ImmunoRes

Question 1

Describe the production mAb

Answer 1

→inject mouse with antigen to produce Bcells
→cell culture myeloma line- lack HAT
→fuse myeloma and Bcells in polyethene glycol
→select hybrid cells using hypoxyanthine-aminopterin-thymidine(HAT)
→propagate desired clones

Question 2

Why is HAT medium used to culture myeloma cells?

Answer 2

→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 3

How long can hybridomas be store for?

Answer 3

→indefinitely

Question 4

What do immunoassays use?

Answer 4

→use polyclonal or monoclonal antibodies

Question 5

What is the common enzyme used to label antibodies?

Answer 5

→horseradish peroxidase
→alkaline phosphatase
→usually colometric

Question 6

What is an example of an immunoassay which uses horse radish peroxidase?

Answer 6

→ELISA

Question 7

What are the two types of ELISA?

Answer 7

→direct/ indirect

→sandwich (capture)

Question 8

What is direct/ indirect ELISA often used for?

Answer 8

→to quantify an antibody

Question 9

What is sandwich ELISA often used for?

Answer 9

→quantify an antigen

Question 10

What is direct ELISA mainly used for?

Answer 10

→screen hybridoma supernatants

→detect exposure to infectious agent

Question 11

What is immobalised on the solid in indirect ELISA?

Answer 11

→antigen

Question 12

Describe indirect ELISA process

Answer 12

→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 13

What is a feature of secondary antibody in indirect ELISA and why is this a benefit?

Answer 13

→polyclonal and so may bind to different epitopes on a primary antibody

→increases sensitivity

Question 14

Describe the process of sandwich ELISA

Answer 14

→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 15

What is Elispot?

Answer 15

→measuring cytokine secretion

Question 16

What can SDS-PAGE and Western blotting be used for?

Answer 16

→1) Can be used to detect antigens or antibodies

→Used to measure size of the protein being analysed

→Can be used to calculate protein concentration

→May show if protein has been degraded

Question 17

Which is method is more useful if protein has been degraded rather than ELISA?

Answer 17

→Western Blot

Question 18

Which chromosome is is the HLA gene found on?

Answer 18

→6

Question 19

How can antibodies to cell surface markers identified?

Answer 19

→flow cytometry

Question 20

What test is used for tracking Tcell responses?

Answer 20

→fluorochrome e.g. phycoerythrin added and visualisation is by flow cytometry

Question 21

What is disease involving neutrophil deficiency?

Answer 21

→chronic granulomatous disease (CGD)

Question 22

How to measure neutrophil function?

Answer 22

→Oxidative burst assay

→green fluorescent compound, which can be detected by flow cytometry

Question 23

What test is used to quantify antibodies?

Answer 23

→Nephelometry

→light-scattering properties of antigen-antibody complexes

Question 24

Why does a skin prick test work?

Answer 24

→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 25

What is a test for allergy diagnosis?

Answer 25

→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 26

What are autoimmune diseases characterised by?

Answer 26

→autoantibodies to nuclear antigens eg DNA, RNA

Question 27

How can SLE be diagnosed?

Answer 27

→Immunofluorescence

→serum added to human cell line

→Then probed with fluorochrome-labelled anti-immunoglobulin antibody.

→Visualised by fluorescence microscopy

Question 28

How can antibodies be used for therapy?

Answer 28

→Intravenous immunoglobulin (IVIG

→eg rabies

Question 29

How do mAbs exert their effects?

Answer 29

→binding and blocking a process

→mediating immune responses such as initiation of complement or antibody-dependent cell mediated cytotoxicity (ADCC)

→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