Antibodies (Seamus Martin Lectures)

Question 1

What are antibodies (immunoglobulins, Ig)?

Answer 1

Proteins produced by B-cells within the immune system that can recognise highly specific regions within other proteins with exquisite specificity.

Question 2

What is an epitope?

Answer 2

The area within another protein (8-10 amino acids) that is the main target region for antibodies.
- Can be multiple epitopes within the same protein (called an antigen in this context)

Question 3

What are the different types of antibodies?

Answer 3

1. IgG
2. IgM (pentamer)
3. IgE
4. IgA (dimer)
5. IgD

Question 4

What is the basic antibody structure?

Answer 4

Comprised of two identical ‘heavy’ chains (~50kDa) and two identical ‘light’ chains (~25kDa) that are assembled into a Y-shaped molecule (linked by disulphide bridges).

Question 5

Give an example of how antibodies work against infectious agents (use SARS-CoV-2).

Answer 5

Antibodies will bind to the spike protein (at the receptor-binding domain) to stop it from entering the host cell by blocking its binding to the human ACE2 receptor.

Question 6

What is endocytosis?

Answer 6

Engulfment of an infectious agent by a macrophage or neutrophil, followed by the destruction within the endolysosome of the macrophage/neutrophil.
- Triggered by an antibody binding the endocytic cell and directing it towards the infectious agent

Question 7

What is meant by the complement cascade?

Answer 7

The triggering of a cascade of proteins (employed by antibodies) that culminates in the formation of pores in the infectious agent, resulting in lysis and destruction.

Question 8

Name the three strategies employed by antibodies?

Answer 8

1. Blocking entry of infectious agent into the host cell (e.g., blocking receptor binding domain of spike protein on SARS-CoV-2, preventing it from entering the ACE2 receptor)
2. Endocytosis
3. The complement cascade

Question 9

What are polyclonal antibodies?

Answer 9

Antibodies that have affinity for the same antigen, but different epitopes.
- Made using several immune cells
- Predominantly, but not exclusively, directed against the immunogen to which they were raised
- Relatively ill-defined response to the antigen (variable specificity)

Question 10

What are monoclonal antibodies?

Answer 10

Antibodies that are specific to a single epitope (highly specific, highly pure).

Question 11

What are hybridomas?

Answer 11

The fusion of normal antibody-forming cells with B-cell tumour line to produce “immortal” clones B-cells (that can be grown in tissue-culture) that can make monoclonal antibodies.

Question 12

How are hybridomas cells selected for in tissue culture?

Answer 12

Using HAT medium - a medium used to select for hybridomas in tissue culture (contains hypoxanthine-aminopterin-thymidine).

Question 13

What are some uses of monoclonal antibodies?

Answer 13

Uses of MONOCLONAL ANTIBODIES…
1. Immunoassay
2. Diagnosis of malignancies
3. Tissue typing
4. Serotyping of microorganisms (a further categorisation of strains)
5. Separation of individual cell types with specific surface markers (e.g., lymphocyte populations)
6. Therapeutic neutralisation of inflammatory cytokines
7. “Magic bullet” therapy (with cytotoxic agents coupled to antitumour-specific antibody)

Question 14

What are fluorescein (FITC) and rhodamine examples of?

Answer 14

Fluorescent dyes that can be coupled to antibodies without destroying their specificity.
- Allow us to explore subcellular localisation of a protein (or other antigen) within a cell or tissue

Question 15

What is horseradish peroxidase an example of?

Answer 15

An example of an enzyme that antibodies can conjugate to that can catalyse the conversion of a colourless substrate into a coloured product.
- Also allows us to explore protein subcellular localisation

Question 16

What are ‘primary antibodies’?

Answer 16

Recognises and binds to a particular antigen.
- Directly used to screen for antigens (in immunofluorescent or immunohistochemical assays)

Question 17

What are ‘secondary antibodies’?

Answer 17

Recognises and binds to the primary antibody.
- Indirectly used to screen for antigens (in immunofluorescent or immunohistochemical assays)

Question 18

How can we visualise immuno-fluorescently labelled cells and tissues?

Answer 18

Scanning confocal microscopy - uses laser light to create sharp images of immunofluorescently-labelled cells and tissues.

Question 19

What is flow cytometry?

Answer 19

Can analyse the fluorescence levels associated with thousands of cells per minute, and thus it is possible to rapidly discriminate between cells that are negative, slightly positive, or positive for a given marker or antigen.
- Most modern cytometers can gather data on the expression of up to four proteins as the cell passes through the flow cytometer
- Can determine whether expression of these proteins is mutually exclusive

Question 20

What methods can be used to detect and quantify an antigen in fluids and cell lysates?

