Lecture 12 Flashcards
What do antibodies secreted from BCR bind?
Native antigens - they are monoclonal
Where are antibodies found?
In the blood
What is a serum?
Plasma once blood clot is removed
What is an antiserum?
Serum from immunised person
What do antiserums not contain?
Cells or clotting proteins
What is a standard way of purifying?
Chromatography based on their molecular weight
What comes out for the column first?
Big things
What are the two types of chromatography used for antibodies?
Gel filtration and affinity
What is purified antiserum sometimes known as?
Polyclonal antiserum
What is the same for all antibodies?
Their MW
How is the filtration column modified?
By using beads
What antibodies will be attached to the beads?
Antibodies that are specific to the antigen
What happens to the antibodies that don’t attach to the beads?
Go through the column
What do patients with multiple myelomas produce?
Large amounts of homogenous antibodies
What are hybridomas?
Cells that are fused by myeloma cells with b cells
When did the generation of monoclonal antibodies start?
1975
What can live in the drug forever?
B cells fusions as they have properties of myelomas
What is a monoclonal antibody?
Has come from a single hybridoma
What do many methods using antibodies rely on?
Rely on labels attached to the antibodies in order to detect them once they are bound to antigens
What should labels not affect?
Antibody or antigen binding
What happens if an antibody has bound to an antigen?
An enzyme is bought in and can detect the present of an antigen antibody
What is the enzyme?
HRP or AP
What do many methods of antibodies also use?
Secondary antibodies to detect the primary antibody binding to its antigen
What does using the same antibody that is not labelled with a secondary antibody that is labelled = ?
More sensitivity
What is an example of a labelled primary antibody?
Mouse IgG monoclonal
How long does it take to collect rat anti-mouse antiserum?
2-6 weeks
What would happen if you were to put one antibody into another animals antibody?
It would make another antibody
What two ways can antibodies be used in?
Research and diagnostics
What ways can antibodies be used in research? 3 ways
Affinity chromatography, immune precipitation and immunofluorescence microscopy
What is affinity chromatography?
Can purify molecules from complex mixtures and use antibodies to pull out proteins from a mixture
What are magnetic bead isolations?
Little beads with antibodies that bind to a Magnet which form a column
What happens when the magnet turns off?
Antibodies will fall off the magnet pulling the proteins with them
What happens in immunofluorescence microscopy?
Identifying the location of a protein within the cell
What happens to the antibodies in fluorescent microscopy?
Antibodies light up different parts of the cell depending on where the protein is being expressed
What does western blotting allow?
Allows you to look at different proteins inside cells
What is a very common technique used in both research and diagnostic?
ELISA
What does ELISA stand for?
Enzyme-linked immunosorbent assay
What does ELISA detect?
Non linear and linear epitopes
What can ELISA be used for in respect to antibodies?
Can be used to quantitate amount of antigen present
What happens in direct ELISA?
Blood is put on a plate and a positive signal is received in the well where the antibody is attached to the antigen
What is sandwich ELISA?
Even more sensitive and uses less of limited sample
What is different to sandwich ELISA compared to direct ELISA?
Only dented the well being positive if the antigen has been captured
What is flow cytometry (FACS)?
A technique used for the characterisation of cells based not their light scattering properties
What can the properties be?
Either natural or induced by pre-incubation of cells with antibodies labelled with dyes
How many cells are able to pass through the lazor at a time?
2000 per second
What are the two ways cells can move through the lazor?
Side scatter and forward scatter
At does forward scatter result from?
A cells size
What does side scatter result from?
A cells granularity
What can be obtained if you add antibodies to the cell before they go through the lazor?
Expression of various molecules
What happened in 1975?
Never ending supply of monoclonal antibodies
