Ligand binding: antibodies

Question 1

What is the difference between the innate and adaptive immune system?

Answer 1

Innate is non-specific and just looks for general non-human features that would indicate a pathogen. The adaptive immune system is specific to particular pathogens

Question 2

What are the two subdivisions of the adaptive immune system?

Answer 2

Cellular and humoral

Question 3

What does the cellular immune system do?

Answer 3

Detects and destroys infected cells through T cell receptors

Question 4

What does the humoral immune system do?

Answer 4

Recognizes and attacks pathogens in body fluids by using antibodies

Question 5

What protein family are antibodies part of?

Answer 5

Immunoglobulins

Question 6

What are the two polypeptide chains in antibodies? What’s the difference between them?

Answer 6

Heavy and light. Heavy chains are longer

Question 7

What are the two domains on one of the chains in an antibody?

Answer 7

Variable and constant

Question 8

How many variable and constant domains are on a heavy chain?

Answer 8

3 constant, 1 variable

Question 9

How many variable and constant domains are on a light chain?

Answer 9

1 constant, 1 variable

Question 10

Which domains make up the antigen binding site on an antibody?

Answer 10

The variable domains of the heavy and light chains

Question 11

How many disulfide bonds are in an antibody?

Answer 11

4

Question 12

Where are the disulfide bonds in an antibody?

Answer 12

2 closer to the top near the antigen binding site on both sides, between the heavy and light chains. 2 in the middle hinge region between the two heavy chains

Question 13

What are the two fragments of an antibody?

Answer 13

Fab (antigen binding) and Fc (crystallizable)

Question 14

How do we break apart the two fragments in an antibody?

Answer 14

Papain

Question 15

What is an antigen?

Answer 15

Molecule that binds selectively to an antibody

Question 16

What is the epitope?

Answer 16

The particular site on the antigen that is recognized by the antibody

Question 17

What are the two types of epitopes?

Answer 17

Linear and conformational

Question 18

What is a linear epitope?

Answer 18

The specific amino acid sequence is recognized, so binding will still occur if the antigen is unfolded

Question 19

What is a conformational epitope?

Answer 19

The 3D structure is recognized, so binding does not occur if the antigen is unfolded

Question 20

How do macrophages recognize pathogens targeted for destruction by antibodies?

Answer 20

Have an Fc receptor that will bind to that region of the antibody and cause the macrophage to engulf the pathogen

Question 21

What is structural motif is present in the constant domains?

Answer 21

Beta sandwich (two antiparallel beta sheets)

Question 22

What structural feature is present in the antigen binding sites of antibodies?

Answer 22

Hypervariable loops

Question 23

How do antibodies improve the complementarity of antigen binding?

Answer 23

Induced fit for shape, charge, hydrophobicity and H bonding

Question 24

What are 4 applications for antibodies?

Answer 24

Western blot, ELISA, immunofluorescence, co-immunoprecipitation

Question 25

What is an ELISA?

Answer 25

Enzyme-linked immunosorbent assay. Provides a quantitative measurement of the amount of antibody of antigen

Question 26

What are the two types of ELISA?

Answer 26

Indirect and sandwich

Question 27

What does indirect ELISA detect?

Answer 27

The presence of the antibody

Question 28

How do you do indirect ELISA?

Answer 28

Coat the well with the antigens, then add the fluid you’re testing. Add secondary antibodies with an enzyme attached then add the substrate for that enzyme. If there were antibodies in the fluid, we would get a colour

Question 29

What does sandwich ELISA detect?

Answer 29

The presence of the antigen

Question 30

How do you do sandwich ELISA?

Answer 30

Coat the well with antibodies, then add the fluid you’re testing. Add the secondary antibodies with the linked enzyme that is also specific to the antigen, then add the enzyme substrate and look for a colour change

Question 31

What do we use immunofluorescence for?

Answer 31

Detection of proteins in living cells

Question 32

What does co-immunoprecipitation detect?

Answer 32

Protein-protein interactions

Question 33

How do you do a co-immunoprecipitation experiment?

Answer 33

Add antibodies attached to resin beads for one of the binding partners, then remove the supernatant and run the pellet on a gel. Do a western blot and probe for the other binding partner