Answer 20

1. Immunoassay of antigen by ELISA
2. Immunoblotting (western blotting)
3. Immunoprecipitation (IP) of protein complexes
4. Chromatin immunoprecipitation (ChIP) assays
5. ChIP-on-ChIP or ChIP-Seq

Question 21

What is an ELISA assay?

Answer 21

Involves immobilisation of the antibody capable of detecting a protein of interest within the plastic wells of a microtiter plate.
- Unbound protein-binding sites are blocked by incubation with an irrelevant protein (such as albumin)

Question 22

What are the different types of ELISA assays?

Answer 22

(i) Direct ELISA
(ii) Indirect ELISA
(iii) Sandwich ELISA (or antigen-capture assay)
(iv) Competitive ELISA

Question 23

What is sandwich ELISA (or antigen-capture assay)?

Answer 23

Commonly used ELISA method which uses a ‘capture antibody’ to capture the antigen in the plastic well, then adds a second ‘detection antibody’ that binds to a different site on the antigen and produces a chemiluminescent reaction product (through bound HRP or alkaline phosphatase) to allow for quantification of the antigen.

Question 24

Name two enzymes that may be conjugated to the detection antibody to produce a chemiluminescent reaction product when bound to an antigen.

Answer 24

Horseradish peroxidase (HRP) or alkaline phosphatase.

Question 25

What are some uses of ELISA?

Answer 25

ELISAs are routinely used to quantify… - Cytokine levels in blood samples and tissues fluids - Viral or bacterial antigens - Drug concentrations in the blood or urine samples

Question 26

Why do we need to "blot" an SDS-PAGE gel onto another positively charged (usually nitrocellulose-based) membrane?

Answer 26

Antibodies are large molecules, and thus cannot readily penetrate the gel matrix. Thus, it is necessary to “blot” the gel onto a positively charged membrane. - This membrane traps charged proteins and immobilised them on the surface of the membrane - Commonly used blotting membranes; polyvinylidene difluoride (PVDF) and nitrocellulose-based membranes - The blot can then be proved with either polyclonal or monoclonal antibodies directed against the protein of interest - Binding of antibody is detected using horseradish peroxidase-conjugated anti-Ig secondary antibodies

Question 27

What can an immunoblot tell us about a protein of interest?

Answer 27

Immunoblot analysis can tell us if a protein is… - Unregulated - Downregulated - Cleaved - Phosphorylated - Glycosylated - Ubiquitinated ; in response to a particular stimulus.

Question 28

What is immunoblotting/western blotting?

Answer 28

A means of assessing a proteins molecular mass, and also allows us to explore its behaviour within a complex mix of proteins.

Question 29

What are two appropriate (and commonly used) blotting membranes?

Answer 29

Polyvinylidene difluoride (PVDF) and nitrocellulose-based membranes.

Question 30

What is immunoprecipitation (IP)?

Answer 30

The purification of a protein from a complex mixture of other proteins using antibodies bound to solid support (such as agarose beads) followed by a centrifugation step. - Immunoprecipitation of a protein (e.g., TLR4) by using a monoclonal antibody against the protein of interest (e.g., anti-TLR4) - Unbound material is washed away by centrifugation in a suitable wash buffer - Immunoprecipitated material can then be applied to an SDS-PAGE for analysis - Has been widely employed in the study of protein-protein interactions

Question 31

What is the main use of immunoprecipitation (IP)?

Answer 31

Has been widely employed in the study of protein-protein interactions (through immunoprecipitation of proteins that are co-expressed within the same cell).

Question 32

What is ChIP?

Answer 32

Chromatin Immunoprecipitation (ChIP) - a modification of standard IP that can analyse the repertoire of gene promoter sequences (or other regions within DNA) that a transcription factor, or other DNA-binding protein is bound to.

Question 33

What is the purpose of the chemical cross-linking step of ChIP?

Answer 33

As a result of this step, our transcription factor will be bound to the region of DNA (e.g., the promoter region to which it was bound) it was actively transcribing when we ended the experiment!

Question 34

What is ChIP-on-Chip or ChIP-Seq?

Answer 34

A further modification of standard ChIP assay!! - Permits a more global analysis of the DNA fragments immunoprecipitated - Looking more objectively at all DNA fragments through hybridisation with a DNA microarray chip (that carried sequences from a huge variety of genes)!