1. Agglutination
Purpose: To understand the
biological basis for, and applications
of, agglutination reactions

2. Serology: Antibody immunology in vitro
So… the use of antibodies to study the
immune system and stuff.
Usually involves Ab-Ag interactions

4. Pentameric
IgM
http://ocw.jhsph.edu/imageLibrary/views/fileLibrary/

5. Agglutination: The action of an antibody when it
cross-links multiple antigens producing clumps of
antigens

6. Agglutination: A Two-Step Process
Step Step
1
Binding of Ab to Ag 2
Formation of aggregates

7. Opsinization: the process by which antibody
binding to both antigen and phagocyte enhances
phagocytosis
http://podcast.scischina.org/groups/drmarkham/public/cd01e.jpg

8. *Ab acts as an opsin and makes the bacterium tastier
for the phagocyte AND clumps bacteria together
allowing the phagocyte to engulf many at once

9. Importance of measuring Ab-Ag
interactions:
•Quantifying Ab
•Identifying Ab targets
•Ab specificity for many targets
• Identification
• Quantification

10. Applications of Ab-Ag interactions:
•ELISAs
•Immunoflorescence
•immunodiffusion
•Immunoprecipitation
•flow cytometry
•Western blots
•magnetic bead column separation

11. Simple Slide: Qualitative (visual)
confirmation of the specificity of an
antiserum to the agglutinated particles.
Example: Blood typing

12. Quantitative determination of the amount
of antibody in the serum (titer)
No
Agglutination
Agglutination
http://nfs.unipv.it/nfs/minf/dispense/immunology/blt.jpg

13. Titer: How far you can dilute the patient’s
serum and still have it contain enough
antibodies to yield a detectable Ab-Ag
interaction.
*Higher titer means higher starting concentration of
Serum/Ab

14. Using agglutination to track disease progression:
What is the titer for this patient?
*Dilute the serum, add a constant amount of antigen- You want
to know how far you can dilute the patient’s serum (titer) and
still have agglutination to determine the concentration of
antibody in the original sample

15. Prozone
effect

16. For today:
Sheep Red Blood Cell = SRBC = Ag
Anti-SRBC antiserum = Ab against
SRBC (rabbit)

17. Add PBS
to wells Add Ab to
1A-12A well 1A
Perform serial
dilution from
well 1A-11A
Add Ag to
wells
1A-12A
*Pipet up and down at least 3 times during serial dilution
*Change tip between each serially dilution
*Avoid contaminating the Ag stock with Ab!