What is cellular immunity?
immunity mediated by T lymphocytes
What is humoral immunity?
immunity mediated by B lymphocytes
What is an antigen (Ag)?
Foreign entities that are recognized by antibodies
Describe the 4 polypeptides that make up antibody
2 identical light chains & 2 identical heavy chains linked together by disulphide bonds
Describe the 3 parts that make up an antibody
2 identical antigen binding sites (Fab - Fragment antigen binding) and Fc (Fragment crystalizable)
What are the five classes of immunoglobulins and how are they differentiated?
They are differentiated based on their heavy chains (C-terminal portions)
Approximately how many B lymphocyte clones circulate throughout the body?
10^5 - 10^8
What are the two fates of a proliferating B cell (when it encounters an antigen)?
- Some progeny differentiate into plasma cells
- Some progeny do not differentiate and are memory cells
How is polyclonal antibodies formed?
1. Injecting antigen into an animal (ie. rabbit)
2. Boost (second Ag exposure)
3. Collect the serum (contain polyclonal Abs -> heterogeneous)
What are hybridomas?
Cell hybrids from the fusing of cancerous B cells with spleen cells from Ag injected animal (B cells provide immortality and spleen cells provide antibody specificity)
What are cancerous B cells?
B lymphocytes that have myeloma (cancer). They grow indefinitely and secrete immunoglobulin (monoclonal).
What are HGPRT- cells used for producing hybridomas?
They only secrete light chains and do not express any immunoglobulins
How is the spleen cells (used to produce hybridomas) obtained?
1. Inject antigen into mouse
2. Kill mouse and dissociate spleen into individual cells
Explain the fusion procedure for producing hybridomas
Fuse the HGPRT- myeloma cells with spleen cells
Grow hybrids on HAT medium
Hybrids are screened for secretion of antibody to the desired region
What are some advantages of monoclonal vs polyclonal antibodies?
- monoclonal antibodies recognize a single antigenic determinant
-polyclonal Abs supply is limited (animal will eventually age and die)
-polyclonal Abs cannot be reproduced exactly in new animals
-Abs need to be highly purified for polyclonal but not for monoclonal
Explain how the ELISA test works
1. Place desired antigen in the well (Ag will stick to the plastic of the well)
2. Add MAb to be tested to the well (will bind to Ag on the surface of plastic)
3. Add Ab-enzyme conjugate with specificity aagianst the first Ab (Fc portion)
4. Detect coloured product produced due to enzyme reaction (read by eye or colorimeter)
Explain how a sandwich ELISA works
1. Place MAb into the well (it will stick onto the plastic)
2. Add Ag to the well (will bind MAb if appropriate)
3. Add MAb-enzyme conjugate with specificity against Ag
4. Detect cooured product produced due to enzyme reaction (by eye or colorimeter)
Explain how MAbs can be used to treat cancer
Tag a chemotherapeutic agent to a MAb directed against a tumor specific antigen (immunotoxin)
**some cancers do not have tumour specific region
Where is passive immunotherapy used?
- organ transplant patients whose immune systems are suppressed
- cancer chemotherapy patients
- immunodeficiency disorders
Explain how protein purification works using MAbs
MAbs of particular protein product is attached to solid support (ie. agarose or polyacrylan beads)
The medium, body lfuid or cell extract is passed over the column (protein is retained)
Elute protein product by passing buffer with different pH