Antibody Technologies

Question 1

What are monoclonal antibody

Answer 1

Ab from the same B cell for the same ag are all the same so are monoclonal (b cells are clonally distributed via allele exclusion)

Question 2

Why are ab easy to purify

Answer 2

They are released from plasma cells in high amounts

Question 3

What do ab bind to with high affinity

Answer 3

Can have ability to bind to any biological molecule = good for research or diagnosis

Question 4

What is the fluid phase of blood called when it is centrifuged to remove blood cells

Answer 4

Plasma (sits on top)

Question 5

What is removed from plasma (after centrifugatjon of blood) to form serum

Answer 5

Blood clotting agents

Question 6

What is serum from a person who’s been immunised with an ag called

Answer 6

Anti sera

Question 7

What does antiserum have

Answer 7

Many soluble proteins and also many types of antibody for the same ag

Question 8

How can anti sera be polyclonal if exposed to only 1 ag

Answer 8

Many B cells react to same ag, produce diff antibodies which bind diff epitopes on the ag

Question 9

How are ab separated from other soluble proteins in anti sera

Answer 9

Gel filtration chromatography (size exclusion)

Or

Affinity chromatography

Question 10

How does gel filtration chromatography separate ab from anti sera proteins

Answer 10

Ab are larger in mw so will elute from column later

Question 11

What is added to column in affinity chromatography to separate ab from anti sera

Answer 11

The ag the person was first immunised with

Question 12

What is anti sera called bc many types of ab

Answer 12

Polyclonal anti sera

Question 13

What is the issue with polyclonal anti sera (reason monoclonal ab produced)

Answer 13

You would want to separate diff types of ab for the ag but can’t separate them bc same mw and also same affinity for ag

Question 14

Which B cell tumours were discovered to grow forever and can’t release of monoclonal ab (further used to produce ma)

Answer 14

Multiple myelomas

Question 15

What was taken from immunised mice and fused with multiple myelomas to produce hybridoma cells

Answer 15

B cells which produce ab for the ag the mouse was immunised with

Question 16

Which ab would be made from the myeloma and immunised B cell mix

Answer 16

The antibodies specific for the ag immunised by mouse, the myelomas used don’t produce any ab but have ability to grow forever and produce them

Question 17

How are myeloma cells killed if they survive after trying to produce hybridomas

Answer 17

They are transferred to media which kills cells which don’t have a specific enzyme producing dna, myelomas don’t have the enzyme

Question 18

Why won’t B cells be present if they haven’t fused with myeloma to form hybridoma

Answer 18

They die in tissue culture

Question 19

How is the ab selected in ma production

Answer 19

The best ab will be tested through binding tests to the ag

Question 20

How are MA when produced for research detected

Answer 20

Via primary or secondary antibodies which are labelled via Fluorochrome or enzyme substrate reaction

Question 21

Why is using secondary antibody immunofluorescence better

Answer 21

Produces a stronger signal because chances of ab binding to are higher

Question 22

What mustn’t labelling affect

Answer 22

Binding ability of ab to the samples

Question 23

How are secondary antibodies produced which bind primary MAs

Answer 23

Antibodies produced from immunised mouse sera are purified from sera eg via GF chromatography

Transferred into a rat for example which is immunised with the antibodies

The rat then produced antibodies for the antibodies injected

The rat antibodies are purified from its anti sera = secondary ab

Question 24

How can ab be used in research to purify / seperate molecules

Answer 24

Affinity chromatography

If ma are made which are specific to the molecule of interest they are bound to column and separate the molecules

Question 25

How does macs work (magnetic bead isolation of molecules)

Answer 25

Magnetic beads with antibodies allow ab to only bind to molecules if a magnet is present The molecules elute when magnet is removed

Question 26

How does immunoprecipation for proteins work using ab

Answer 26

Proteins are radiolabelled due to Met aa, Cells lyse and release these proteins Ab are added and then removed , the protein bound is run on sds gel And visualise under light

Question 27

How does immunofluorescence work to find if cells have ag

Answer 27

Using microscopy you bind ab to a cover slip and the ab has fluorochrome on it The light will show where ab has bound on the cells or if it has. = cell has ag

Question 28

How is western blot used for diagnostics or to detect level or protein (similar to immunoprecipitation)

Answer 28

Sds done first Anti sera then added and ab will detect the protein of interest via binding on a membrane Ab usually seen by light due to enzyme (in immuoprecipitation the protein is radiolabelled and run on gel after )

Question 29

Why is Elisa used

Answer 29

To detect proteins with a low sample size

Question 30

What are the 3 types of Elisa

Answer 30

Direct (using primary ab) Indirect (using secondary ab too) Sandwich (binds capture ab then sample added then a secondary antibody used)

Question 31

What are the labels used for Elisa (same as western blot)

Answer 31

Enzymes Substrate colour change

Question 32

Which technique is used to characterise cells from eg a blood sample

Answer 32

Flow cytometry

Question 33

What does the laser measure when cells move through column in flow cytometry

Answer 33

The patterns of light scattering

Question 34

What does forward scatter mean

Answer 34

The scatter which depends on the size of a cell Eg macrophages are large so have a large forward scatter to others

Question 35

What does side scatter measure in cells

Answer 35

Their granularity levels (low in lymphocytes but high in eg neutrophils)

Question 36

What type of graph separates the cells depending on the ss and fs results

Answer 36

A dot plot

Question 37

What is the term used to describe the focus on one type of cell in flow cytometry after the initial Fs and ss analysis

Answer 37

Gating using a computer eg gate lymphocytes from macrophages and neutrophils (Low fs and ss)

Question 38

What is added after the first gating to fully characterise cells

Answer 38

Antibodies with fluorochrome Antibodies will be specific for cell type specific molecules eg cd19 ab binding = it’s a B cell Cd3 antibody binds both t and B cell

Question 39

Is just one ab used

Answer 39

No , different ab used with diff fluorochrome colours so fluoresce at diff wavelengths of light to detect diff cell types from diff laser detectors So could use two at a time in first gate eg both cd3 and cd19 antibodies added

Question 40

Another gate is done after ab binding. How

Answer 40

You gate positive cells ie cells which ab has bound to Eg cd3 antibody will have positive B cells only in the second gate

Question 41

What is the issue with using diff ab fluorochrome at a time

Answer 41

Fluorochromes have cross over at some wavelengths. This can give false positive or negative results for cells

Question 42

What is the solution to cross over of fluorochromes

Answer 42

Compensation - A control is done where cells only bind 1 ab at a time which should be picked up by 1 laser detector. The levels of defection by detector 2 are deleted because this is false (The control is done with the other detector fluorochrome too)

Question 43

Why can’t discontinuous / conformation epitomes be used in immunoprecipitation or western blot

Answer 43

Because the proteins are denatured on the sds page removing secondary structures so ab won’t be able to bind on gel