Immunology in the clinic and research lab

Question 1

Selection of Hybridomas - explain

Answer 1

Selection of Hybridomas
In HAT medium, myeloma cells die as they cannot make nucleotides due to lack of HGPRT gene. B cells die as they have a short life span. Only hybridomas grow and proliferate

Question 2

Hybridomas - describe storage

Answer 2

Hybridomas can be stored indefinitely and grown to produce monoclonal antibody when required

Question 3

What allows ABs to be engineered for different applications

Answer 3

Antibody genes can be cloned from the hybridomas which allows antibodies to be engineered for different applications

Question 4

Anti-isotypic antibodies - define

Answer 4

Polyclonal or monoclonal antibodies can be produced which bind to Fc regions of particular antibody classes e.g. to IgG’s, IgA’s etc. These are called anti-isotypic antibodies

Question 5

Immunoassays - define

Answer 5

immuno
uses antibody-antigen interaction (one of which is “labelled” or “tagged” to allow its detection)

assays
measures (amount, concentration) of antibody or antigen

very sensitive and specific
hence, widely used in research and analytical labs

Question 6

Immunoassays: the label - describe

Answer 6

originally radioactive
radioimmunoassay (RIA)
commonly now enzyme e.g. horseradish peroxidase or alkaline phosphatase -usually detected by coloured product (colorimetric)
enzyme-linked immunosorbent assay (ELISA)
other alternatives are luminescent

Question 7

Solid phase immunoassays: e.g. ELISA - function of in/direct vs sandwich

Answer 7

Direct/Indirect
Often used to quantify an antibody

Sandwich (Capture)
Often used to quantify an antigen

Question 8

The concentration of analyte (antibody or antigen) in the sample can be calculated by

Answer 8

Using these assays, the concentration of analyte (antibody or antigen) in the sample can be calculated by comparison to analyte standards of known concentration

Question 9

Direct ELISA - describe process

Answer 9

antigen immobilised on solid support
test antibody solution covalently linked to enzyme (e.g. Horseradish peroxidase or alkaline phosphatase for colorimetric ELISA) added
Enzyme substrate added, coloured product produced which can be measured by absorbance

Question 10

Direct ELISA - list uses

Answer 10

uses

• screen hybridoma supernatants
• detect exposure to infectious agent

Question 11

Indirect ELISA - describe process

Answer 11

antigen immobilised on solid support
primary antibody which binds to antigen is then added
Secondary antibody covalently attached to enzyme is subsequently added. Secondary antibody binds to Fc region of primary antibody
Enzyme substrate added, colour measured by absorbance

Question 12

Indirect ELISA - explain how the sensitivity of the test is increased

Answer 12

Secondary antibody is often polyclonal and so may bind to different epitopes on a primary antibody. This allows multiple secondary antibodies to bind to the same primary antibody thereby amplifying the signal and increasing the sensitivity of the test

Question 13

Sandwich (Capture) ELISA - describe process

Answer 13

Antigens may be present in low concentration. Because antibodies have high affinity for antigen this technique can concentrate the antigen
need two antibodies reacting with different epitopes on the antigen
one antibody immobilised on solid support
test antigen solution added, incubated and non-bound removed by washing
bound antigen detected by incubation with the other antibody, which has been labelled, and non-bound removed by washing

Question 14

Immunoassays: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western Blotting - uses

Answer 14

1) Can be used to detect antigens or antibodies
2) Used to measure size of the protein being analysed
3) Can be used to calculate protein concentration
4) May show if protein has been degraded

Question 15

What is used alongside ELISA

Answer 15

SDS PAGE/Western blotting often used alongside ELISA

Question 16

WB - describe measurement of [protein]

Answer 16

In WB, protein concentration can be measured by comparing intensity of band we are detecting to band from a protein standard of known concentration

Question 17

ELISA - steps if protein is degraded and why

Answer 17

If protein is degraded it may be more useful to use WB to calculate protein concentration,

as some of degradation fragments may contribute to signal in ELISA if both coating and detecting antibody are able to bind to them

Question 18

Antibody-antigen interaction: Flow cytometry and Fluorescence-activated cell sorter (FACS) analysis - describe process

