W12L21 - Monoclonal Antibodies

Question 1

Ag-Ab Reactions

Answer 1

Paratope (antigen binding site) on an Ab binds to an epitope (antigenic determinant) on an Ag
This complex known as Primary reaction

Question 2

Affinty

Answer 2

Describes the binding of a single paratope to a single epitope
Strength of attraction between complimentary shapes
High affinity = close fit

Question 3

Specificity

Answer 3

Ability of Ab to distinguish between Ags
Greater the difference - the more specific the Ab
Ab that reacts with antigen X but not with related antigens Y and Z is specific (anti-X antibodies)

Question 4

Cross-Reactivity

Answer 4

Can occur with unrelated antigens where structural similarities exist with an epitope

Question 5

Polyclonal vs Monoclonal Abs

Answer 5

Polyclonal abs directed to a number of epitopes and are less specific
They have varying affinities for epitopes and may cross react with epitopes not specific for antigen = false positive results
Monoclonal are directed to one epitope and are very specific

Question 6

Production of Monoclonal Abs

Answer 6

Infect animal with antigen
Remove spleen and take out B cells
Mix B cells with Myeloma cells (cancer cells)
- PEG
- caused fusion of membranes
- B cell/myeloma mix cells called Hybridoma
Remove PED and screen cells for those producing Abs to Ag
Culture cells and freeze

Question 7

Selecting Hybridomas

Answer 7

When fusing cells you get:
- B cell/B cell fusion
- cancer cell/cancer cell fusion
- B cell/cancer cell fusion (hybridoma)
Nucleotides in a cell produced by:
1. De novo pathway
- inhibited by aminopterin
2. Salvage pathway
- requires source of hypoxanthine and thymine
- incorporation requires hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymine kinase (TK)
Myeloma cells don't produce HGPRT
- myeloma cells cant use salvage pathway 
Grow cells on HAT media
- hypoxanthine, aminopterin, thymidine
Aminoprotein stops de novo pathway
Cancer cell fusions wont grow as they lack HGPRT
B cell fusions will die (not immortal)
Hybridomas will grow

Question 8

Uses of Monoclonal Abs

Answer 8

Specific and good as primary antibodies in assays
ELISA, RIA, Western Blot, Flow cytometry
Detecting tumor antigens
CD identification
Bacteria and virus detection/identification

Question 9

Monoclonals as Therapy

Answer 9

Can selectively target an epitope
- specifically target cell type or microbe
- tag a toxin onto a monoclonal Ab for specific delivery
- tag with enzyme to activate drugs
Problems
- mouse monoclonal Ab dont activate human complement or opsonise in humans
- foreign antibodies quickly eliminated
- difficult to get human monoclonals

Question 10

Strategies for Human Monoclonals

Answer 10

Human lymphocytes fused with murine myeloma
- less efficient
Transform human lymphocytes with EBV
Genetic engineered mouse-human hybrid antibodies

Question 11

Rituximab

Answer 11

Chimeric monoclonal Ab directed against CD20
CD20 expressed on B cells
Used to remove B cells
- leukaemia
- transplant rejection
- autoimmune disorders