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CBI - UOE > Monoclonal antibodies > Flashcards

Monoclonal antibodies Flashcards

1
Q

antibodies can be used to

A

exploit immune mechanisms we use naturally

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2
Q

antibodies which have been refined for specificity and affinity can be used to

A

track diseases in vitro and in vivo

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3
Q

specific antibodies can be used to also

A

turn immune systems on to kill cancer cell

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4
Q

most antigens have

A

many epitopes
- animals injected with a single antigen will produce a complex mixture of antibodies- each made by a different clone of cells

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5
Q

polyclonal response

A

when body becomes exposed to an antigen, a mixture of antibodies will be produced due to the fact antigens have many epitopes
- does not discriminate between different antigens

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6
Q

to discriminate between different antibodies

A

antibodies need to be identified that bind to epitopes specific to individual antigens

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7
Q

monoclonal anitbodies

A

bind to epitopes specific to individual antigens

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8
Q

monoclonal antibodies will not

A

cross react with conserved antigens- due to the antigen being focused on being specific to that antigen

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9
Q

need to find a B cell

A

producing antibodies specific to antigen-monoclonal

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10
Q

two ways of creating monoclonal antibodies

A

Hybridisation

Phage Display

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11
Q

hybridisation is brought about using

A

hybridoma technology

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12
Q

hydrbidoma technology allows

A

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

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13
Q

Hybridoma simple-

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)

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14
Q

B ells activated within

A

mouses immunised with unique antigen- to create an immune response- with hyper mutations- class switching to produce highly specific monoclonal antibodies

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15
Q

B cell is a

A

Ig+ spelnocyte with finite lifespan

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16
Q

Myeloma cell

A

cancerous Ig- B cell with infinite lifespan

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17
Q

basic principle of hybridoma

A

fuse a mortal cell with immortal cell 9which can live outside the body)to produce an immortal B cell hypridoma- producing monoclonal antibodies

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18
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 the first few days: activates low affinity multivalent IgM
4) as you carry on immunising (i.e. primary infection, secondary injection, tertiary injection, multiple boosts), the immune response builds up by genetic switching- making antibodies more and more specific to the antigen- increasing affinity
5) then you cut the mouse’s tai and the serum of the blood that comes out can be tested for response to target antigen
4) if response is large enough- the mouse will be euthanised and spleen removed
5) B cells isolated
6) fuse spleen cells (splenocyte) with myeloma in vitro
7) hybrid cell

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19
Q

spleen is

A

a secondary lymphoid organ

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20
Q

in the mix produced after B cells and myeloma are fused

A

there will also be many unfused myeloma and splenocytes in the the mix

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21
Q

there will also be many unfused myeloma and splenocytes in the the mix- how do we get around this

A

Isolation of hybrid cells using HAD

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22
Q

Isolation of hybrid cells using HAD medium

A

-kocks out splenocytes and B cells which havens been fused- therefore only left with hybrids

23
Q

what is used to isolate hybrid cells using HAD medium

A

20, 96 well culture plates

- each could have a hybrid cells which is producing the correct monoclonal antibody

24
Q

what is used to identify if a well contains the hybrid cell producing correct monoclonal ntibody

A

EISA

25
Q

if you want to produce larger quantities of antibodies

A

hybridisation is carried out within a rabbit or goat

26
Q

unfused myeloma cells dies in

A

HAT mediaum

27
Q

unfused B cell

A

dies naturally

28
Q

hybridoma cells are

A

immortal and survive in HAT

29
Q

mechanism of hybrid cell election in HAT medium

A

within HAT medium there is a component called Aminopterin (blocks the main biosynthetic pathways for nucleic acids- stops cell producing RNA and DNA).

  • Within the cells there are two salvage pathways- which rely on two enemies (hypoxanthine and thymidine kinases)- which slaavege the nucleic acid biosynthesis
  • both cells types have thymidine kinase (so can make DNA)
  • only cells with HGPRT can make RNA
30
Q

spleen B cell is HGPRT

A

positive

31
Q

Myeloma cell is HGPRT

A

negative

- therefore not able to use the slavge pathway for RNA synthesis

32
Q

hybridoma cells

A

has the HGPRT from the spleen cell (RNA synthesis) and immortality from myeloma cells- so able to grow in HAT medium

  • providing there is an outside supply of H and T
33
Q

What is used to then isolate the hybridoma in the wells which have survived

A

Plate-trapped-antigen- enzyme- Linked immunosorbent assay

34
Q

Plate-trapped-antigen- enzyme- Linked immunosorbent assay

A

PTA-ELISA

35
Q

PTA-ELISA process allows identification of antibody of interest

A

1) on wall of the well you have your Ag of interest
2) mAb specific to Ag will bind to the epitome via Fab domain
3) then rabbit anti-mouse enzyme conjugate added which binds to the Fc region of mAb
4) this antibody is bound to an enzyme- which causes colour change when substrate is added

36
Q

rabbit anti-mouse enzyme

A

a second antibody- raised in mice, using mouse antibodies which have been injected into rabbits which ave made antibodies agains the mouse antibody)

37
Q

If colour change occurs in PTA-ELISA

A

monoclonal antibody is present

38
Q

which technique allows you to get away from using a live host to produce monoclonal antibodies

A

phage display

39
Q

phage display

A

taking a phage which infects bacteria and getting them to display antibody fragments on their surface

40
Q

phage display is a techniue

A

in which peptides and proteins (antibody fragments) are expressed as a fusion with a coat protein or bacteriophage

  • fuse proteins are displayed not eh surface of VIRION- while the DNA encoding fusion resides within the virion
41
Q

example of a virus which is used to express antibodies

A

M13

42
Q

M13

A

ssDNA genome- integarre DNA inro genome of host cell to express antibodies

43
Q

making antibody combinatorial libraries using phage display

A

1) B cell source (immunised animal)
2) production of mRNA
3) production off cDNA from mRNA
4) PCR to amplify each of the V-gene families (VL?VH)
5) assembles the VH and VL genes ar random
6) clone as scFv onto PIII phage

44
Q

scFV

A

VL and VH domains of an immunoglobulin linked by a peptide spacer

45
Q

scF are expressed

A

on surfaces of phages

46
Q

up to ….. ca be pressed on a phage

A

5 copies

47
Q

fusion pteoteins form between

A

scFV and PII minor coat proteins to allow expression of scFV on surface of the phage

48
Q

expression of Fab fragments on filamentous phage

A

Fab fragments of antibodies can be expressed on the surface of phage as fusion proteins, with PIII

49
Q

expression of Fab garments on filamentous phage

A

Fab fragments can also be expressed on the surface of phage as fusion proteins with pVII- multiple copies expressed along the length of the phage particle
- useful for isolation of low affinity antibodies

50
Q

if you want to epees slow affinity antibodies

A

multiple copies can be expressed along the length of the phage particle- rather than on the PII

51
Q

isolating antibodies using phage technology

A

take a population of phage and do ELISA this allows you to pull out the phage producing the antibody of interest

52
Q

phage selection

A

1) need to eliminate phages not producing antibody of interest
2) to get rid of all phages not of interest big UV
3) these phages are then eluted and used to infect E.coli
4) e.coli used to bulk u population of phage and is lysed

53
Q

phage display and selection means

A

never have to back to immunization- have combinatorial library

CBI - UOE (61 decks)

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