Antibody Structure and Function IV

Describe the structure of IgE

1. 2 identical epsilon heavy chains

2. 2 identical kappa or lambda light chains

Exists as a monomer

Is there large or small amounts of IgE in the serum? What about during allergy or parasitic infections?

Small amounts but large amounts during allergy and parasitic infections

What does IgE bind to?

High affinity Fc(epsilon) receptors on mast cells and basophils

What does cross-linking membrane-bound IgE cause?

Mast cell and basophil degranulation leading to acute inflammation and atopic (type I) allergic responses

What is the important role of IgE?

In host defence against certain species of parasitic worms

What does cross linking mean?

When the receptor binds to its antigen

What does IgE resemble?

IgG but the disulfide bonds are different

Describe the structure of IgD

1. 2 identical delta heavy chains

2. 2 identical lambda or kappa light chains

Exists as a monomer

Is IgD found in low or high concentrations in the serum? Why?

Low concentrations because of its susceptibility to proteolysis

Where is most IgD located?

On the surface of naive B cells

Where can IgD be found?

It is a mucosal Ig so it is present in the airways, saliva and tear fluid

What does IgD activate? (immune cells)

Basophils and mast cells

What is IgD similar to in terms of function? Structure?

Function: IgA and IgE

Structure: IgG but disulfide bond placement is different

What is the most abundant immunoglobulins in serum?

IgM, IgA, and IgG1

What is the half-life of IgM?

5 days

What is the half-life of IgD?

3 days

What is the half-life of IgG1-4?

21 days, 20 days, 7 days, 21 days

What is the half-life of IgA1-2?

6 days for both

What is the half-life of IgE?

3 days

What do many molecules associated with immune function exhibit? Are they associated with the immunoglobulin superfamily?

The immunoglobulin-fold domain structure. 110 amino acid residues arranged in antiparallel sheets of B-pleated strands. Yes they are

What are the members of the immunoglobulin superfamily?

1. Antibodys

2. Iga/Ig-B

3. TCR

4. Fc receptors

5. CD4

6. MHC

7. Various cell adhesion molecules

What is the immunoglobulin family of genes though to have originated by?

A mutation and duplication from a single primordial gene encoding a polypeptide of 110 amino acids

What does sequencing of genes and proteins allow for?

That the immunoglobulin superfamily members are used for a variety of receptors in the immune system

What is CD2 involved in? Is it part of the immunoglobulin superfamily?

Cell adhesion and yes

What is CD3 involved in? Is it part of the immunoglobulin superfamily?

Involved in signalling of the T cell receptor and yes

What is CD4 and CD8 involved in? Is it part of the immunoglobulin superfamily?

They help the T cell bind to MHC whether they are cytotoxic or not and yes

What does MHC have in terms of structure?

They have immunoglobulin domains but have more of a variable region

What is an example of a Fc receptor? What does it bind to?

CD64 and it binds to IgG

What are monoclonal antibodies? Can we make them in a lab?

They are derived from a single B cell clone and are therefore specific for a single epitope and yes we can make them in a lab

What are polyclonal antibodies?

They are produced by many different B cell clones and can bind to many different epitopes

How are monoclonal antibodies produced?

1. A B cell that has been immunized with a specific antigen is fused with a myeloma cell that has been transformed (is cancerous)

2. A hybridoma cell is fused with the antigen-specific B cell which makes it immortal

3. The hybrid cells are selected by growth in special tissue culture medium that only allows the hybridoma cells to grow

4. The hybridoma cells that produce a monoclonal antibody are propagated in this tissue culture using bioreactors

What are monoclonal antibodies used for?

1. Diagnostics

2. Imaging

3. Therapeutic agents

(Can use them for cancer)

Describe the steps involved in the production of a mouse monoclonal antibody

1. Mouse is immunized with the antigen of interest

2. The mouse will see this antigen as non-self and will produce antibodies against it through plasma cells

3. The plasma cells are harvested by taking the spleen or lymph node (spleen is usually taken because it is bigger and has more antibodies)

4. The antibodies are fused with myeloma cells

5. Fusion results in a hybridoma to form which is immortal

6. The hybridoma can keep secreting the antibody as long as you continuously feed the culture because it contains the genome for the antibody

7. Grow the hybridoma in the presence of a drug to get rid of the ones that don’t make that antibody

What process can be used to clone one specific hybridoma?

Single cell cloning

How does single cell cloning work?

1. Dilute the hybridoma cell into a volume where you get one per each well

2. Plate that specific volume in each well

3. Harvest the soup and then see which ones will make the antibody you want through an agglutination reaction

4. Grow that specific one

Can monoclonal antibodies be treatments for disease?

Yes

What organism were monoclonal antibodies first made in?

A mouse

Describe how the mouse monoclonal antibody was made in summary

1. Injected mice with human antigens such as components of cancer cells

2. Mouse made plasma cells against these antigens

3. The plasma cells were immortalized and they made huge amounts of the antibody which they harvested and purified

4. Injected the antibodies into the human to neutralize and the kill the cancer cell

5. Mouse antibody is non-self so the human will make antibodies against it

Describe how the chimeric monoclonal antibody was made

1. In the next generation, they genetically swapped all the constant regions (were made human)

2. The mouse region was the variable region which made it better at activating complement and ADCC

3. Still had a portion that was non-self so human made antibodies against it

Describe how the humanized monoclonal antibody was made

1. In the next generation, they replaced most of the regions with the human sequences that didn’t encode the specificities (hyper variable regions were kept mouse)

2. A portion was still non-self so the human made antibodies against it

Describe how the fully human monoclonal antibody was made

1. All the genetics of the mouse antibody were replaced with human sequences

2. Insert this antibody in a mouse and it will see this as non-self and make antibodies against it

3. The antibody can then be injected into a human because it is fully humanized

4. Can clone it via hybridoma technology