L13 - Monoclonal antibodies and their uses

Question 1

Antibodies: why are they good detection agents and what modifications must be made for their optimal use?

Answer 1

Very specific reagents - hypervariable regions

Large proteins - easily “tagged” - identify where they bind to an antigen and show what antigen they bind to

Antibodies are colourless - often tagged by fluorescent markers e.g. FITC, metal ions, enzyme markers that change colour when a substrate is added e.g. peroxidase

Question 2

Antibodies: what is the typical process behind making them?

Answer 2

Inject small samples of antigens (usually in mg as a protein) into an animal, combined with an ‘adjuvant’

Adjuvant - a substance that alerts the immune system

This occurs several times (usually over 10 weeks) which boosts the immune response, causing the maximum levels of antibodies within the serum which is now enriched

Question 3

Polyclonal antibodies: why are they formed and how often are they formed during the immune response?

Answer 3

Antigens usually contain many epitopes - several different b cells can recognise them

Every time - can be useful, helps to still destroy the pathogen even if it changes slightly

Question 4

Anti-toxins: how are they formed?

Answer 4

Inject animals with sub-lethal doses of toxins - anti-toxins are made

Question 5

Anti-venom: how are they formed?

Answer 5

Inject animals with sub-lethal doses of venom - anti-venoms are made

Question 6

Convalescent plasma: what is it, where is it obtained, and what can it do?

Answer 6

Blood plasma from people who have formed a defence against a specific pathogen

Blood of people who are recovering from an infection

Confer passive immunity

Question 7

Monoclonal antibodies: what are they, how are they formed, what are their strengths, what are their possible weaknesses, and where are they used?

Answer 7

The production of only a single type of antibody

Forming a hybridoma of a cancer cell with a b cell

• High specificity
• Low cross-reactivity
• Standardised worldwide
• Unlimited supply
• Can be bioengineered (solving some weaknesses) ie derive from different types of organism to circumvent immunity, creating Fab fragments, adding a delivery system to target cells, etc
• Can trigger immune responses in vivo
• May cause off-target effects
• May not access sites needed

Used in immunoassays, diagnosis, tissue typing, separation of cell types, and clinical therapeutic agents

Question 8

Monoclonal antibodies

Answer 8

• Affinity chromatography
• ELISA (indirect/direct)
• ELISpot
• Agglutination *Immunohistochemistry/immunofluorescence
• western blotting
• Flow cytometry
• Immunoisolation

Question 9

How do monoclonal antibodies exclusively get produced?

Answer 9

Cells cultured with growth medium containing HAT (hypoxanthine, aminopterin, and thymidine) - myeloma cells cannot survive and, since spleen cells are not immortal, only the single ideal hybridoma cell survives

After this, different hybridoma cells are screened against each other and the best one is chosen

Question 10

Affinity chromatography

Answer 10

Molecule added to material, antibody binds to antigen, everything else gets washed away, pure left

Often used to purify antigen/protein - ie for inoculum/treatment

Question 11

Magnetic Separation of cell populations - immuno-isolation

Answer 11

Antibody causes ideal cells to be attracted to magnetic charge, when charge is applied, cell gets stuck while the others pass through

Often used in the lab to obtain purified cell populations

May be used clinically e.g. cellular immunotherapy

Question 12

Agglutination: what does it do, where is it commonly used, and when can it be used?

Answer 12

Identify if an antibody is present for an antigen by causing clumping

Commonly used in blood typing

• Also to diagnose if a patient has HAD an infection - e.g. Typhoid fever- mix serum with a culture of Salmonella typhi - clumping indicates the patient either had or has an S. typhi infection.

Question 13

ELISA: what is it, what does it do, and when can it be used?

Answer 13

Clinical test that is typically used for liquids - serum, urine, culture supernatant, etc - but can also be cells (using lyses)

• Viral diagnostics
• Levels of hormone
• Antibody

Question 14

ELISA process: what is it and can it be increased?

Answer 14

1 - Antibodies bound to the plastic
2 - Sample added
3 - Unbound antigens washed away
4 - absorbance of light is monitored after adding an enzyme which causes colourisation

The signal from antigen-antibody interactions can be amplified - usually done with an anti-antibody – a secondary antibody, allows you to detect low levels through amplification

(preg test core)

Question 15

Secondary antibody: what is it?

Answer 15

An antibody generated against the first antibody

Secondary antibody interacts with many epitopes on the primary antibody, thus amplifying the signal

Question 16

Indirect ELISA: what does it do, what is its process, and how is it measured?

Answer 16

Uses secondary antigen - allows for twice or more of the detection

Same as direct but uses 2°Ab

Substrate added and amount of 2°Ab (absorbance) measured by ELISA reader

Question 17

ELISpot: what is it, what does it do, and what is its process?

Answer 17

Enzyme-linked Immunospot

Highly sensitive assay often used to assess frequency of specific T cell responses or frequency of antibody secreting cells in an infection or vaccine response - may measure frequency of cytokine-secreting cells at the single-cell level, etc

• Cells are cultured and +/- stimuli on a surface with capture antibody
• Uses a substrate with a precipitating product to create visible spots on the surface.
• After the incubation period, cells are removed
• Visualisation of antibody formed, the cells producing them, how much, how many cells, etc

Can be used for any secreted protein

Also used to monitor the breadth of antibodsy response (polyclonality - how ideal of a response you have)

Question 18

Immunohistochemistry and Immunofluorescence: what do they do, what cells do they act against, and what applications are there?

Answer 18

Shows localisation of antigen, can be quantitative

Fresh, frozen, fixed tissues; cultured cells etc.

Applications include:
* DNA sequences on chromosomes
* Spatial-temporal patterns of gene expression within cells/tissues.

Question 19

Western Blotting: what are they, what do they do, and when are they used?

Answer 19

Qualitative and quantitative analysis of protein expression - tells you sizes/charges of antibodies (SDS-PAGE stuff)

e.g. from tissue homogenates or cultured cells.

Can be used diagnostically

e.g. Identify if pathogen present e.g. Lyme disease, HIV

Question 20

Flow cytometry: what is it, what does it do, what information does it detect and what uses does it have?

Answer 20

Uses lasers to detect fluorescent antibodies bound to cells - detecting light scatteration can be used to gather information

e.g. different cell types in a sample of blood or tissue

• Levels of cell activation
• Cell viability
• Cytokines secreted
• Rare cell types

Clinical uses common

e.g. Diagnosis/staging of patients with a haematological neoplasm, detecting rare tumour cells, monitoring cell numbers (e.g. CD4 cells in HIV)

Question 21

Monoclonal antibodies: uses in the home?

Answer 21

Pregnancy test

Test utilises “ELISA” technology to test for the hormone human chorionic gonadotropin (hCG) which is released from placenta very soon after conception.

Question 22

Monoclonal antibodies: uses in the clinic

Answer 22

You could block just one cytokine and prevent cells from then releasing other inflammatory cytokines and thus interrupt the cycle of inflammation

And the success was remarkable leading to the adoption of anti-TNF therapy in many other conditions (TNF = tumour necrosis factor(?))

Question 23

Not rrl relevenant, more a ITC thing

Answer 23

Blockade of immune checkpoints to enhance T cell responses. After T cell activation, T cells express immune checkpoints such as CTLA-4 and PD-1. This may be further driven by tumours actively inhibiting the reponse. The immune checkpoint inhibitors block this negative interaction and allow the T cells to stay switched on and kill tumour

There are many other examples of antibody therapy include alzheimers, asthma etc etc