immunoaffinity

Question 1

define epitope

Answer 1

the targeted molecular structure of a specific antibody

Question 2

Describe the structure of antibodies

Answer 2

.Heavy chains inside, light chains
.Biological activity in base
.2 Fab fragments for binding-terminated by N terminals called variable regions
.A disulfide bond hinge region
. Divalent ( can bind two antigens)

Question 3

effect of papain and pepsin on antibody

Answer 3

.papain cleaves hinge region to leave two detached fab fragments and constant region
. pepsin cleave below hinge region leaving attached fab fragments. Fragments the constant region

Question 4

How is diversity in antibodies produced?

Answer 4

.Somatic recombination of genes coding for VDJ region.

. RNa splicing

Question 5

Polyclonal antibody

Answer 5

a collection of antibodies from different B cells that recognise multiple epitopes on the same antigen

Question 6

Monoclonal antibody

Answer 6

derived from a single antibody producing B call- only bind to one unique epitope

Question 7

Considerations of using polyclonal antibodies

Answer 7

.cheap to produce

.tolerant of small changes in protein structure

Question 8

Considerations of using monoclonal antibodies

Answer 8

.Expensive to produce

.Highly specific for protein form

Question 9

What is the purpose of excess antibody with respect to antigens in immuno affinity chromatography?

Answer 9

Favors monovalent binding-why is this good? Monovalent bonding means that majority of antigen is bound.

Question 10

characteristics of good binding matrix

Answer 10

.Provide mechanical/physical/chemical stability
.Acceptable pressure drop
. Exhibit minimal non-specific binding
.Provide surface area of Ab-An interactions.

Classes:
.natural polymers
.Synthetic polymers
.inorganic materials

Question 11

Describe mechanism of low pH elution strategy for IAC

Answer 11

Disrupts ionic bonds

comment: proteins have to be pH stable

Question 12

Describe mechanism of chaotropic salts elution strategy for IAC

Answer 12

interferes with stabilizing intra-molecular interactions. High salt concentration disrupts water molecules around the affinity interaction

Question 13

Describe mechanism of organic solvent elution strategy for IAC

Answer 13

Hydrophobic effects- signifcant elution of smaller molecules, efficient column regeneration

Question 14

Describe mechanism of organic solvent elution strategy for IAC

Answer 14

Hydrophobic effects- significant elution of smaller molecules, efficient column regeneration

Question 15

Describe mechanism of displacer elution strategy for IAC

Answer 15

competition- exploits high affinity interactions. Can promote bio-specific interactions because of mild conditions. disadvantage: longer elution time.

Question 16

Describe mechanism of displacer elution strategy for IAC

Answer 16

competition- exploits high affinity interactions. Can promote bio-specific interactions because of mild conditions. disadvantage: longer elution time.

Question 17

What does ELISA stand for

Answer 17

Enzyme linked immunosorbent Assay

Question 18

examples of foreign antigens

Answer 18

foreign proteins, viruses, environmental pollutants ( tattoo ink, smoke..), bacteria and parasites, foreign transplanted tissue

Question 19

Why did we stop using radio immuno assay

Answer 19

.safety
.produces radioactive waste
.short shelf life

Question 20

indirect ELISA method

Answer 20

1. Bind known antigen to wells of microtiter plate
2. Block unnocupied are bound by solution of non-interacting proteins.
3. Continue…

Question 21

Difference between direct and indirect ELISA

Answer 21

in direct elisa, the antibody that binds to the target antigen in enzyme linked- whilst in indirect elisa, a secondary antibody binds to the primary antibody, and the secondary enzyme is the one that is enzyme linked

Question 22

advantage of indirect elisa over primary elisa

Answer 22

.to identify an antigen, the primary antibody must be specific, hence monoclonal antibodies are used, which are expensive to manufacture. in indirect antibody, these monoclonal antibodies can be reused, as the “single-use” enzyme reaction is done by a secondary antibody.
.Because multiple secondary antibodies can bind to one antibody, this leads to signal amplification. Hence, indirect is more sensitive than direct.

Question 23

purpose and method of sandwich ELISA

Answer 23

Sandwich ELISAs capture and detect antigens. Antigen sample is loaded into antibody coated well. enzyme-bound antibody is then added.

Question 24

Advantages of sandwich ELISA

Answer 24

sample doesn’t require prior purification, 2-5 times more sensitve than direct/indirect ELISA

Question 25

structure of enzyme-bound Ab

Answer 25

biotin bound to enzyme label via avidin or streptavidin in a tetravalent manner. enzyme labels:
.Horse radish peroxidase
. Alkline phosphatase

Question 26

List 4 uses of antibodies in biotech

Answer 26

1. ELISA
2. Immunofluorescence microscopy
3. western blotting
4. Immunoprecipitation

Question 27

What does immunofluorescence microscopy allow us to visualize?

Answer 27

The abundance and distribution of antigens. The use of different colours can identify different antigens in a biological system

Question 28

What does western blotting allow us to do?

Answer 28

1. Verify the expression of a protein
2. determine the relative amount of a protein present in different samples
3. Analyze protein-protein interactions.

Question 29

Western blot method

Answer 29

1. separate proteins via SDS or native PAGE electrophoresis
2. Transfer proteins to the membrane
3. block non-specific sites on membrane
4. incubate with primary Ab, wash\
5. incubate with secondary antibody, wash
6. detect secondary antibody

Question 30

Reaction with enzyme to produce light

Answer 30

HRP catalyses breakdown of H202 to H20 and O2
luminol is oxidized and emits light
captured on film

Question 31

Main purpose of protein complex immunoprecipitation

Answer 31

identification of protein interaction partners ( ie other proteins, ligands, co-factors…) to a protein of interes. Non-ionic denaturation is required as to not disrupt these interactions

Question 32

Purpose and functioning of chromatin immunoprecipitation

Answer 32

Used to determine the location of specific protein-DNA binding sites on the genome.
uses a bead bound to an IgG specific to a putative DNA binding protein, can precipitate out Dna segment of interest