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cellular basis of immunity > 13 - monoclonal antibodies > Flashcards

13 - monoclonal antibodies Flashcards

1
Q

inject 1 antigen into a mouse - what kind of response?

A

polyclonal

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2
Q

polyclonal response

A

complex mixture/pool of antibodies produced
many different B cells switched on
no discrimination between antigens

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3
Q

why do we get a polyclonal response

A

1 antigen carries many different epitopes

each antibody specific for a different epitope

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4
Q

what does the monoclonal response allow

A

recognition of just the 1 B cell producing the antibody of interest
no cross reaction with other epitopes

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5
Q

relevance of Kohler and Milstein 1975

A

hybridoma technology

production of monoclonal antibodiees

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6
Q

what does hybridoma technology allow

A

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

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7
Q

method of hybridoma technology

A

immunise animal and allow immune response to build up
B cells become activated by specific antigen causing disease
B cells produce monoclonal antibodies
fuse splenocyte with myeloma cell
results in immortal hybridoma cell

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8
Q

splenocyte

A

antibody producing B cell from the spleen with a finite lifespani

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9
Q

myeloma cell

A

non antibody producing B cell with infinite life span

produces immortality in hybridoma cells

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10
Q

3 methods to immunise/inject the mouse

A

intrasplenically
into tail vein
into peritoneum

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11
Q

most common way to immunise mouse

A

inject antigen into peritoneum

antigen can enter bloodstream quickly to amount immune response

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12
Q

time course of immunisation of mouse

A

after a few days
- build up and activation of multivalent, low affinity IgM antibodies

after 2 weeks

  • immunise again
  • allow build up of immune response then drop off

after multiple boosts
- build up of bivalent, higher affinity IgG

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13
Q

method to test which cells are fully fused hybridoma cells

A

tail vein removed from mouse and serum from blood tested for immune response
if +ve, then booster injected
spleen then removed from mouse and cells fused with myeloma cells in vitro to produce hybridoma cells

isolation

  • HAT medium: knocks out un-fused splenocytes and myeloma cells
  • ELISA used as screening assay to identify well producing antibody of interest
  • expand cell
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14
Q

hybrid cell selection using HAT medium

A

hybridoma cells have HGPRT from spleen and immortality from myeloma cells
therefore they are the only cells that will grow in the HAT medium

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15
Q

HAT medium =

A

Hypoxanthine-Aminopterin-Thymidine medium)

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16
Q

aminopterin

A

blocks main biosynthetic pathways for nucleic acids

prevents cells from making RNA/DNA

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17
Q

importance of adding Hypoxanthine and Thymidine

A

they can salvage the biosynthetic pathways for making nucleic acid

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18
Q

importance of HGPRT

A

only cells that have HGPRT can make RNA via the salvage pathway

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19
Q

use of ELISA

A

assay used by immunologists that allows identification of antibody of interest

20
Q

method of ELISA

A

microtitre well with antibody attached
expose hybridoma solution to the well
add antibodies to bind to Fab region of antigen
add secondary antibodies to target Fc domain of primary antibody
colour reaction allows identification

21
Q

example secondary antibody used in ELISA

A

rabbit-antimouse enzyme conjugate

22
Q

phage display technology

A

takes antibody fragments and expresses them as fusion proteins on the outside of bacteriophage

23
Q

method of phage display technology

A

take B cell source and make a combinatorial library
convert mRNA to cDNA
PCR –> amplifies V-gene families
reassemble V-genes into new combinations
phage then expresses new combinations on surface as antibody fragments

24
Q

V-gene families

A

variable light and heavy genes of antibody

25
Q

scFv

A

single chain variable fragment

a fusion protein of the variable regions of the heavy and light chains of immunoglobulins

26
Q

where to bacteria express scFv

A

on their terminal coat protein

27
Q

pII protein

A

allows Fab fragments to also be expressed on the phage as an antibody fragment

28
Q

if antibody fragments are expressed along the length of the phage

A

then there is low affinity of antibody expression

29
Q

isolating antibodies using phage technology

A

use ELISA on phage population

process called biopanning

30
Q

limitations of biopanning

A

very expensive and fiddly

31
Q

method of biopannign

A

expose antigen to phage population
wash off non-binding antigens
identify bound phages using reporter molecule
use UV to eliminate non-bound
elute/remove bound phages and increase population using E.Coli

32
Q

3 methods of in vitro diagnositc assays using monoclonal antibodies

A

1 - ELISA
2 - Lateral-flow assay (LFA)
3 - Proximity Ligation Assay (PLA)

33
Q

PLA - proximity ligation assay

A

use PCR from antigen instead of from nucleic acid

34
Q

uses of monoclonal antibodies

A
  • diagnostic pathology to identify cancerous cells
  • in vitro diagnostic assays
  • diagnostics to track pathogens
  • affinity purification
  • characterisation of antigens
35
Q

recent surge in ability to use mouse monoclonal antibodies (mAbs) in vivo due to

A

phage display and hybridoma technology

36
Q

problems with monoclonal antibody use

A

need to “humanise” monoclonal antibodies otherwise the immune system recognises them as foreign and the mouse antibdy will be destroyed quickly (HAMA reactions)

37
Q

xenogenic

A

antibodies coming from another species

38
Q

HAMA reaction

A

Human Anti-Mouse Antibody reaction

39
Q

4 methods of humanisation

A

1 - produce human hybridomas
2 - create chimeric antibody
3 - CDR grafting
4 - make a transgenic mouse

40
Q

limitations to the production of human hybridomas

A

low efficiency

ethical issues - source of human B cell pool?

41
Q

production of chimeric antibodies

A

replace constant region of mouse antibody with humans

42
Q

CDR grafting

A

replace CDR of human antibody with mouse CDR

43
Q

advantages of transgenic mouse

A

fully humanised

44
Q

disadvantages of transgenic mouse

A

expensive

45
Q

transgenic mouse method

A

knock out all mechanisms to create mouse antibodies
replace with humans
only human antibodies expressed

46
Q

Rituximab

A

chimeric antibody drug

cellular basis of immunity (21 decks)

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