Hybridoma technology

Question 1

who founded hybridoma technology

Answer 1

George Kohler and Cesar Milstein

Question 2

George Kohler and Cesar Milstein won the

Answer 2

nobel prize in physiology of medicine in 1984

Question 3

aim of hybridoma technology

Answer 3

to produce monoclonal antibodies

Question 4

hybridoma technology allows

Answer 4

the identification and culture of cells secretion identical antibodies with pre-defined specificity

Question 5

basic mechanism of hybridoma technology

Answer 5

A single clone of cells secreting a single
antibody is made by fusing a B cell (Ig+ splenocyte
with finite lifespan) with a myeloma cell (cancerous
Ig- B cell with infinite lifespan)

The resulting hydrid cells (HYBRIDOMAS) each
make a specific monoclonal antibody

Question 6

B cells

Answer 6

Ig+ splenocyte with finite lifespan

Question 7

myeloma cell

Answer 7

cancerous, Ig- B cell with infinite lifespan

Question 8

hybridisation process takes how many injection of specific antigen?

Answer 8

4

Question 9

the 4 injections cause

Answer 9

antibody switching, meaning antibodies become more and more specific to the antigen
- affinity increasing as immunisation continues

Question 10

Hybridisation process

Answer 10

1) inject mice with antigen of interest (directly into spleen)
2) antigen goes into blood stream and activates B cells
3) within first few days activation of multivalent IgMs
4) as immunising continues (every 2 week) the immune response builds up: IgM- IgG
5) then blood serum retrieved from mouse by cutting tail
6) serum tested for response to target antigen
7) if response large enough, the mouse it euthanised and spleens removed
8) B cells isolated for hybridisation
9) spleen B cell (splenocyte) fused with myeloma cells in vitro
10) left with a mixture of useful hybrid cells and un-useful unfused myeloma and splenocytes

Question 11

within the first few days

Answer 11

activation of multivalent IgMs

Question 12

how long between each injection

Answer 12

2 weeks

Question 13

IgG

Answer 13

best form of antibody for therapy and diagnostics

Question 14

splenocyte

Answer 14

spleen B cell

Question 15

myeloma

Answer 15

cancer cells which are immortalised and Ig-

Question 16

what is used to isolate hybrid cells

Answer 16

HAT medium

Question 17

HAT medium used to

Answer 17

isolate hybrid cells by knocking out splenocytes and B cells which haven’t fused

Question 18

hybrids produced through this process

Answer 18

will produce different antibodies (polyclonal response)

Question 19

single cel hybrids put into a

Answer 19

96 well culture plate

Question 20

what is used on the 96 well culture plate

Answer 20

ELISA to identify wells of interest

Question 21

myeloma cells are

Answer 21

immortalised and unable to produce antibodies

Question 22

what do myeloma cells not have

Answer 22

HGPRT

Question 23

without HGPRT myeloma cells are not able to

Answer 23

survive in the HAT medium

Question 24

HAT medium is used to

Answer 24

kill all unfused cells

Question 25

why does the HAT medium kill myeloma cells

Answer 25

within the HAT medium is Aminopterin which blocks the main biosynthetic pathways for nucleic acid - stops cells from producing RNA and DNA. Two salvage pathways exist, which rely on two enzymes (hypoxanthine and thymidine kinase. Both cells types have thymidine kinase, so both can make DNA. Only cells with with HGPRT can make RNA. Myeloma cells do not have HGPRT

Question 26

what blocks the biosynthetic pathways for nucleic acid

Answer 26

aminopterin

Question 27

which enzymes are needed for salvage nucleic acid biosynthesis

Answer 27

hypoxanthine and thymidine kinase

Question 28

both cells have

Answer 28

thymidine kinase

Question 29

spleen cells are are

Answer 29

HGPRT +

Question 30

myeloma cells are

Answer 30

HGPRT -

Question 31

myeloma has thymidine kinase but not

Answer 31

hypoxanthine - cannot make RNA- dies

Question 32

myeloma cells are HCPT - therefore

Answer 32

not able to use salvage pathway for RNA synthesis- therefore dies

Question 33

why do spleen cells die in HAT culture

Answer 33

not able to grow i long-term culture (not immortal

Question 34

hybridoma cells can grown in HAT medium because

Answer 34

HGPRT from the spleen cell (RNA synthesis) and importability from myeloma cells

Question 35

although hybridoma cells can grow in HAT medium what they do need

Answer 35

an exogenous supply of H and T.

Question 36

single hybrid cells are then out into a

Answer 36

single well

Question 37

hybridoma cells continue diving in vitro due to

Answer 37

immortal nature of myeloma cells- give ability to be able to live outside the body

Question 38

hybridoma cells divide every

Answer 38

12 hours

Question 39

each well will be producing the same

Answer 39

B cell and therefore antibodies

Question 40

how can hybridomas be stored indefinitely

Answer 40

by freezing in liquid nitrogen

Question 41

when you want more antibodies

Answer 41

you allow them to thaw- will start diving again, producing more monoclonal antibodies