Lecture notes
B cells and their products part 2
- Module
- Immunology
- Institution
- Bournemouth University Talbot Campus (BUTC)
Detailed notes regarding B cells and their products, part 2
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Lecture 7 – B Cells and their Products Part 2Uses of Monoclonal Antibodies
Diagnostics (pregnancy tests).
Targeted drug delivery (cancer treatment).
Assays for the purification of proteins.
Clinical treatments (like Herceptin).
In Vivo imaging.
Assay Techniques
ELISA.
Flow cytometry.
Western blot.
Immunofluorescence staining.
Immunohistochemistry.
Immunoprecipitation
- Bringing antibodies and antigens out of solutions.
- Depends on the valency of antibody and antigen.
- Immunoprecipitation depends on the concentration of antibodies and antigens.
- Soluble antigen/antibody in either antibody excess or antigen excess.
Assay= tool used to address multiple complex questions.
Valency= number of bonds an atom can form as part of a compound.
- Immunoprecipitation in gel not solution= easy to quantify:
o Punch holes in the agar in a petri dish and fill with the antibody or antigen. Over
time they will diffuse out and meet. If there is a zone of equivalence a line will
appear which is called the precipitin line.
o You can add lots of antibodies for different antigens, antigens are different sizes, so
they diffuse at different rates, meaning you get precipitin lines at different points.
o Antibodies also have different diffusion rates.
- Differences in diffusion rates- affecting immunoprecipitation:
o Relative concentrations.
o Size of protein.
o Protein shape.
o Temperature.
Double Diffusion.
Radial Immunodiffusion
Determines the concentration of an antigen.
- Antibody in gel.
- Antigen (A-G) in wells.
Single radial diffusion= Mancini reaction.
, Ouchterlony Reaction
Double radial immunodiffusion.
- Antibody in middle and antigens are around the outside.
1) Confirm antigen – antibody binding.
2) Test cross the reactivity of antibodies for multiple antigens.
3) Confirm relative concentrations of 2 antigens.
Method
Agarose gel in petri dishes.
Punch holes at equidistant points.
Add antibodies and antigens to wells.
Incubate for 12-48 hours in a moist chamber.
Visualise lines of perception.
Interpreting
1
1 2.4 1
1.2
1.3
Ab
Ab
Ab
IDENTITIY NON-IDENTITY PARTIAL IDENTITY
Prescence of ‘3’ has Polyclonal antibody Polyclonal antibody
no effect as mix binds to 2 antigens mix binds to 2
concentrations of ‘1’ and at nonoverlapping antigens but
in each case are epitopes. epitopes overlap.
equivalent.
Identity= antigens are identical, or antibodies bind to same epitope on each antigen (cross-
blocking).
ELISA
Enzyme linked immunosorbent assay.
- Detection/quantification of an antigen or antibody.
- Can be qualitative or quantitative.
- Fast method.
- Different approaches but they all rely on antibody-antigen binding and detection with
enzymes.
- Enzymes catalyse the colour change which can be quantified.
- ELISA test done in 96 well plates.
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