Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

SEM 4: Immunology > Immunology in The Clinic and Research Lab > Flashcards

Immunology in The Clinic and Research Lab Flashcards

1
Q

Summarise structure of IgG antibody

A
  • 2 heavy chains and 2 light chain held by disulphide bridges
  • Upper part = Fab (variable chain) which binds to the antigen
  • VL in the light chain binds to the specific epitope on the antigen via the CDR.
  • Fab region has:
    • > Antibody repertoire: total number of antibody binding sites
    • > Affinity: the strength of the interaction between the antibody and the epitope.
    • > Avidity: sum of different affinities; some Ab are multimeric, so have several different binding sites.
  • Lower part = Fc (constant region) that binds to various parts of the immune system.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are the important aspects of the Fc region?

A
  • Antibody dependent cell mediated cytotoxicity (ADCC)
  • Antibody dependent cellular phagocytosis (ADCP)
  • Complement dependent cytotoxicity (CDC)
  • Pharmacokinetics half-life
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is the pharmacokinetics half life?

A

The amount of time the antibody will spend in the body.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Describe the polyclonal antibody response model

A
  1. When infected with a pathogen, a polyclonal antibody response occurs,
  2. In the body, there are different B cells, each specific for a certain epitope.
  3. When the pathogen was injected, the specific B cells complementary to the epitopes were activated.
  4. The activated B cells start to proliferate and produce polyclonal B cells which are identical B cell, which will then secrete antibodies.
  5. The antibodies will have the same specificity of binding.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Why are monoclonal antibodies important?

A

They bind to a single epitope and are very important in medicine and immunology.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How are monoclonal antibodies produced through a hybridoma culture?

A

They are produced in a hybridoma culture.

  1. Inject a mouse with antigen which is specific for the desired antigen.
  2. After 2 weeks, harvest the B cells which make the antibody.
  3. The B cells are then fused with myeloma cells via a chemical.
  4. Once the fusion has occurred, there is a mixture of B cells, myeloma cells and a fusion of both.
  5. Only the fused ones are selected via hypoxanthine-aminopterin-thymidine (HAT) selection.
  6. These fused cells are then collected and diluted to a single cell.
  7. This single cell is cultured, and will divide to form a clone of the cells, which will produce monoclonal antibodies of all the same specificity.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are myeloma cells?

A

They are immortal and are derived from a B cell tumour but do not produce antibodies themselves. They also lack the gene HOPRT.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the hypoxanthine-aminopterin-thymidine (HAT) selection?

A

A selection medium stops the cells from making DNA the normal way. The myeloma cells do not have the HOPRT genes, so they die off. The B cells do have the gene, however, they are naturally short lived, so they die off.
The fused cells have the gene and immortality, so they survive.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Why are the monoclonal antibodies of the same specificity at the end of selection?

A

This is because all the cells have come from a single cell.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What happens to the hybridomas after they are collected?

A
  1. Once they are collected, they can be stored indefinitely and grown to produce monoclonal antibody when required.
  2. Antibody gene can then be cloned to form the hybridomas which hallows antibodies to be engineered for different applications.
  3. The polyclonal or monoclonal antibodies are produced which binds Fc regions of particular antibody classes e.g. IgG and IgA. These are called anti-isotypic antibodies.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are immunoassays?

A

Use antibody antigen interaction, one of which is labelled or tagged to allow detection. Then this measures the amount or concentration of antibody or antigen. Polyclonal or monoclonal antibodies can be used.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Why are immunoassays used?

A

They are sensitive and specific and hence used in research and analytical labs.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is the new types of immunoassays?

A

Now commonly used is an enzyme, which is detected by coloured product such as horseradish peroxidase or alkaline phosphatase, used in ELISA. Other alternatives are luminescent.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are direct ELISA used for?

A

Used to quantify an antibody

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Describe the process of a direct ELISA

A
  1. Antigen immobilised on solid support such as stuck on the bottom of the well.
  2. Test antibody solution covalently linked to enzyme added.
  3. The enzyme substrate is then added, and if both the antigen and the antibody are present, coloured product will be produced.
  4. This can be measured by absorbance.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are the uses of direct ELISA?

