Immunology 4- Antibodies

Question 1

What is meant by an antibody

Answer 1

an antibody is a protein that is produced in response to a foreign molecule (antigen), and has the property of binding specifically to that antigen

Question 2

What class of proteins are antibodies part of

Answer 2

antibodies form the class of proteins known as immunoglobulins, and are a large family of soluble glycoproteins

Question 3

Which immune cell produces antibodies

Answer 3

B lymphocytes

Question 4

What secondary functions do antibodies initiate after binding

Answer 4

complement activation
opsonisation (promotion of phagocytosis)
cell activation via specific antibody-binding receptors (Fc receptors)

Question 5

How many different antibodies does each individual posses

Answer 5

each individual has a large number (>107) of different antibodies

Question 6

Why do we need different types of antibodies

Answer 6

To be able to defend against a wide variety of pathogens

Question 7

What is the antibody-bearing fraction of serum referred to as

Answer 7

Immunoglobulin

Question 8

Why is a highly heterogeneous spectrum of proteins in the blood

Answer 8

Because normal serum immunoglobulins are the heterogeneous products of many clones of B cells, the immunoglobulins in serum constitute a highly heterogeneous spectrum of proteins rather than a highly than a single molecular species.

Question 9

Why are biochemical studies of immunoglobulins difficult.

Answer 9

Antibodies that arise in response to a single complex antigen, such as bacterial protein with multiple epitopes, are heterogeneous in chemical structure and specificity because they are formed by several different clones of B cells, each expressing an immunoglobulin capable of binding to a different epitope on the antigen. Electrophoresis helped to overcome this difficulty

Question 10

What does a well-defined spike on electrophoresis of immunoglobulins show

Answer 10

The monoclonal B cells all produce exactly the same immunoglobulin molecule ( same molecular weight and charge)- sign of B-cell malignancy (myeloma)

Question 11

Describe the basic molecular structure of an antibody

Answer 11

Antibodies consist of 2 identical heavy chains and 2 identical light chains. The light and heavy chains are linked together by intermolecular disulphide bonds. Heavy chain (50kDa). Light chain (25kDa). Disulphide bonds are also present within each of the chains. LHHL.

Question 12

Explain how we know that immunoglobulins contain molecular parts with distinctive functions

Answer 12

limited digestion of -globulin with purified papain
produced 3 fragments in equal amounts, each 50 kDa
2 fragments had antigen binding activity (Fab), the other did not
this fragment formed protein crystals (Fc)- it does not bind to antigens but can activate complement- can bind to Fc receptors on macrophages and other cells

Question 13

What does the fact that Fc fragments crystallise show

Answer 13

All amino acid sequences in that protein are the same, hence must be the same in all antibodies. Hence it is known as the constant region.

Question 14

Are Ig molecules symmetrical

Answer 14

Yes- axis of symmetry lies in the middle of the Y-shaped structure.

Question 15

Which region of the Ig molecule is responsible for flexibility and why is this flexibility important

Answer 15

The hinge region. Allows antibodies to bind to widely spaced cell surface determinants as well as closely spaced cell surface determinants.

Question 16

What can light chains and heavy chains be divided into

Answer 16

Constant and variable regions. Constant regions play a role in biologic effector functions, the variable regions play a role in antigen binding.

Question 17

What has additional 3-D determination of Ig molecules demonstrated

Answer 17

Ig molecules are composed of folded, repeating segments called domains. L chains consist of one variable domain and one constant domain. However, H chains consist of one variable domain and 3 or more constant domains. These regions of hypervariability are known as Complementarity Determining Regions.

Question 18

Describe the forces involved in antigen-antibody binding

Answer 18

Non-covalent interactions, each of which is individually relatively weak. These interactions are made up of hydrogen bonds, ionic bonds, hydrophobic interactions and van der waal interactions.

Question 19

Describe the antigen binding site

Answer 19

The antigen binding site is a relatively large, approximately flat surface with undulations, and is made from the 3 hypervariable regions within the Variable domains that form the complementary determining regions

Question 20

What is meant by antibody affinity

Answer 20

Defined as the strength of the total noncovalent
interactions between a single antigen binding site
and a single epitope on the antigen

Question 21

Describe the equilibrium constant for antibodies

Answer 21

Ab + Ag — Ab-Ag

K constant varies from 104 to 1011 L/mol

Question 22

What is meant by antibody avidity

Answer 22

Defined as the overall strength of multiple interactions
between an antibody with multiple binding sites and
a complex antigen with multiple epitopes:

Question 23

Which is a better measure of binding capacity in biological systems

Answer 23

Antibody avidity

Question 24

What forms the walls of the combining site of the antibody formed by

Answer 24

The amino acid residues of the hypervariable regions.

Question 25

What is meant by the epitope

Answer 25

Part of the antigen which the antibody binds to

Question 26

Describe antibody cross-reactivity, giving examples

Answer 26

Antibody elicited in response to one antigen can sometimes recognise a different antigen of similar structure: 1. Vaccination with cowpox induces antibodies which are able to recognise smallpox 2. ABO blood-group antigens (glycoproteins on r.b.c.) Antibodies made against microbial antigens on common intestinal bacteria may cross-react with carbohydrates on r.b.c.

Question 27

Ultimately, what does antibody cross-reactivity demonstrate

Answer 27

That antibodies are not 100% selective

Question 28

What is responsible for the differences between the different classes of antibodies

Answer 28

Different constant regions of their heavy domains.