Answer 18

Individual cells within a mixed 
population are tagged by treatment
with monoclonal antibodies which bind
to surface molecules and are labelled
with fluorescent dyes

Mixed cells are then forced through a
nozzle to form stream of single cells

Individual cells pass through a laser
beam which scatters light and causes dye to fluoresce and
provides information on bound antibody and cell surface protein

Question 19

Types of sample that are analysed by biomedical scientists

Answer 19

Blood serum 
Blood cells
Urine 
Synovial fluid
Saliva 
Mucus
Cerebrospinal fluid

Question 20

Types of disease

Answer 20

Transplant compatibility
Immunodeficiency
Autoimmunity
Allergy
Malignancy

Question 21

MHC alleles of donor and recipient are identified by

Answer 21

MHC alleles of donor and recipient are identified by Polymerase Chain Reaction

Question 22

X-linked agammaglobulinemia (XLA). X-linked disorder - define

Answer 22

X-linked agammaglobulinemia (XLA). X-linked disorder- inability to generate mature B cells. B cells have CD19 surface protein which is a co-receptor for the B cell receptor

Question 23

Flow cytometry in the clinic - monitoring HIV infection

Answer 23

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 a FACS machine

The results are reported as percentages and absolute counts

Question 24

Tracking T cell responses – fluorescent MHC complexes for antigen-specific T lymphocytes - describe process

Answer 24

MHC-peptide complexes made which bind specifically to the T cell receptor of appropriate MHC-peptide specific T cells. A fluorochrome e.g. phycoerythrin added and visualisation is by flow cytometry

By creating MHC complexes loaded with HIV antigen we can calculate for example what proportion of CD 8+ T cells will bind to the antigen

Question 25

Neutrophil deficiencies - explain generation of pus

Answer 25

Neutrophils are found in acutely inflamed tissue
Ingest pathogens and kill using reactive oxygen species
Rapidly die after phagocytosis which generates pus

Question 26

Deficiency in neutrophil numbers - effect

Answer 26

Deficiency in neutrophil numbers – neutropenia – high rate of infection

Question 27

Deficiency in phagocyte function - effect

Answer 27

Deficiency in phagocyte function; chronic granulomatous disease – patients cannot form reactive oxygen species, succumb to bacterial and fungal infections

Question 28

Measuring neutrophil function: Neutrophil Oxidative burst assay

Answer 28

This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA – phorbol myristate acetate) can be oxidized by reactive oxygen species (ROS),

produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry.

Question 29

Quantitation of antibodies: Nephelometry - describe

Answer 29

Electrophoresis
Nephelometry:an automated and rapid method used to measure serum immunoglobulin levels; it relies on the light-scattering properties of antigen-antibody complexes

Question 30

Quantitation of antibodies: Nephelometry - use

Answer 30

Nephelometry often used to study the amount of antibody from different classes present in serum e.g. IgG, IgA etc

Question 31

Skin Prick Test: - describe

Answer 31

Skin Prick Test: In allergic individual, IgE binds to allergen and via the Fc region of the antibody binds to receptors on mast cells.

This causes mast cells to degranulate causing the release of mediators (e.g histamine) which causes reddening and swelling of skin

Question 32

RAST (RadioAllergoSorbent Test) - describe

Answer 32

RAST (RadioAllergoSorbent Test)

The suspected allergen is bound to an insoluble material and the patient’s serum is added.

If the serum contains antibodies to the allergen, those antibodies will bind to the allergen.

Radiolabeled anti-human IgE antibody is added where it binds to those IgE antibodies already bound to the insoluble material.

The amount of radioactivity is proportional to the serum IgE for the allergen

Question 33

Immunofluorescence: - describe process

Answer 33

Immunofluorescence: serum added to human cell line. Then probed with fluorochrome-labelled anti-immunoglobulin antibody. Visualised by fluorescence microscopy

Question 34

Monoclonal antibodies can have their effects either by what

Answer 34

Monoclonal antibodies can have their effects either by binding and blocking a process or by mediating immune responses such as initiation of complement or antibody-dependent cell mediated cytotoxicity (ADCC)

Question 35

What do monoclonal antibodies bind to

Answer 35

Molecules such as toxins or radionuclides can be joined to monoclonal antibodies: antibody binds to cancer cell which is then killed by toxin or radioactivity