Polyclonal and monoclonal antibodies

Question 1

How do B cells generate antibodies? (3)

Answer 1

Somatic rearrangement
Affinity maturation
Class switching

Question 2

Applications of antibodies (5)

Answer 2

Physiological role- fighting disease

Research - detecting “invisible substances”, blocking function, etc

Diagnosis- detecting disease markers, blood types, pregnancy hormones

Therapeutics- anti-venom, passive vaccination, immunotherapy

Industrial uses- abzymes, detectors

Question 3

What are polyclonal antibodies? (2)

Answer 3

Those produced by many different B cells responding to the same antigen.
Polyclonal preparations contain a mixture of antibodies targeting different parts of the protein.

Question 4

What are monoclonal antibodies? (2)

Answer 4

Produced by a population of identical clonal - B cells
They are homogeneous and all recognise the same epitope

Question 4

Describe the process of polyclonal antibodies produced? (6)

Answer 4

Immunisation of animal
Normally immunised with purified protein plus an adjuvant
Adjuvant needed to elicit local immune response
Recruitment of cells, slow release of antigens, also activate APCs via pattern recognition receptors- release
of cytokines, T cell help needed for effective antibody production CD40-CD40L interaction etc)
A small hapten requires a carrier protein to elicit an immune response
Whole sera (liquid fraction of clotted blood) is collected and purified

Question 5

Why are multiple boosts required? (2)

Answer 5

Multiple boosts generates higher titre, higher affinity antibodies

Question 6

Advantages of polyclonal antibodies (3)

Answer 6

Technically easy to obtain
Antibodies are against numerous epitopes
Allows more effective crossing-linking/neutralisation also higher chance of cross-reactivity)

Question 7

Give example of use of polyclonal antibodies (3)

Answer 7

Examples- passive protect against rabies, neutralisation of snake venom, Anti-D

Question 8

What is the Haemolytic Disease of the Newborn? (3)

Answer 8

Rh- mother is sensitised to Rh+ foetus during first pregnancy/birth
Production of IgG against Rh by memory
B cells during second pregnancy destroys foetus (IgG is transfered across placenta)

Question 9

Treatment for Haemolytic Disease of the Newborn (1)

Answer 9

Therapeutic anti-Rh Ab before birth prevents memory response

Question 10

Disadvantages of polyclonal antibodies (4)

Answer 10

Polyclonal antibodies are a complex mixture of antibodies directed against different epitopes and that differ in their affinity for the antigen. (can not easily manipulate via recombinant means)

Each antisera preparation differs in specificity, average affinity, crossreactive specificies, etc.

Not optimised for application

Supply is limited.

Question 11

How are monoclonal antibodies generated? (3)

Answer 11

Immunisation of animal and inject with adjuvant and antigen
Get poly-sera
Harvest the spleen for B cells
B cells produce antibodies
Immortalise B cells by fusing with cancer cells to prevent B cells from dying
Continued production of antibodies - hybridoma - mixture of B cells and cancer cells
Separate B cells from cancer cells by growing cancer cells in a selected media

Question 12

Uses of monoclonal antibodies

Answer 12

Research
Diagnostic/prognostic indicators
Therapeutic antibodies

Question 13

What are ELISAs? (3)

Answer 13

Allows for specific, sensitive and quantitative detection of soluble molecules within a
complex mixture. eg blood, saliva, tissue culture growth media (in both research and diagnostics)

Multiplex ELISAs can be performed (ie measuring multiple analytes and the same time)

Question 14

What is Western blot analysis (2)

Answer 14

Allows specific detection from complex mixtures (eg cell lysate)
Not particular quantitative, but provides size information

Question 15

What is flow cytometry? (4)

Answer 15

Depends on specificity of antibody
Defines individual cells from a mixture of cells- allows analysis of multiple antigens simultaneously on millions of cells.
Sensitive, quantitative, versatile

Question 16

What is immunohistochemistry/Immunofluorescence (2)

Answer 16

Specific antibody linked to enzyme or fluorophore
Used to look at individual cells and localisation of cells
Look at components of cells

