14 - Immunology Based Techniques in Research & Medicine - Partridge

Question 1

Generally, how do Abs protect against infectious material?

Answer 1

label pathogens and infectious material for ELIMINATION

Question 2

Why can Abs used in therapeutics? Give 5 reasons

Answer 2

• diverse. > 10^9 specificities
• robust and stable domain structure which facilitates structural engineering. member of the Ig gene superfamily
• effector properties. can exploit the natural functions of Abs eg in activating complement, binding to Fc receptros on surface of phagocytes and NK cells
• specific and high affinity. Kd = 10^-8 > -9 M
• multivalent with high avidity allowing cross-linking

Question 3

What is antisera?

Answer 3

serum containing high levels of Abs specific to certain antigen

Question 4

Draw a graph of [Ab] against time that highlights how antisera is produced

Answer 4

311 -14 word

Question 5

When Abs are purified, they can be labelled with certain tags name the 5 types they can be labelled with and how these labels can then be identified

Answer 5

• gold particles; immuno-electron microscopy
• enzyme; adding a S -> coloured product, ELISA, immunohistology
• fluorescence eg fluorescin; FACS (fluorescence activated cell sorting), immunofluorescence microscopy
• radioisotope; radioimmunoassay imaging of eg tumours
• sepharose; Abs bound to stationary matrix. immunoprecipitation or affinity purification

Question 6

Name an Ab that Partridge created and name its target

Answer 6

LP9 Ab targetting the CD63 TM protein

Question 7

How are polyclonal Abs generated? Draw a diagram to help

Answer 7

antigen with many varying epitopes used to raise many Abs against it
different subsets of B cells bind to varying epitopes on it and produce varying Abs
polyclonal antisera is generated w/ a mix of different Abs

Question 8

What are the advantages of polyclonal antisera?

Answer 8

• cheap
• robust (can recognise partially unfolded/denatured antigen because likely at least some Abs will recognise the 1ry sequence)
• form immune complexes well because there are high numbers of them that can cross Link

Question 9

What are the disadvantages with polyclonal antisera?

Answer 9

• purified antigen is needed to immunise. do not want to get Abs specific to unwanted product ->. contamination
• poly-specific. if trying to distinguish between v conserved antigens then wont be useful because many different Ab types
• difficult to standardise. at certain times, the no.s & types of different Abs present in serum will differ

Question 10

What are the 3 main uses of polyclonal antisera?

Answer 10

• 2ndry Ab used for immunoassays. instead of having to label many different monoclonal Abs, can raise Abs against this original Ab (eg IgG). these 2ndry ones can be labelled with ease
• can identify relationships between molecules eg Immunoglobulins. eg Abs will recognise different isotypes and allotypes
• gene product identification. eg couldn’t isolate protein from dystrophin gene. knew sequence -> protein sequence -> peptide. Abs raised against this theoretical protein sequence to purify it (human genome project was helped using this method)

Question 11

Describe the type of specificity of monoclonal Abs and where they are derived from

Answer 11

single specificity

derived from single B cell type

Question 12

describe how monoclonal Abs are produced

Answer 12

• mouse immunised with antigen
• B cells isolated from organisms (contains both specific B cells and other Ab-producing B cells)
• fused to myeloma cells (using PEG - polyethylene glycol) to give hybridoma cells
• hybridoma cells (fused cells) selected for by growing on drug-containing medium. unfused cells selected against
• hybridoma cells producing correct Ab selected for and isolated
• clone selected myeloma cells and purify Ab

Question 13

Who won the Nobel prize for this technique in 1984?

Answer 13

Kohler and Milstein

Question 14

What are the advantages of monoclonal Abs?

Answer 14

• highly specific
• pure antigen not needed to immunise. this is because myeloma cells grow indefinitely in tissue culture therefore produce large amounts of the same Ab
• can be standardised

Question 15

What are the disadvantages of monoclonal Abs?

Answer 15

• does not form immune complexes as well as polyclonal antisera
• expensive
• conformation sensitive. will only work if protein has correct conformation

Question 16

How can monoclonal Abs be used in diagnostics?

Answer 16

• detect/quantitate diagnostically important molecules eg Down’s syndrome, fertility/pregnancy testing
• serotyping pathogens (of closely related strains). therefore can be used to assign certain antibiotics

Question 17

How are mAbs used in defining cell surface molecules?
Give an example
(1st reason on pp)

Answer 17

• CD (cluster of differentiation) classification system of human leucocytes (>300 CD numbers now)
  eg CD3; defining cell types
  CD8/4; defining subsets
  defining stages of differentiation

eg LP9 identifying CD63 - used to clone this gene - member of the tetraspanin superfamily

Question 18

List the 3 other ways in which mAbs can define cell surface molecules

Answer 18

• biochemical analysis/purification - eg immunoprecipitation, SDS-PAGE (purifying proteins)
• functional studies - binding to surface structure may activate/inhibit cell functions. may mimic the natural ligand
• gene cloning - identifying gene products, screen expression libraries

Question 19

Why are Abs described as magic bullets?

Answer 19

Abs can be used to target cell surface structures found only on cancer cells

Question 20

Name the man who won the first Nobel prize for developing serum therapy - passive immunisation

Answer 20

Emil von Behring

Question 21

What diseases was serum therapy effective against and describe how it works

Answer 21

• tetanus and diphtheria
• serum from immunised animals was an effective treatment against these diseases
• serum effective because of the Abs it contained

Question 22

What were some of the downsides of serum therapy?

Answer 22

people developed immune response to horse serum

serum sickness

Question 23

Name a rodent-based Ab that can be used to induce an immune response in humans

Answer 23

HAMA; human anti mouse antibody

Question 24

Describe the 2 types of antibody engineering and some of their disadvantages

Answer 24

ANTIBODY CHIMERAS;

• mouse V region combined with human C region
• mouse v regions were still immunogenic

“HUMANISED” ABs;

• human framework regions and mouse CDRs (CDR grafting)
• relies on the immunoglobulin structure being stable therefore can be modified
• may lose affinity/specificity - sometimes framework regions can be damaged
• also v time consuming

Question 25

Describe how we obtain Abs from gene libraries

Answer 25

• isolate mRNA from Ab producing cells eg lymphoid tissue, blood, bone marrow
• amplify Fab fragment by PCR -> cDNA
• clone and express the cDNA using a vector into bacteria/phage -> Ab library
• in bacteria, Ab expressed as soluble proteins. in phage expressed as surface proteins on filamentous phage particles
• screen Ab phage display library against solid phase antigen (panning)

Question 26

Draw a diagram and explain how we obtain fully human Abs from a gene library through selection by phage display

Answer 26

• 311 - 14 word

- obtaining the Fab fragment (fully human) we can fuse to another human C region

Question 27

Describe how Greg Winter won the Nobel prize for Chemistry in 2018

Answer 27

• synthetic/synthetic human Ab gene library
• used human V regions and randomised CDRs giving > 10^9 specificities
• CDRs are continually mutated to give rise to more specific Abs against a particular target

Question 28

Name and describe 2 further strategies for generating human Abs

Answer 28

1) SCID MICE reconstituted w/ human leucocytes
- because they have no adaptive immune system, can tolerate human lymphocytes.
- can be immunised to generate fully human Abs
- but repertoire is limited to that of the human cells used

2) TRANSGENIC mice expressing human immunoglobulin genes.
- these introduced using yeast artificial chromosome (YAC)
- mouse Ab genes replaced w/ human Ab genes (xenomouse)
- mice can be immunised to generate human Abs through conventional monoclonal or phage display techniques