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B cells and their products part 2

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  • February 24, 2022
  • 5
  • 2021/2022
  • Lecture notes
  • Sarah buchan
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biomedicalscience4
Lecture 7 – B Cells and their Products Part 2
Uses 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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