Question 29

Describe the molecular properties of each of the Ig classes

Answer 29

IgG Heavy chain: gamma Ch Domains: 3 Light chain: kappa or lambda IgE Heavy chain: epsilon Ch domains: 4 Light chain: Kappa or lambda IgM mu 4 kappa or lambda IgA alpha 3 kappa or lambda IgD delta 3 kappa or lambda

Question 30

Describe the 2 different types of light chains

Answer 30

Light chains can be divided into kappa or lambda. They are found in all 5 classes of Ig, however any one antibody can only contain one type of light chain.

Question 31

Describe the differences in IgA and IgG subclasses

Answer 31

Class; IgG Sub-classes: IgG1, IgG2, IgG3, IgG4 Heavy chains: gamma 1, 2,3,4 Class IgA Sub-classes: IgA1, IgA2 Heavy chains: Alpha 1, 2

Question 32

Describe the characteristics of the IgG antibody

Answer 32

 heavy chain Most abundant immunoglobulin Occurs as a monomer, but 4 subclasses variability mainly located in hinge region and effector function domains Actively transported across the placenta and due to its comparatively long half life it can protect the new-born baby for up to 3 months. Blood and extracellular fluids Major activator of classical complement pathway (mainly IgG1 and IgG3) Neutralises toxins and viruses Most important in defence generally, the other Ig classes have a more specialised role

Question 33

What are a number of the functions of IgG dependent on

Answer 33

The nature of its Fc. When IgG binds to a bacterium or other infectious agent, macrophages or neutrophils recognise the Fc ( they have Fc receptors) and this leads to opsonisation, facilitating phagocytosis. In the same situation complement activation can take place, promoting phagocytosis or lysis of the bacterium.

Question 34

Describe the variations in each of the IgG sub-classes

Answer 34

1(70%), 2(20%),3(7%),4(3%) 1 and 3 are more active. Different Fc receptors Antibodies binding to bacterial carbohydrates are often Ig2.

Question 35

Describe the characteristics of IgA

Answer 35

 heavy chain Second most abundant immunoglobulin after IgG Occurs as a monomer (blood) and as a dimer (secretions) Major secretory immunoglobulin Protects mucosal surfaces from bacteria, viruses and protozoa

Question 36

Describe the structure of secretory IgA

Answer 36

A dimer together with a J-chain and secretory component.

Question 37

Describe the formation of secretory IgA

Answer 37

Dimeric IgA produced by plasma cell in submucosa. IgA binds to poly Ig receptor on epithelial cell- endocytosed into epithelial cell. Enzymatic cleavage- IgA remains, secretory component added which prevents against degradation in the lumen Secretory IgA can neutralise toxins. Moreover, by binding to infectious agents it can block their infectivity- often by preventing the adherence of agents to epithelial cells.

Question 38

Describe the variations in the 2 sub-classes of IgA

Answer 38

1 predominates in the blood, 2 is more strongly represented in the secretions.

Question 39

Describe the characteristics of the IgM antibody

Answer 39

 heavy chain large pentameric molecule 5 monomers joined by J chain (10 x Fab) mainly confined to blood (80%) first Ig synthesised after exposure to antigen primary antibody response multiple binding sites compensate for low affinity efficient at agglutination activates complement Due to its large size, it is mainly found in the blood.

Question 40

Describe the characteristics of the IgD antibody

Answer 40

 heavy chain extremely low serum concentrations surface IgD expressed early in B cell development involved in B cell development and activation membrane bound antibody - B cell receptor

Question 41

Describe the characteristics of the IgE antibody

Answer 41

 heavy chain present at extremely low levels produced in response to parasitic infections and in allergic diseases binds to high affinity Fc receptors of mast cells and basophils cross-linking by antigen triggers mast cell activation and histamine release

Question 42

Describe selective Ig distribution

Answer 42

IgG & IgM in blood IgG in extracellular fluid Dimeric IgA in secretions across epithelia, including breast milk Maternal IgG in foetus via placental transfer IgE with mast cells below epithelia

Question 43

Describe the consequences of antibody binding to antigen

Answer 43

``` Neutralisation Agglutination Opsonisation Complement activation Bound by cells expressing Fc receptors (predominantly cells of innate immunity: phagocytes, NK cells) ```

Question 44

Describe the different uses of antibodies

Answer 44

``` In defence targeting of infective organisms recruitment of effector mechanisms neutralisation of toxins removal of antigens passive immunity in the new born In medicine levels used in diagnosis and monitoring pooled antibodies for passive therapy/protection monoclonal antibodies for treatment of cancer In laboratory science vast range of diagnostic and research applications ```

Question 45

What is meant by a monoclonal antibody

Answer 45

Antibodies of a single selectivity | Problem with using antibodies as drugs- can lead to cancer.

Question 46

What is the role of the carbohydrate that coats the constant regions

Answer 46

To prevent against proteolytic degradation.

Question 47

What is the difference between a linear and discontinuous epitope

Answer 47

Linear - amino acids residues are adjacent in the polypeptide chain Discontinuous- created from amino acid residues located in different parts of the polypeptide chain.

Question 48

What is meant by the immunoglobin superfamily

Answer 48

Other molecules of the immune system have similar folded polypeptide domains, giving rise to the term immunoglobin superfamily to describe this group of related proteins