Lecture 16; Immunoassay

Question 1

What is an antigen?

Answer 1

A substance with which the antibody reacts

Question 2

What is an immunogen?

Answer 2

Substance used to produce an immune response - could be larger than an antigen

Question 3

What is a B cell?

Answer 3

Lymphocytes that produce antibodies

Question 4

What is required to produce antibodies?

Answer 4

B Cells and T Cells recognition of the antigen

Question 5

Describe the production of antibodies;

Answer 5

1) Antibodies are triggered when B cell encounters its matching antigen
2) B Cell takes in antigen and digests it
3) Displays antigen fragments on its MHC molecules
4) Antigen fragments and MHC attracts help of mature T cell
5) Lymphokines secreted by the T Cell allows the B cell to multiple and make antibody producing plasma cells
6) Antibodies are released into the blood stream and bind antigen

Question 6

What happens to antigen-antibody complexes?

Answer 6

They are eliminated either by the compliment cascade, liver or spleen

Question 7

Describe the specificity of Plasma B cells;

Answer 7

Each B cell produces only one type of antibody molecule.. i.e specific for one antigen..

But antigens can closely resemble one another and antibodies can be cross reactive

Question 8

What is Cross reactivity?

Answer 8

Antibodies may bind multiple antigens because they are very similar.

Question 9

Whats the implication of cross reactivity?

Answer 9

When using antibodies experimentally this must be taken into consideration

Question 10

Whats an example of cross reactivity?

Answer 10

If an antibody binds to the alpha chain of hCG it will also cross react with the alpha chain of LH

Question 11

What could be targeted instead of hCG alpha chain to reduce cross reactivity?

Answer 11

beta hCG has a c terminal extension of 30 amino acids that is not in the beta LH chain. Antibodies directed towards this are specific for bhCG

Question 12

Write some short notes on antibodies;

Answer 12

• Antibodies area type of protein called immunoglobulins

- Most common is IgG

Question 13

Describe IgG structure;

Answer 13

• Compromised of two main structural and functional regions
• Two heavy chains specific for antibody class and two light chains shared by all classes
• heavy + light chain variable binding regions create antigen binding site
• Held together by disulphide bonds

Question 14

What is Fab and fc?

Answer 14

Fab = antigen binding site, varies between antibodies

Fc = Antibody constant region, consistant amongst antibody class

Question 15

What are the five immunoglobulin classes?

Answer 15

IgG
IgM
IgA
IgE
IgD

There are subclasses within each class

Question 16

Describe basic immune response to infection;

Answer 16

First exposure;

Latent period then; IgM followed by larger IgG response

Secondary response;

No latency!
- huge IgG and some IgM together!

Question 17

What are epitopes?

Answer 17

An epitope is that part of an antigen that interacts with an antibody i.e the antibody-binding site.

Question 18

What are the types of epitopes?

Answer 18

Conformational and linear

Question 19

What is a conformational epitope?

Answer 19

A binding site that is several parts of the protein causes by its conformation

Question 20

What is a linear epitope?

Answer 20

Binding sites in succession

Question 21

Describe antigen-antibody interactions;

Answer 21

Non-covalent and reversible i.e H bonds, hydrophobic interactions, electrostatic and van der waal forces

Question 22

What do affinity and avidity describe?

Answer 22

Strength of interaction between antigen-antibody complex

Question 23

What is affinity;

Answer 23

Measure of the strength of an antibody binding to an epitope

Question 24

What is avidity;

Answer 24

Measure of the stability of an interaction between antibodies and antigen

Question 25

What does avidity measure?

Answer 25

1) Affinity of Ab for epitope
2) Valency of AB antigen (n. antigen AB can bind)
3) Geometric arrangment of complex

Question 26

What are polyclonal responses?

Answer 26

• Large antigens contain many hundreds of epitopes
• An organism will produce antibodies for all of these epitopes
• Microbes may have thousands of epitopes

Question 27

What is a monoclonal antibody?

Answer 27

One antibody for one antigen

Polyclonal is many antibodies for different epitopes on one antigen

Question 28

How are monoclonal antibodies produced?

Answer 28

produced in mice by intreoducing the antigen then cloning the B cell that produces the specific antibodies.

Monoclonal antibodies are good for reducing non-specific binding

Question 29

What are the types of immunoassays?

Answer 29

• Precipitation
• Agglutination
• Immunohistochemistry
• Sandwich assays
• Antibody Capture assays
• Antigen Capture assays

Question 30

Describe precipitation;

Answer 30

• Polycloncal antisera bind insoluble antigens
• Then polyclonal antibodies that are soluble diffuse towards soluble antigen and precipitate is formed (at optimal concentrations)

Question 31

Describe agglutination;

Answer 31

• Polycloncal antisera bind insoluble antigens
• Typically antigens are RBC or beads/ latex particles
• Antserum cross links the particles causing them to agglutinate. (this can be visualised)

Question 32

Describe sandwhich assay (two antibody)

Answer 32

1) Bond antibody to solid phase (bottom of well)
2) Bind antigen to antibody
3) Bind second antibody to antigen
4) Quantitate second antibody

Question 33

Describe antigen capture assay;

Answer 33

1) Bond antibody to solid phase (bottom of well)
2) Bind antigen to antibody
3) Quantitate antigen

Question 34

Describe antibody capture assay;

Answer 34

1) Bond antigen to solid phase (bottom of well)
2) Bind antibody to antigen
3) Quantitate antibody

Question 35

Describe IHC;

Answer 35

• Thin section of tissues placed on histology slides.
• Probed with antigen specific antibodies (primary antibody)
• Enzyme conjugated secondary antibody is introduced
• Reagent is added that is converted by the enzyme into insoluble precipitate

Question 36

What can IHC be used for?

Answer 36

This technique can be used to identify particular cell types in a tissue i.e tumor typing or to locate the expression pattern of a particular antigen

Question 37

Given an example of amplification compound

Answer 37

Biotin streptavin