A
  • Screen hybridoma supernatants

- Detect exposure to infection antigen

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What are indirect ELISA used for?

A

Used to quantify an antigen

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Describe the process of indirect ELISA

A
  1. The antigen gets immobilised on solid support.
  2. Primary antibody which binds to antigen is then added.
  3. Secondary antibody covalently attached to enzyme is subsequently added. This antibody binds to Fc region of primary antibody.
  4. Enzyme substrate is added, and the colour changes is measured by absorbance.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What is unique about the secondary antibody used in indirect ELISA?

A

Often polyclonal so it can bind to different epitopes on a primary antibody which allows multiple secondary antibodies to bind to the same primary antibody which amplifies the signal and increases the sensitivity of the test.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What is sandwich capture ELISA?

A

Antigens may be present in low concentration and because antibodies have high affinity for antigens, they can be used to concentrate them. For this to occur 2 antibodies reacting with different epitopes on the antigen are needed.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Describe the process of sandwich-capture ELISA

A
  1. One antibody will be immobilised on a solid surface.
  2. The test antigen solution will then be added, incubated, then washed so any non-bound antigens get removed.
  3. Second antibody is then added which is attached to the enzyme, and will bind to the antigens (already bound to the first antibody)
  4. The substrate is then added and will change colour.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What is an elispot assay?

A

An immunoassay used in labs to detect cytokines given off by cells.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Describe the process of elispot assay

A
  1. Antibodies specific for the cytokine to be detected are used.
  2. These antibodies are immobilised in the bottom of the plate.
  3. Activated T cells are then added to plate, which will secrete the cytokines.
  4. This cytokine if complementary will bind to the antibody.
  5. An enzyme conjugated antibody is then added.
  6. Substrate to that enzyme is added and a coloured product should be seen.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What is SDS PAGE and Western Blotting used for?

A

It can be used to:

  • Detect antigen and antibody
  • Measure size of protein being analysed
  • Calculate protein concentration
  • Show if the protein has been degraded.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Explain the process of SDS PAGE/Western Blotting

A
  1. Start of with sample containing protein to be detected.
  2. The sample is then boiled with sodium dodecyl sulphate, which will bind to the protein and give it a negative charge.
  3. Then place the protein in on a gel, which will run the protein according to its size.
  4. The gel is then taken and blotted on nitrocellulose
  5. This is then labelled with specific enzyme conjugated antibodies.
  6. When the substrate for the enzyme is added, a band will form on the nitrocellulose paper.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

What is SDS PAGE/ Western Blotting used alongside with?

A

Used alongside ELISA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

How is WB protein concentration measured?

A

They are measured by comparing intensity of band compared to a band of known concentration. If the protein is degraded, it may be more useful to WB to calculate protein concentration. This is because some of degradation fragments may contribute to signal in ELISA if the coating and detecting antibodies are able to bind to them.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What is a technique used for antibody-antigen interaction?

A

Antibody coated magnetic beads - antibodies can be used to purify immune cells.

29
Q

Describe the antibody coated magnetic beads technique

A
  1. The antibodies are coated with microscopic magnetic beads.
  2. These antibodies will bind to certain proteins on the surface of the cells that are being insolated.
  3. Once bound, cells are applied to machine that has mesh and magnetic fields.
  4. Only the cells that are coated with magnetic beads will bind to the mesh and the rest of the cell will wash out.
  5. The magnetic field in then switched off and the cells can be extracted.
30
Q

What is FACS?

A

Fluorescence activated cell sorter analysis

31
Q

Define the FACS technique

A
  1. During this individual cells (desired cells) within a mixed population are tagged with monoclonal antibodies which will bind to surface molecule.
  2. These are then labelled with fluorescent dyes.
  3. The mixed cells are then forced through a nozzle to form a stream of single cells.
  4. Individual cells pass through a laser beam which scatters light and causes dye to fluorescence and provides information on bound antibody and cell surface protein.
  5. A readout will be produced and will be in the form of a dot plot.
32
Q

What are the types of samples analysed?

