Lecture 16; Immunoassay Flashcards
What is an antigen?
A substance with which the antibody reacts
What is an immunogen?
Substance used to produce an immune response - could be larger than an antigen
What is a B cell?
Lymphocytes that produce antibodies
What is required to produce antibodies?
B Cells and T Cells recognition of the antigen
Describe the production of antibodies;
1) Antibodies are triggered when B cell encounters its matching antigen
2) B Cell takes in antigen and digests it
3) Displays antigen fragments on its MHC molecules
4) Antigen fragments and MHC attracts help of mature T cell
5) Lymphokines secreted by the T Cell allows the B cell to multiple and make antibody producing plasma cells
6) Antibodies are released into the blood stream and bind antigen
What happens to antigen-antibody complexes?
They are eliminated either by the compliment cascade, liver or spleen
Describe the specificity of Plasma B cells;
Each B cell produces only one type of antibody molecule.. i.e specific for one antigen..
But antigens can closely resemble one another and antibodies can be cross reactive
What is Cross reactivity?
Antibodies may bind multiple antigens because they are very similar.
Whats the implication of cross reactivity?
When using antibodies experimentally this must be taken into consideration
Whats an example of cross reactivity?
If an antibody binds to the alpha chain of hCG it will also cross react with the alpha chain of LH
What could be targeted instead of hCG alpha chain to reduce cross reactivity?
beta hCG has a c terminal extension of 30 amino acids that is not in the beta LH chain. Antibodies directed towards this are specific for bhCG
Write some short notes on antibodies;
- Antibodies area type of protein called immunoglobulins
- Most common is IgG
Describe IgG structure;
- Compromised of two main structural and functional regions
- Two heavy chains specific for antibody class and two light chains shared by all classes
- heavy + light chain variable binding regions create antigen binding site
- Held together by disulphide bonds
What is Fab and fc?
Fab = antigen binding site, varies between antibodies
Fc = Antibody constant region, consistant amongst antibody class
What are the five immunoglobulin classes?
IgG IgM IgA IgE IgD
There are subclasses within each class
Describe basic immune response to infection;
First exposure;
Latent period then; IgM followed by larger IgG response
Secondary response;
No latency!
- huge IgG and some IgM together!
What are epitopes?
An epitope is that part of an antigen that interacts with an antibody i.e the antibody-binding site.
What are the types of epitopes?
Conformational and linear
What is a conformational epitope?
A binding site that is several parts of the protein causes by its conformation
What is a linear epitope?
Binding sites in succession
Describe antigen-antibody interactions;
Non-covalent and reversible i.e H bonds, hydrophobic interactions, electrostatic and van der waal forces
What do affinity and avidity describe?
Strength of interaction between antigen-antibody complex
What is affinity;
Measure of the strength of an antibody binding to an epitope
What is avidity;
Measure of the stability of an interaction between antibodies and antigen
What does avidity measure?
1) Affinity of Ab for epitope
2) Valency of AB antigen (n. antigen AB can bind)
3) Geometric arrangment of complex
What are polyclonal responses?
- Large antigens contain many hundreds of epitopes
- An organism will produce antibodies for all of these epitopes
- Microbes may have thousands of epitopes
What is a monoclonal antibody?
One antibody for one antigen
Polyclonal is many antibodies for different epitopes on one antigen
How are monoclonal antibodies produced?
produced in mice by intreoducing the antigen then cloning the B cell that produces the specific antibodies.
Monoclonal antibodies are good for reducing non-specific binding
What are the types of immunoassays?
- Precipitation
- Agglutination
- Immunohistochemistry
- Sandwich assays
- Antibody Capture assays
- Antigen Capture assays
Describe precipitation;
- Polycloncal antisera bind insoluble antigens
- Then polyclonal antibodies that are soluble diffuse towards soluble antigen and precipitate is formed (at optimal concentrations)
Describe agglutination;
- Polycloncal antisera bind insoluble antigens
- Typically antigens are RBC or beads/ latex particles
- Antserum cross links the particles causing them to agglutinate. (this can be visualised)
Describe sandwhich assay (two antibody)
1) Bond antibody to solid phase (bottom of well)
2) Bind antigen to antibody
3) Bind second antibody to antigen
4) Quantitate second antibody
Describe antigen capture assay;
1) Bond antibody to solid phase (bottom of well)
2) Bind antigen to antibody
3) Quantitate antigen
Describe antibody capture assay;
1) Bond antigen to solid phase (bottom of well)
2) Bind antibody to antigen
3) Quantitate antibody
Describe IHC;
- Thin section of tissues placed on histology slides.
- Probed with antigen specific antibodies (primary antibody)
- Enzyme conjugated secondary antibody is introduced
- Reagent is added that is converted by the enzyme into insoluble precipitate
What can IHC be used for?
This technique can be used to identify particular cell types in a tissue i.e tumor typing or to locate the expression pattern of a particular antigen
Given an example of amplification compound
Biotin streptavin
