Chapter 12 - Animal Cell Culture Products: Proteins - Antibodies

Question 1

What is cellular immunity?

Answer 1

immunity mediated by T lymphocytes

Question 2

What is humoral immunity?

Answer 2

immunity mediated by B lymphocytes

Question 3

What is an antigen (Ag)?

Answer 3

Foreign entities that are recognized by antibodies

Question 4

Describe the 4 polypeptides that make up antibody

Answer 4

2 identical light chains & 2 identical heavy chains linked together by disulphide bonds

Question 5

Describe the 3 parts that make up an antibody

Answer 5

2 identical antigen binding sites (Fab - Fragment antigen binding) and Fc (Fragment crystalizable)

Question 6

What are the five classes of immunoglobulins and how are they differentiated?

Answer 6

IgM

IgA

IgD

IgG

IgE

They are differentiated based on their heavy chains (C-terminal portions)

Question 7

Approximately how many B lymphocyte clones circulate throughout the body?

Answer 7

10^5 - 10^8

Question 8

What are the two fates of a proliferating B cell (when it encounters an antigen)?

Answer 8

• Some progeny differentiate into plasma cells
• Some progeny do not differentiate and are memory cells

Question 9

How is polyclonal antibodies formed?

Answer 9

1. Injecting antigen into an animal (ie. rabbit)
2. Boost (second Ag exposure)
3. Collect the serum (contain polyclonal Abs -> heterogeneous)

Question 10

What are hybridomas?

Answer 10

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)

Question 11

What are cancerous B cells?

Answer 11

B lymphocytes that have myeloma (cancer). They grow indefinitely and secrete immunoglobulin (monoclonal).

Question 12

What are HGPRT- cells used for producing hybridomas?

Answer 12

They only secrete light chains and do not express any immunoglobulins

Question 13

How is the spleen cells (used to produce hybridomas) obtained?

Answer 13

1. Inject antigen into mouse
2. Kill mouse and dissociate spleen into individual cells

Question 14

Explain the fusion procedure for producing hybridomas

Answer 14

Fuse the HGPRT- myeloma cells with spleen cells

Grow hybrids on HAT medium

Hybrids are screened for secretion of antibody to the desired region

Question 15

What are some advantages of monoclonal vs polyclonal antibodies?

Answer 15

• 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

Question 16

Explain how the ELISA test works

Answer 16

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)

Question 17

Explain how a sandwich ELISA works

Answer 17

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)

Question 18

Explain how MAbs can be used to treat cancer

Answer 18

Tag a chemotherapeutic agent to a MAb directed against a tumor specific antigen (immunotoxin)

**some cancers do not have tumour specific region

Question 19

Where is passive immunotherapy used?

Answer 19

• organ transplant patients whose immune systems are suppressed
• cancer chemotherapy patients
• immunodeficiency disorders

Question 20

Explain how protein purification works using MAbs

Answer 20

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