A 
Blood serum 
Blood cells 
Urine 
Synovial Fluid 
Saliva 
Mucus 
CSF
33
Q

What are the types of diseases analysed?

A 
Transplant compatibility 
Immunodeficiency 
Autoimmunity 
Allergy 
Malignancy
34
Q

What is histocompatibility?

A

The genetic difference between individuals is detected by the immune system and leads to rejection of non-self organs.

35
Q

When are the best transplant results seen?

A

The best transplant results are seen when the donor and the recipient MHC are similar.

36
Q

Summarise the binding and what cells recognise MHC Class I

A
  • Fragments of intracellular proteins

- T cell receptor on Cytotoxic T cells, with assistance from CD8

37
Q

Summarise the binding and what cells recognise MHC Class II

A
  • Fragments of proteins which have been taken up by endocytosis
  • T cell receptor on helper T cells, with assistance from CD4.
38
Q

What is the MHC complex known as? Where is it located in genes?

A
  1. In humans, the MHC complex is known as HLA.
  2. It is located on chromosome 6 and contains 3 MHC I proteins and 3 MHC II proteins.
  3. It is highly polymorphic with 100s of different variants, in order to bind to different antigens.
39
Q

How are MHC typing for transplant compatibility matched?

A

Try to match donor and recipient by MHC alleles.

The alleles are identified by PCR.

40
Q

How are immunodeficiencies such as X-linked agammaglobulinemia identified?

A

They are unable to generate mature B cells. B cells have a CD18 surface protein that is a co-receptor for the BCR. This can be tested via flow cytometry by investigating antibodies to cell surface markers. This shows that the XLA patient doesn’t have the CD19 cell surface protein and therefore no antibodies bound so no B cells. However, they will have CD3 which is a cell surface protein on T cells.

41
Q

How can flow cytometry be used in the clinic to monitor HIV infection?

A
  • Lymphocyte subset estimations are performed using monoclonal antibodies to CD3, CD4 and CD8 on whole blood and analysed by flow cytometry.
  • The percentages of cells in each subset is determined using FACS machine.
  • The results are reported as percentages and absolute counts.
42
Q

How are T cell responses tracked?

A

Through fluorescent MHC complexes for antigen-specific T cells

43
Q

Describe the creation of fluorescent MHC complexes for T cell tracking

A
  1. The MHC molecules which are bound to a specific peptide in the groove and in the C terminal of the MHC molecule is bound to a vitamin called biotin.
  2. This vitamin binds to a molecule called streptavidin.
  3. Streptavidin is bound to a molecule which gives off light.
  4. When mixed all together, a tetramer is formed.
  5. This tetramer will bind to the TCR on a cytotoxic T cell. This tetramer structure means the binding is strengthened.
  6. Once the binding has occurred, the fluorescence can be measured.
  7. The fluorescence measurement can tell us the proportion of cytotoxic T cells which can bind to the antigen.
44
Q

Describe the life cycle of a neutrophil briefly

A

They are found in acutely inflammed tissues
They ingest pathogens and kill using reactive oxygen species
They rapidly die after phagocytosis which generates pus

45
Q

What is neutropenia?

A

It is deficiency in neutrophil numbers which causes high rate of infection.

46
Q

What causes granulomatous disease?

A

It is caused by deficiency in phagocyte function

47
Q

What is granulomatous disease?

A

When the patient cannot form reactive oxygen species, so they succumb to the bacterial and fungal infection.

48
Q

How is neutrophil function measured?

A

By carrying out neutrophil oxidative burst assay

49
Q

Describe the neutrophil oxidative burst assay procedure

A
  1. Nonfluorescent DHR 123 becomes rhodamine 123.
  2. This occurs when nonfluorescent DHR 123 is phagocytosed by normal activated neutrophils.
  3. This causes the ROS during the activated neutrophil respiratory oxidative burst to change it to rhodamine.
  4. This is a green fluorescent compound which can be detected via flow cytometry.
50
Q

What are the two ways antibody levels are analysed?

