CMB2004/L12 Antibody-Based Technologies

Question 1

Where are antibodies found following immunisation with a pathogen or purified Ag?

Answer 1

Fluid phase of blood/Plasma

Question 2

What is serum?

Answer 2

Plasma once the blood clot is removed

Question 3

What is antiserum?

Answer 3

Serum from immunised person/animal
Contain antibodies that bind Ag and other soluble blood components (GF, proteins)
No cells or clotting proteins

Question 4

How can multiple different antibodies bind the same Ag?

Answer 4

Bind different regions (epitopes) of same Ag

Question 5

Give 2 methods used to purify different antibodies away from other serum proteins.

Answer 5

Gel filtration chromatography (on MW)
Affinity chromatography

Question 6

Give 2 limitations of using purified/polyclonal antiserum.

Answer 6

Individual Ab separation would be better
Once used, another individual needs immunising and Ab generated won’t be exactly the same

Question 7

How can individual plasma cells be kept in culture to continuously produce a single antibody? (3)

Answer 7

Patients with myelomas (plasma cell tumour) produce lots of homogenous Ab with unknown specificity
Mouse myeloma cells (don’t make Ab anymore) fused with mouse plasma cells that make Ab of known specificities
Generate hybridomas

Question 8

How were monoclonal antibodies generated in 1975? (6)

Answer 8

Spleen cells producing antibody from mouse immunised with antigen A
Myeloma cells (immortal) lacking antibody secretion and enzyme HGPRT
Mix and fuse cells with PEG
Transfer to HAT medium: hybridomas proliferate; mortal spleen cells and unfused HGPRT-myeloma cells die
Select hybridoma that makes Ab for antigen A
Clone selected hybridoma

Question 9

Why are labels attached to antibodies?

Answer 9

To detect them once bound to Ag
Detection of a labelled or fluorochrome Ab allows confirmation of presence of Ag A

Question 10

What is the effect of using a secondary antibody to detect primary antibodies binding to Ag?

Answer 10

Increases sensitivity

Question 11

How would rat anti-mouse polyclonal antibodies be collected? (6)

Answer 11

Production of secondary Ab
Collect mouse sera
Purify mouse Ab
Immunise rat 2-3x
Collect rat anti-mouse antiserum
Purify rat anti-mouse polyclonal antibodies from other rat serum proteins

Question 12

Why are antibodies particularly useful in research and diagnostics?

Answer 12

Specific
Make excellent tools for purifying, isolating and identifying biological molecules of interest

Question 13

How would biological molecules be purified from a mixture (affinity chromatography)? (4)

Answer 13

Ab to Ag A bound to beads
Add mixture of molecules
Wash away unbound molecules
Elute specifically bound molecules

Question 14

Which method would be used to identify the location of a protein within a cell?

Answer 14

Immunofluorescence microscopy

Question 15

Describe Western blotting. (5)

Answer 15

Dissociate HIV in SDS (lyse virus)
Use SDS-PAGE to separate viral proteins based on MW
Transfer separated proteins to membrane and incubate with monoclonal Ab
Detect bound antibody with enzyme-linked anti-Ig

Question 16

What is ELISA used for?

Answer 16

Enzyme-linked immunosorbent assay
Detecting presence of specific biological material in wide range of complex samples
Very reliable, sensitive, easily automated for scaling up
Works with polyclonal sera but better with Mabs

Question 17

Describe direct ELISA. (3)

Answer 17

Biological sample containing Ag added to well in plastic plate designed to bind proteins
Add labelled Ab specific for Ag
Add substrate for enzyme label

Question 18

Describe indirect ELISA. (4)

Answer 18

Biological sample containing Ag added to well in plastic plate
Add unlabelled primary antibody specific for Ag
Add labelled secondary then substrate for enzyme label
Measure substrate conversion (colour)

Question 19

Describe sandwich ELISA. (4)

Answer 19

Unlabelled antibody specific for Ag added to plate (capture antibody)
Add sample
Add a different Ag specific labelled secondary then substrate
Measure substrate conversion (colour)

Question 20

Describe flow cytometry (FACS). (2)

Answer 20

-Technique used for characterisation of cells based on light scattering properties
-Properties either natural or induced by pre-incubation of cells with antibodies labelled with dyes that fluoresce
Measures how single cells in population affect laser beam as they pass through
Very rapid
Can measure multiple parameters at once

Question 21

Give 2 natural effects of cells passing through a laser beam.

Answer 21

Forward scatter results from cell’s size
Side scatter from a cell’s granularity

Question 22

How can immune cells be identified? How is this useful?

Answer 22

By expression of various molecules
Antibodies specific for these molecules labelled with fluorescent dyes and used to identify antibody positive cells in mixed population
Dye emits light when stimulated by flow cytometer laser

Question 23

How is data generated from a flow cytometer? (3)

Answer 23

Analyse labelled cells for FSC/SSC then set gate for cells of interest
Level of FITC fluorescence measured (log10) just on cells in FSC/SSC gate
Second gate used to define sub-population of cells based on fluorescence