Lecture 8

Antibody

• Refers to the protein that binds to an antigen

• E.g. Anti-cholera toxin antibody

Immunoglobulin

• Refers to the antibody as a protein

• Regardless of what antigen it binds to

Antigen

• Substance that causes the body to produce specific antibodies or sensitized T cells

IgG

Main Function:

• Long-term immunity, opsonization

Location:

• Blood & tissues

Characteristics:

• 20% of all plasma proteins

• Only antibody that crosses placenta

• Also found in milk and colostrum

IgA

Main Function:

• Protects mucosal surfaces

Location:

• Saliva, tears, breast milk, mucus

Characteristics:

• Large amounts in body secretions
  • Has 2 forms: Blood and secretory form

IgM

Main Function:

• First antibody produced in infection

Location:

• Blood - as a pentamer

• B cell surface – as a monomer

Characteristics:

• 1st antibody secreted into blood during early stages of infection – primary response.

• Is synthesized by the fetus.

• Indicates recent/acute infection

IgE

Main Function:

• Allergic reactions, parasite defense

Location:

• Body fluids: bound to basophils

• Skin/tissues: bound to mast cells

Characteristics:

• The fab fragments are free to bind ag to which humans cand develop allergies

• Responsible for allergies & anaphylaxis

IgD

Main Function:

• B-cell receptor function

Location:

• Surface of B cells

Characteristics:

• Main role is B-cell activation

• Co-expressed with IgM

Main Function of B cells

1. Antibody production

• When they come across an antigen they differentiate into plasma cells
  which release large amounts of antibodies to fight infection

2. Memory formation

• Some B cells differentiate into memory B cells

3. Antigen presentation

• B cells can act as antigen-presenting cells (APCs)

Where do we have B cells?

• Blood lymphocytes

• Lymph nodes
  • Filter the lymph

• Spleen
  • Filters the blood

B cell Activation

1. Antigen Binds to B cell Surface Ig (BCR)

2. B cell becomes activated

3. B cell Proliferates

4. B cell differentiates into a Plasma Cell

5. Plasma Cell produces antibodies

How do we activate B cells?

1. Antigen binding to B cell surface Ig
   • Only activates few cells with antibody specific for that antigen

2. Adding Antibodies to the B cell Igs
   • Adding anti-mouse IgG to mouse B cells
   • Two binding sites on added Ab bind B cell BCR to each other
   • Activates most or all B cells

3. Polyclonal Activators
   • Agents that can activate whole groups of cells, regardless of what antigen they are specific for.
   a. Lipopolysaccharide - Activates B cells
   b. Concanavalin A & Phytohemagglutinin - Activate T cells
   c. Pokeweed Mitogen - Activates B and T cells

Lipopolysaccharide

• Found on the Outer membrane of Gram-Negative Bacteria
  • E. coli, Salmonella, Shigella
  • Made of lipid and sugars bound together

• Nonspecifically activates B cells
  • Binds to the Toll-Like Receptors (TLRs)

• Only activates IgM B cells
  • Results in IgM secretion
  • Very little effect on IgG

Why do we need to activate B cells?

• Most B cells are Resting (G0 phase)

• To study how B cells are activated to produce antibodies

• Study how T cells govern B cell activation and antibody production
  • T cells produce cytokines (IL-4, IL-6, etc.)
  • T cells and B cells interact through cell surface proteins

• To study how added agents can alter B cell activation and antibody production
  • How T cell factors enhance B cell proliferation
  • How T cell factors enhance antibody production
  • Effect of drugs

Experiment 8

1. Harvest mouse spleen

2. Tissue Dissociation

3. Cell Collection

4. Centrifugation and Red Blood Cell lysis

5. Final Processing:

• Count the cells using trypan blue – as in previous labs

• Prepare cell suspension of mouse spleen cells - Dilute the cells in RPMI TCM to 5 x 106 cells/ml in a total of 2.5 mL

• Culture cells with LPS

Perform differential leukocyte counts on mouse Spleen

1. Dilute the cells to 8 x 10⁵ cells/mL in 2 ml of Wash Medium (2% BCS PBS). Add 1 ml to a 5 ml round bottom centrifuge tube to give 8 x 10⁵ cells/tube

2. Follow the procedure in the Lab manual to prepare the cells

3. While waiting, clean and Label a microscope slide with "Spleen" and your group name.

4. Set up 1 Cytospin cytocentrifuge holder with 2 filters

5. To each slide holder, add 0.25 ml of the spleen cells you prepared in #2 to give 2 x 10⁵ cells/slide.

6. Centrifuge the cells at 800 rpm for 10 minutes.

7. Stain the cells with the Leukostat Stain as in the the previous experiment.

8. Counts cells.