A
  1. This is done via electrophoresis by looking at the sample through a gel.
  2. Also done via nephelometry
51
Q

What is nephelometry?

A

An automated and rapid method used to measure serum antibody levels. It relies on light scattering properties of antigen-antibody complexes.

52
Q

Describe the process of nephelometry

A
  1. To measure IgG in serum, an anti-IgG is added to bind to it.
  2. When these bind, they will form a complex.
  3. When light is shined on the serum containing the complex, light will be defracted.
  4. The amount of defraction will be measured which will correspond to the amount of complexes present and hence the amount of IgG present.
53
Q

Which immunoglobulin mediates allergy?

A

IgE

54
Q

What are some common allergies?

A
  • House dust mites
  • Cat and dog
  • Trees and grass
  • Mould
  • Eggs, milk, cod, soya and peanuts
55
Q

Describe what happens when a person is first exposed to an allergen

A

They will produce IgE which will bind to receptors on mast cells.

56
Q

Describe what happens when a person is exposed to an allergen following previous exposure

A
  1. The allergen will bind to IgE, leading to clustering on the surface of the mast cell.
  2. This will cause a signal in the cell to degranulate its contents including histamine which will cause the allergic reaction.
57
Q

How is allergy measured?

A
  • By doing the skin prick test

- By doing the radioallergosorbent test (RAST)

58
Q

What is the skin prick test?

A
  1. Apply a certain allergen on the skin of an individual

2. If the allergic reaction is stimulated, a bump will form on the skin.

59
Q

What is the radioallergosorbent test?

A
  1. The suspected allergen is bound to an insoluble material.
  2. The patient’s serum is then added to the allergen.
  3. If the serum contains IgE antibodies to the allergen, they will bind to it.
  4. Radiolabelled anti-human IgE antibody is then added.
  5. This will bind to those IgE already bound IgE antibodies.
  6. This radioactivity can then be measured and will be proportional to the serum IgE for the antigen.
    Sometimes fluorescence can be used instead of radioactive.
60
Q

What are the key characteristic of autoimmune diseases?

A

Characterised by autoantibodies to nuclear antigens such as RNA and DNA

61
Q

Why is detection of autoantibodies useful?

A

It is useful for diagnosis and monitoring disease activity

62
Q

What is systemic lupus erythematous (SLE)?

A

An autoimmune disease characterised by production of autoantibodies causing a range of symptoms, often dermatological.

63
Q

How are autoimmune antibodies detected?

A

Using SLE immunofluorescene and ELISA in parallel to increase sensitivity

64
Q

What is SLE immunofluorescene test?

A
  1. Serum is added to the human cell line
  2. It is then proven to exist with fluorochrome labelled anti-immunoglobulin antibody.
  3. This is then visualised by fluorescence microscopy.
65
Q

What is polyclonal antibody therapy?

A

It is a technique used to treat patients with antibody deficiencies. It uses intravenous immunoglobulins (IVIG) that are a blood product purified from the serum of between 1000-15000 people. At high doses, it is used as an immunomodulatory agent in a number of immune and inflammatory disorders.

66
Q

Describe the use of polyclonal antibody therapy in rabies

A

After a bite from a suspected animal, polyclonal antibodies are isolated from the serum of immunised indiviuals with the rabies vaccine and injected into the wound site.

67
Q

What is monoclonal antibody therapy?

A

Treatment that involves monoclonal antibodies either binding and blocking a process or mediating immune responses such as intimation of complement or antibody dependent cell mediated cytotoxicity (ADCC).

68
Q

How many monoclonal antibodies are licensed to treat which diseases?

A

45-50 licensed to treat diseases such as cancer, chronic inflammatory diseases, transplantations, infectious diseases and cardiovascular medicine.

69
Q

Which molecules can be combined with monoclonal antibodies in therapy and why is this useful?

A

Toxins or radionuclides can be joined to monoclonal antibodies. For example in cancer, the antibody will bind to the cancer cell which is then killed by a toxin or radioactivity.

SEM 4: Immunology (15 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions