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Immunity- last few lectures > Hybridoma technology > Flashcards

Hybridoma technology Flashcards

1
Q

who founded hybridoma technology

A

George Kohler and Cesar Milstein

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2
Q

George Kohler and Cesar Milstein won the

A

nobel prize in physiology of medicine in 1984

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3
Q

aim of hybridoma technology

A

to produce monoclonal antibodies

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4
Q

hybridoma technology allows

A

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

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5
Q

basic mechanism of hybridoma technology

A

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

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6
Q

B cells

A

Ig+ splenocyte with finite lifespan

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7
Q

myeloma cell

A

cancerous, Ig- B cell with infinite lifespan

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8
Q

hybridisation process takes how many injection of specific antigen?

A

4

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9
Q

the 4 injections cause

A

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

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10
Q

Hybridisation process

A

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

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11
Q

within the first few days

A

activation of multivalent IgMs

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12
Q

how long between each injection

A

2 weeks

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13
Q

IgG

A

best form of antibody for therapy and diagnostics

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14
Q

splenocyte

A

spleen B cell

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15
Q

myeloma

A

cancer cells which are immortalised and Ig-

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16
Q

what is used to isolate hybrid cells

A

HAT medium

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17
Q

HAT medium used to

A

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

18
Q

hybrids produced through this process

A

will produce different antibodies (polyclonal response)

19
Q

single cel hybrids put into a

A

96 well culture plate

20
Q

what is used on the 96 well culture plate

A

ELISA to identify wells of interest

21
Q

myeloma cells are

A

immortalised and unable to produce antibodies

22
Q

what do myeloma cells not have

A

HGPRT

23
Q

without HGPRT myeloma cells are not able to

A

survive in the HAT medium

24
Q

HAT medium is used to

A

kill all unfused cells

25
Q

why does the HAT medium kill myeloma cells

A

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

26
Q

what blocks the biosynthetic pathways for nucleic acid

A

aminopterin

27
Q

which enzymes are needed for salvage nucleic acid biosynthesis

A

hypoxanthine and thymidine kinase

28
Q

both cells have

A

thymidine kinase

29
Q

spleen cells are are

A

HGPRT +

30
Q

myeloma cells are

A

HGPRT -

31
Q

myeloma has thymidine kinase but not

A

hypoxanthine - cannot make RNA- dies

32
Q

myeloma cells are HCPT - therefore

A

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

33
Q

why do spleen cells die in HAT culture

A

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

34
Q

hybridoma cells can grown in HAT medium because

A

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

35
Q

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

A

an exogenous supply of H and T.

36
Q

single hybrid cells are then out into a

A

single well

37
Q

hybridoma cells continue diving in vitro due to

A

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

38
Q

hybridoma cells divide every

A

12 hours

39
Q

each well will be producing the same

A

B cell and therefore antibodies

40
Q

how can hybridomas be stored indefinitely

A

by freezing in liquid nitrogen

41
Q

when you want more antibodies

A

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

Immunity- last few lectures (12 decks)

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