Lecture 12: Uses of monoclonal antibodies Flashcards
Why are antibodies good biological tools?
Secreted in high amounts from differentiated B cells/plasma cells
Bind almost all biological materials
Binding well characterised and often high affinity (SHM)
What were the early approaches with purifying antibodies?
Purified antigen antibodies found in plasma
What is serum?
The plasma once blood clot removed
From immunised person/animal known as antiserum
Antiserum does not contain cells or clotting proteins
What will antiserum contain?
Many different antibodies secreted by different B cells and bind same antigen (different parts of pathogen)
How do antibodies bind different parts of the antigen?
As they are epitopes
How can different antibodies be purified from other serum proteins?
Using gel filtration chromatography (based on molecular weight)
Or
Affinity chromatography (binds to bead which filters out the other proteins)
What are the imitations of antisera?
The individual antibodies are separated out
Another individual has to be immunised however the antibodies will not be the same
What was the method devised by George Kohler, Cesar Milstein and Niles Jerne?
Fusing of mouse myeloma cells with mouse plasma cells making antibodies of know specificity to generate hybridomas
Why are myelomas useful in antibody purification?
They produce large amounts of homogenous antibodies
How are monoclonal antibodies produced?
Immunise organism (with specific antigen)
Harvest spleen
Spleen produces antibody and myeloma cells are produced
Cells mixed and fused
Transfered to HAT medium
Select hybrid making specific antibody
Clone
How can antibodies be detected once separated?
Using labels once bound to antigen which should not affect antibody/antigen binding
Why do methods using antibodies use secondary antibodies?
To detect primary antibody as it increases sensitivity
How are secondary antibodies produced?
Collect mouse sera
Contains mouse antibodies
Purify mouse antibodies and immunise rat
collect rat anti-mouse antiserum
Purify rat anti-mouse polyclonal antibodies
What are uses of antibodies in research?
Purifying biological molecules from a mixture (affinity chromatography)
Identifying the location of a protein within a cell using immunofluorescence microscopy
What are the techniques using antibodies in diagnostics?
Western blot:
HIV dissociate in SDS (lyse virus)
SDS-PAGE (separate proteins based on molecular weight)
Transfer proteins to membrane and incubate with monoclonal antibody
Detect bound antibody with enzyme-linked anti-IgG
ELISA:
Linear and non-linear epitopes
Sensitive for specific biological material in wide range of complex samples
Quantitate amount of antigen present
Flow cytometry:
Characterisation based on light scattering properties
Natural or induced by pre-incubation of cells with antibodies labelled with dyes
Rapid and measures multiple parameters from each individual cell
How does ELISA work?
Enzyme linked immunosorbent assay
Direct: biological sample with antigen
add labelled antibody for specific antigen
add substrate for enzyme label
Indirect: biological sample containing antigen
add unlabelled antibody specific for antigen
add labelled secondary and substrate for enzyme label
Sandwich: unlabelled antibody specific for antigen added to plate
sample added
add different antigen specific labelled secondary then substrate
How does flow cytometry work?
Mixture of cells goes up a thin tube a laser pointed then measured forward (FSC) and side scatter (SSC)
What do FSC and SSC measure in flow cytometry?
FSC - cells size
SCC - cells granularity
How is flow cytometry data presented?
On a dot plot
How can extra information be obtained using antibodies in flow cytometry?
Identified by expression of various molecules
Antibodies specific can be labelled and identify number of positives in mixed population
What allows further definition of cells in a flow cytometer?
Fluorescently (FITC)- conjugated antibody
How does Fluorescently (FITC)- conjugated antibody allow further definition?
Analysis of labelled cells for FSC/SSC then set gate for cells of interest
Level of fluorescence measures just on cells in gate
Second gate used in analysis to define sub-population based on fluorescence
How can cross over with different labelled antibody happen in flow cytometry?
Wrong colour software removes fluorescence
How can compensation be performed in flow cytometer?
First labelled antibodies are analysed so false detection of second labelled antibody can be removed by software
