Ag-Ab Interactions

Front 1

How strong is Ag-Ab interaction?

Back 1

Not very; it's a weak interaction of multiple non-covalent bonds.

Front 2

What type of configuration is the Ag-Ab interface?

Back 2

Complementery

Front 3

Definition of Affinity:

Back 3

the strength of the total noncovalent interactions between a SINGLE epitope and antigen-binding site.

Front 4

Is affinity a good measure of Ag-Ab interaction?

Back 4

No; there can be multiple binding sites.

Front 5

What is the Ka?

Back 5

Ratio of Ag-Ab complex to concentrations of each

Front 6

What is the Kd?

Back 6

1/Ka; the dissociation constant.

Front 7

Strong interaction is indicated by high or low Ka?

Back 7

HIGH. Low Kd.

Front 8

What is the preferred parameter for describing affinity?

-What is high, what is low?

Back 8

Kd.

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


Kd < 10^-9 is High.

Front 9

What assay detects the Ka/Kd?

Back 9

Equilibrium dialysis

Front 10

What is a Scatchard plot?

What does it show?

Back 10

A plot of r/c versus r.



Shows
The NEGATIVE slope = Ka
X intercept = valency of the Ab.

Front 11

What is Avidity?

Back 11

A measure of the combination of valency and affinity.

Front 12

What is the valency of IgM?

Back 12

10

Front 13

wait, it's five

Back 13

lkj

Front 14

What is Cross-reactivity?

Back 14

Binding of an antibody to an antigen it's not actually specific for, but shares a same or similar epitope with the actual antigen.

Front 15

2 examples of cross-reactivity:

Back 15

-Ag on RBC and intestine bacteria (HUS in e. coli)

-Streptococcus Ag and heart muscle --> post strep complications -> rheumatic fever

Front 16

8 applications of Ab-Ag interactions:

Back 16

1. Precipitation reactions
2. Agglutination
3. Radioimmunoassay
4. ELISA
5. Western blot
6. Immunoprecipitation
7. Immunoflourescent/electron microscopy
8. Flow cytometry, cell sorting.

Front 17

What are the requirements of a Precipitation Reaction?

Back 17

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

Front 18

What does optimal precipitation reaction require?

Back 18

Good balance between Ag/Ab concentrations.

Front 19

2 types of precipitation reactions:

Back 19

-radial immunodiffusion.

-double immunodiffusion.

Front 20

What is an Agglutination reaction?

2 uses:

Back 20

Similar to precipitation, but Ag is clumped.

Hemagglutination - to determine blood type

Agglutination inhibition - pregnancy test.

Front 21

Principle of the competitive agglutination pregnancy test:

Back 21

-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.

Front 22

What is Radioimmunoassay for?

Back 22

detecting trace amounts of hormones, drugs, or vitamins in a sample.

Front 23

How do you run a Radioimmunoassay?

Back 23

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.

Front 24

One thing forgot in the Radioimmunoassay:

Back 24

Label the Ag of interest that you add so you can count how much is free.

Front 25

Principle of Indirect Elisa:

Back 25

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.

Front 26

Principle of Sandwich ELISA:

Back 26

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.

Front 27

Principle of Competitive Elisa:

Back 27

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

Front 28

What does a western detect?

Back 28

-Amount
-Size
-Presence

of protein of interest.

Front 29

process of Western:

Back 29

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.

Front 30

What is immunoprecipitation for?

Back 30

purifying a protein from a sample.

Front 31

What is immunoflourescent or immuno-electron microscopy for?

Back 31

to localize a protein of interest -> to see if it is even PRESENT.

Front 32

What does flow cytometry and cell sorting allow?

Back 32

selection of a specific cell you want.

Front 33

What are the 3 most sensitive techniques?

Back 33

-Radioimmunoassay
-Elisa
-Western