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Ag-Ab interactions


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How strong is Ag-Ab interaction?
Not very; it's a weak interaction of multiple non-covalent bonds.
What type of configuration is the Ag-Ab interface?
Definition of Affinity:
the strength of the total noncovalent interactions between a SINGLE epitope and antigen-binding site.
Is affinity a good measure of Ag-Ab interaction?
No; there can be multiple binding sites.
What is the Ka?
Ratio of Ag-Ab complex to concentrations of each
What is the Kd?
1/Ka; the dissociation constant.
Strong interaction is indicated by high or low Ka?
HIGH. Low Kd.
What is the preferred parameter for describing affinity?

-What is high, what is low?

Kd >> greater than 10^-9 is LOW
(like 10^-8)

Kd < 10^-9 is High.
What assay detects the Ka/Kd?
Equilibrium dialysis
What is a Scatchard plot?

What does it show?
A plot of r/c versus r.

The NEGATIVE slope = Ka
X intercept = valency of the Ab.
What is Avidity?
A measure of the combination of valency and affinity.
What is the valency of IgM?
wait, it's five
What is Cross-reactivity?
Binding of an antibody to an antigen it's not actually specific for, but shares a same or similar epitope with the actual antigen.
2 examples of cross-reactivity:
-Ag on RBC and intestine bacteria (HUS in e. coli)

-Streptococcus Ag and heart muscle --> post strep complications -> rheumatic fever
8 applications of Ab-Ag interactions:
1. Precipitation reactions
2. Agglutination
3. Radioimmunoassay
5. Western blot
6. Immunoprecipitation
7. Immunoflourescent/electron microscopy
8. Flow cytometry, cell sorting.
What are the requirements of a Precipitation Reaction?
Antibody must be bivalent or multivalent.
-Fab does not work -> univalent.

Antigen must be bivalent or multivalent.
-what good is one valency for a bivalent antibody? won't get any CLUMPS
What does optimal precipitation reaction require?
Good balance between Ag/Ab concentrations.
2 types of precipitation reactions:
-radial immunodiffusion.

-double immunodiffusion.
What is an Agglutination reaction?

2 uses:
Similar to precipitation, but Ag is clumped.

Hemagglutination - to determine blood type

Agglutination inhibition - pregnancy test.
Principle of the competitive agglutination pregnancy test:
-Urine cells have hormone from being pregnant.

-Coat beads w/ hormone.
-Put beads in urine.
-Add Antibody to the hormone.
-If lots of hormone in the urine, it will compete for the Ab with the beads. The beads won't clump as result.
What is Radioimmunoassay for?
detecting trace amounts of hormones, drugs, or vitamins in a sample.
How do you run a Radioimmunoassay?
1. Generate the Ag you're looking for, and the antibody to it.

2. Add known amt of Ab to sample.
3. Add known amt of Ag of interest to sample.
4. The amt of Free Ag floating shows how much Ag in the sample bound to the Ab.
One thing forgot in the Radioimmunoassay:
Label the Ag of interest that you add so you can count how much is free.
Principle of Indirect Elisa:
1. Immobilize Ag -> stick it to a plate.
2. Add serum to be tested.
3. Add antibody to the serum; this is a 2ndary Ab conjugated to enzyme.
Principle of Sandwich ELISA:
1. Immobilize Monoclonal Antibody -> stick to plate.
2. Add Sample with Ag of interest -> must be bi-epitoped so you can put the top slice of bread on.
3. 2ndary Ab to other epitope. Conjugated to enzyme to see reaction.
Principle of Competitive Elisa:
1. Immobilize Ag to well
2. Add Ag-containing sample mixed with Ab.
3. Wash off the native Ag-Ab complexes still in the well.
4. Add 2ndary Ab; look for color.

Amt of original Ag you were looking for is the difference
What does a western detect?

of protein of interest.
process of Western:
1. SDS page seperates proteins based on size and charge.
2. Transfer proteins to nitrocellulose membrane.
3. incubate membrane w/ Ab to Ag.
4. wash primary Ab. Put on 2ndary.
5. color or luminescence shows how much antigen was there.
What is immunoprecipitation for?
purifying a protein from a sample.
What is immunoflourescent or immuno-electron microscopy for?
to localize a protein of interest -> to see if it is even PRESENT.
What does flow cytometry and cell sorting allow?
selection of a specific cell you want.
What are the 3 most sensitive techniques?

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