L8 - Antibody-based technologies

Question 1

What are antibodies?

Answer 1

Are secreted forms of BCRs

Bind free Ag

Are monoclonal – have a unique BCR – clonal selection

Question 2

What can antibodies bind?

Answer 2

They can bind almost all biological materials – proteins, CHO, nucleic acids

Their binding can be well characterised & often of very high affinity

Question 3

What is plasma?

Answer 3

The fluid phase of the blood

Question 4

What is serum?

Answer 4

Serum is the clotted fluid phase of the blood

Question 5

What is antiserum?

Answer 5

Serum from immunised person/animal known as antiserum

Question 6

What does antiserum contain?

Answer 6

Antiserum will contain antibodies that bind the Ag (as well as those that were present before the immunisation), along with other soluble blood components but doesn’t contain cells or clotting proteins

An antiserum may contain many different antibodies that bind the same Ag – even if the Ag is a single purified protein

Question 7

How can you purify different antibodies away from other serum proteins?

Answer 7

Gel filtration chromatography – separates based on MW

Affinity chromatography – separates using beads with antigens on
• Antibodies we want to purify will bind & be retained in the column
• Can then change the conditions to remove the antibodies off the beads & out the column

Question 8

Limitations of using antisera

Answer 8

Once the antiserum has been used, more antibodies generated will never be exactly the same

Question 9

How can we overcome the fact that antibodies produced by antisera will never be the exact same?

Answer 9

What if the individual B cell clones could be kept in culture to continuously produce a single antibody

Patients with multiple myelomas were known to produce large amounts of homogenous antibodies, although the specificity of the antibodies were unknown

Georges Kohler, Cesar Milstein & Niles Jerne devised a method for fusing mouse myeloma cells with B cells that were making antibodies of known specificity

MONOCLONAL ANTIBODIES

Question 10

Generation of monoclonal antibodies

Answer 10

1) Take a mouse
2) Immunise it with an antigen you want to have an antibody against (antigen A)
3) Harvest the spleen once the B cells have made antibodies to the antigen (after 2-6 weeks)
4) Fuse spleen cells with tumour cells with PEG - produces cells with 2 nuclei
5) Need a selection technique to select fusions which were from a B cell & an immortal B cell – the hybrids
6) Need to find the hybrid that’s making the correct antibody for the antigen
8) Can now make cell that grows forever & makes the antibody we want (hybridoma)

Question 11

Where can monoclonal antibodies be stored?

Answer 11

In liquid nitrogen

Question 12

What is phage display?

Answer 12

Even though animals can make up to a possible 10^11 different antibodies (predicted number based on number of heavy VDJ and light VJ/combinations/somatic possibilities), recombinant DNA technology now means that this number can actually be made using a technique called phage display

Question 13

How does phage display work?

Answer 13

Can take genes out of the B cells & put them into artificial systems

Uses phages to generate all the possible antibodies that can be made – artificial way to make even more antibodies

Question 14

What do methods involving antibodies rely on?

Answer 14

Many methods using antibodies rely on labels attached to the antibodies in order to detect them

Label should not affect antibody/Ag binding

Gives a signal when it interacts with a substrate

Many methods using antibodies use secondary antibodies to detect the primary antibody binding to its Ag

Question 15

Why are secondary antibodies used?

Answer 15

This increase the sensitivity

Has a multiplication effect

Same signal with half as much antigen – can detect smaller amounts of antibodies

Question 16

Production of secondary antibodies

Answer 16

1) Take mouse sera to act as the antigen
2) Purify the antibodies from the sera (from other mouse serum proteins)
3) Then inject them into another animal to immunise them (eg. rat)
4) Rat will make antibodies against the mouse antibodies
5) After 2-6 weeks collect & purify the rat anti-mouse antiserum
6) Now have rat anti-mouse polyclonal antibodies

Question 17

Uses of antibodies

Answer 17

In research & diagnostics – as antibody/interactions are specific, they make excellent tools for purifying, isolating and identifying biological molecules of interest

• Affinity chromatography
• Immunoprecipitation
• Immunofluorescence microscopy

Question 18

Affinity chromatography

Answer 18

Purifying biological molecules from a mixture

1) Can make columns with beads that have antibodies attached
2) If you put a mixture of proteins down the column, the antibodies will bind the protein you want to purify
3) All other proteins get washed through the column
4) Changing the pH allows antigen to be released
5) In an experiment the antigen can be the protein you’re working on, as the antibody recognizes it – doesn’t necessarily cause an immune response

Question 19

Immunoprecipitation

Answer 19

Isolating biological molecules from more limited supply of a mixture (eg. from metabolically labelled cells)

1) Take cells you think are making the protein & give them radioactive methionine
2) All the proteins the cells make becomes radioactive
4) Smash open the cell & put in an antibody that recognizes the protein
5) Binds the protein very specifically
6) All others don’t bind – can spin out the beads
7) Can run it on a gel – will give you a band on a piece of film as its radioactive

Question 20

Immunofluorescence microscopy

Answer 20

Identifying the location of a protein within a cell

1) Can put fluorochromes on them
2) If the fluorochrome is attached to the cell, when the laser hits the cell the fluorochrome will emit light which is picked up by a detector

Question 21

Flow cytometry (FACS)

Answer 21

Cells expressing different markers can be identified, distinguished & separated from each other

1) Take populations of mixed cells & add fluorescent antibodies to the cells
2) Go through a flow cytometer
3) If the cells have antibodies attached to them, the cells will fluoresce in the machine & be detected by the detector

Question 22

Magnetic bead isolation (MACS)

Answer 22

1) Label cells with antibodies
2) Instead of fluorochromes you attach metal beads to the antibodies
3) Put them down a column with magnets on
4) Cells bound to antibodies stick to the magnet
5) Magnetic field is removed to release the coupled cells from the column

Buy beads coated with antibodies to help purify the cells

Question 23

Western blotting

Answer 23

A commonly used research technique, where antibodies are required to recognize unfolded, denatured proteins is following their separation (based on MW differences) using SDS PAGE

Question 24

Enzyme-linked immunosorbent assay (ELISA)

Answer 24

Plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones

• Very common technique used both in research & diagnostic/clinical labs
• Works well for detection of both 3D epitopes & linear epitopes
• Very reproducible, easily automated for scaling up to screen many samples simultaneously
• Several different ELISA formats exist, that all rely on the ‘capture’ of Ag onto a solid surface

Question 25

Direct ELISA

Answer 25

1) Biological sample containing Ag added to well in plastic plate designed to bind proteins
2) Add labelled antibody specific for Ag
3) Add substrate for enzyme label
4) Measure substrate conversion (colour)

Question 26

Indirect ELISA

Answer 26

More sensitive

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

Question 27

Sandwich ELISA

Answer 27

Even more sensitive, uses less of limited sample

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