Question 17

Therapeutic uses of monoclonal antibodies (3)

Answer 17

Rheumatoid arthritis, Crohn’s disease - Anti-TNF alpha (Infliximab), anti-CD11a (Efalizumab)
B-cell leukaemia anti-CD20 (Rituximab)
Breast cancer- anti-HER2/neu receptor (Herceptin)

Question 18

What are the problems with monoclonal antibody therapy (3)

Answer 18

Repeat doses are often immunogenic, resulting in neutralising anti-antibody antibodies
Side-effects mediated via Fc region, eg complement activation, FcR activation, ADCC
scFv have different pr biological half lives, greater tissue penetrance (important for tumours/delivery to the CNS).

Question 19

What is radioisotope arming method? (1)

Answer 19

DNA damage, can penetrate solid tumour

Question 20

What is cytotoxic drug arming method? (1)

Answer 20

Cytotoxicity. Drug must be internalized

Question 21

What is toxin arming method? (1)

Answer 21

Cytotoxicity. Toxin must be internalized

Question 22

What is Antibody dimer arming method? (1)

Answer 22

More effective engagement of physiological killing mechanisms

Question 23

What is enzyme arming method? (1)

Answer 23

Activation of prodrug

Question 24

What is Bispecific antibody arming method? (1)

Answer 24

Engagement of cytotoxic T cells or natural killer cells

Question 25

Advantages of monoclonal antibodies (5)

Answer 25

Consistent Limitless supply of specific reagent More easily tested for cross-reactivity Can optimise for application Can be manipulated via recombinant technologies

Question 26

Disadvantages of monoclonal antibodies (2)

Answer 26

technically more difficult and time consuming, less likely to neutralise/cross react

Question 27

Problem with mouse antibodies (2)

Answer 27

They’re mouse: immunogenic, need to house and kill mice, once antibody is generated one needs to clone the DNA sequences before manipulation. Can’t generate antibodies against very homologous (similar) proteins

Question 28

Solution for mouse antibodies problem (2)

Answer 28

In vitro generation, phage display technology

Question 29

Why are antibodies good at eliminating pathogens? (4)

Answer 29

Specificity, high affinity, stability and their effector Fc region provides an ideal mechanism for binding and eliminating antigens

Question 30

Polyclonal antibodies can be used to treat which disease? (1)

Answer 30

Haemolytic disease of newborn

Question 31

Cloning hybridoma (5)

Answer 31

Spleen cells and cancer cells mixed Polyethylene glycol to allow fusion Separate out hybridomas and place at limiting dilution Allow clones to expand Expand positive well and test for production of antibody of desired specificity

Question 32

Which tests are used for research? (3)

Answer 32

Tissue staining Western blot ELISA

Question 33

Which tests are diagnostic/prognostic indicators? (3)

Answer 33

Immunohistochemistry Flow cytometry Confocal microscopy

Question 34

What are the therapeutic antibodies? (2)

Answer 34

Toxin Abzymes

Question 35

How to work out the concentration of an unknown protein with ELISA? (4)

Answer 35

Standard amount of protein Generate a standard curve Plot a graph Annulytes and read off the graph

Question 36

Describe Western Blot Analysis (11)

Answer 36

SDS page gel - separate proteins based on size Stain Only works with pure proteins Use specificity of antibodies Separate proteins using gel electrophoresis Transfer Probe with antibody Visualisation See proteins of certain sizes See isoforms of proteins See degrading proteins Can be fluorescence

Question 37

Lateral flow tests

Answer 37

Test zone has an antibody against a specific hormone

Question 38

Phage display (5)

Answer 38

Rearranged variable-region gene segments are randomly combined through the use of recombinant molecular biology techniques to yield a large phage ‘library’ of antibodies/scFvs Bacteriophage library is used to infected E. Coli which display the “antibody” fragment on the surfaces of the generated phage Binding phage are then isolated and used to re-infect E. Coli Totally human-derived antibody fragment with known sequence, ready for manipulation

Question 39

Alternatives for antibodies (2)

Answer 39

Antibodies are composed of Ig domains VLR genes from jawless vertebrates consist of LRRs