure of the overall strength of the Ab-Ag complex.
Affinity of the antibody for epitope.
Valency (no of binding sites) of both antibody and Ag.
Structural arrangement of parts that interact
Why can antibody cross-reactivity be positive or negative?
It is negative if antibody is needed to bind specifically to only one antigen, but positive if variety of species and animal models are being studied, e.g. mouse and human p53 being recognised by same antibody.
What are four general uses of antibodies?
In diagnosis for identification of a disease biomarker via IHC, WB OR ELISA.
Characterisation of target: what does gene X do/where is it located in cell/tissue? via IF, IHC, WB, IP, ChIP.
Cell identification/sorting: e.g. identifying types of B cell pop via FACS (fluorescence activated cell sorting)
Therapeutics: e.g. antibody based therapies, develop novel Ab. (phage display, Ab-drug conjugate, disease/pathway targets).
Describe how primary and secondary antibodies are used to detect Ab-Ag binding
The primary antibody binds and detects the antigen.
Then a secondary antibody is added which detects the primary antibody. Species specific and does not interfere with Fab regions so Fc regions are labelled with a reporter.
How do direct and indirect detection methods differ?
In direct, the label/reporter is attached to the primary antibody. Indirect, the label is attached to a secondary antibody.
How can antibodies be labelled?
Enzymatic detection via colourimetic or chemiluminescence, or fluorescence.
How does colorimetric detection of antibody labelling work?
Enzyme tagged to antibody reacts with a substrate to generate an insoluble coloured product, which is observed to confirm location of antibody.
Commonly used HRP, and AP, which catalyse conversion of substrates to various colours.
How does chemiluminescence detection of antibody labelling work?
Enzyme converts substrate to product and produces light as a by-product. Light signal can be captured on x-ray film or by CCD imager.
How does fluorescence detection of antibody labelling work?
Antibody conjugated to fluorochrome which emits light when excited by light of a shorter wavelength. immunofluorescence (IF) commonly used for simultaneous visualisation of multiple cellular targets.
What is the basis of how aggulutinaton/precipitation assays function?
Ability of antibody-antigen binding that can clump or precipitate out of solution.
What is the basic procedure of ELISA?
Coating: unknown Ag attached to multi well plate. Blocking: unbound sites are coated with blocking agent. Wash. Incubation: Enzymes labelled antibody that can detect the antigen that are added to the wells. Wash away unbound antibody.
Detection: Chromogenic substrate added for detection.
