Monoclonal Antibodies

Question 1

Define immunogen and antiserum

Answer 1

Immunogen - Ag that raises immuno response
Antiserum - serum with Ab raised by immunisation

Question 2

Isotypes (5) and valent

Answer 2

bivalent: G, D, E
tetravalent: A
decavalent: M

Question 3

Describe Ab:
-2 regions and their functions (variable or not)
-2 identical chains (where? light or heavy)
bonds that link chains

Answer 3

Fab: binds to Ag - variable end (different Ag)
Fc (fragment crystallizable region)- triggers immune response and effector binding to cells of immune system - non variable end

Y shape- 2 identical heavy chains (inside)
Outside of Fab - 2 identical light chains

disulfide bonds

Question 4

functions of antibodies

Answer 4

Ag binding
effector mechanism interaction with cells and receptors

Question 5

Monoclononal Ab - origin and specificity

Answer 5

B cells- one Ab specific (recognizes) one Ag epitope

Question 6

Monoclonal Ab vs Antiserum
- epitope specificity
- sensitivity to changes in Ag
- Ab affinity
- immunogen purity required
- genetically manipulable
- cost
-quantites per preparation
- time

Answer 6

Monoclonal
- one per Ag
- high
-medium
-low
-yes
-high
- infinite
-long

Antiserum
-several
-low
-high
-high
-no
-low
-finite
-short

Question 7

Mab production (4 steps)

Answer 7

1- immunization and isolation splenocytes from mice
2- hybridoma: splenocytes + myelonomas
3- screening of clones with desired Ab
4- clonal expansion

Question 8

1970 - Milstein and Kohler

Answer 8

Milstein - Ab hypermutation: using Ig-producing myeloma grown in culture & developed methods for fusing myeloma cells
Kohler: How H and L chains combine to generate antigen specificity – difficult to get enough AB as B cells die in culture

Conclusion: Fusing Ag specific B cells with myeloma cells to grow selective AB producing cells indefinitely

Question 9

Recovering and maintaining Ab producing B cells, how? Selection of hybrids (B cells, mylenomas and hybridomas)

Answer 9

HAT (hypoxanthine, aminopterin and thymidine) medium

Aminopterin blocks DNA synthesis (glutamine and aspartate) pathways requiring salvage pathways where HGPRT and thymidine kinase (TK) are used to produce DNA. If cells don´t have those two, will die.
- B cells can´t replicate
- mylenomas don´t have HGPRT, die
- hybridomas have the HGPRT from spleen cells and survive

Question 10

Applications (5)

Answer 10

Affinity purification Ag (McAb that only binds to AgA)
Immunohistochemistry (location of cells in a tissue labelled with HRP)
immunofluorescence
ELISA
Western Blotting