Week 4 - Immunological methods

Question 1

what is the immune system

Answer 1

• protective systems in mammals
• reacts to infective challenges

Question 2

what are B cells

Answer 2

• they are derived from stem cells in the bone marrow
• they produce antibodies in response to the presence of foreign (‘non-self’) substances in tissues

Question 3

what are antibodies used for

Answer 3

antibodies are used to:
- purify proteins
- locate proteins within a cell
- quantify protein levels

Question 4

why are antibodies labelled with fluorescent molecules

Answer 4

antibodies can be labelled with fluorescent molecules to locate proteins within:
- tissues ( immunohistochemistry)
- cells (immunocytochemistry)

Question 5

what is the definition of an antibody immunoglobulin

Answer 5

antibody immunoglobulin definition:
protein synthesised by an animal in response to an antigen

Question 6

how specific are antibodies and how do antibodies recognise antigens

Answer 6

antibodies are highly specific and they have a high affinity for the corresponding antigen.
antibodies have a paratope that recognises an epitope on a antigen.
an epitope typically has 15 amino acids. an epitope is a group of amino acids on an antigen

Question 7

what is the definition of antigen

Answer 7

antigen definition:
a substance producing an immune response that produces specific antibodies to it

Question 8

what type of cells are antigens and what usually attacks it

Answer 8

antigens are:
- polypeptides
- proteins
- polysaccharides

other parts of the immune system will destroy antibody-labelled antigens, e.g.
T lymphocytes attack/destroy antibody-labelled cells

Question 9

what is the structure of an antibody

Answer 9

antibody consists of 4 chains:
- 2 heavy chains
- 2 light chains

heavy and light chains are linked by disulphide bonds
heavy and light chain combine = Fab domains
antigen-binding sites are at the end of Fab domains
2 heavy chains form the FC domain
Fab domains linked to FC domain by flexible linkers

Question 10

how do antibody-antigen interact

Answer 10

antigens bind to Fab domain end on antibody
antibody and antigen have complementary shapes, this allows large surface area interaction

Question 11

What are the four types of bonding types between antigens and antibody

Answer 11

• hydrogen bonds
• electrostatic interactions
• Van Der Waals forces
• hydrophobic interactions

Question 12

what is the definition of affinity and specificity

Answer 12

affinity: strength of interaction between antibody and antigen

specificity: degree of complementation between antibody and antigen

Question 13

what is the affinity of antibodies to antigen

Answer 13

the affinity of antibody (Ab) to antigen (Ag)
1 epitope to 1 paratope

Ag + Ab ⇌ AgAb

Question 14

what is the equilibrium constant K equation

Answer 14

K = [AgAb]/ [Ag] x [Ab]

Question 15

what is the definition of auidity/functional affinity

Answer 15

the sum of individual affinities within an antibody

Question 16

how is antibody produced

Answer 16

1- antigen injected in
to host cells
2- increased production of B lymphocytes in lymph nodes and spleen
3- lymphocytes produce antibody to antigen
4- primary response - the first time the animal has encountered the antigen
5- secondary response - antigen has already been encountered previously
6- blood is removed from the immunised animal and the blood is centrifuged. the centrifuge separates the blood cells from the serum. the antiserum contains antibodies produced by animals.
7 - target antibodies purified from antiserum using affinity chromatography
8- resulting antibody is donated by saying the source of the antibody and the source of the antigen. e.g. rabbit anti-mouse

Question 17

what are polyclonal antibodies

Answer 17

polyclonal antibodies are when B lymphocytes produce many different antibodies for the same antigen, meaning each antibody binds to a different epitope

Question 18

what are monoclonal antibodies

Answer 18

monoclonal antibodies are identical antibodies to the antigen
they are produced by a clone of cells all derived from a single B lymphocyte

Question 19

who developed monoclonal antibody production

Answer 19

it was developed by:
- Georges Kohler
- Casar Milstein
- Niels Jerne

they obtained large amounts of homogenous antibodies by fusing an antibody-producing cell with an immortal myeloma cell

Question 20

what is PEG
what is a fused cell

Answer 20

PEG: A fusogen
Fused cell: Hybrifoma

Question 21

where are cells that produce target antibodies purified and grown

Answer 21

cells producing target antibodies are purified and grown in vivo or in vitro

Question 22

what is the step to step process on how antibodies are made

Answer 22

Antigen injected in a rat cell-culture myeloma line
↓ ↓
Spleen cells myeloma cells
↳ Fuse in polyethene glycol ↲
↓ select and grow hybrid cells
↓ select cells making antibodies of desired
specificity
propagate desired cells ⇆ freeze/thaw
↙ ↘
grow in mass culture inject induce tumours
↓ in rat
Antibody ↓
Antibody

Question 23

where are monoclonal antibodies used

Answer 23

• chromatography
  >affinity columns for protein purification
• clinical/medical
  > blood for transfusion
  > immunotherapy for cancer
  -research/diagnostics
  > ELISA
  > western blot assay
  > immunocytochemistry/immunohistochemistry
  > fluorescent microscopy
  > flow cytometry

Question 24

how is antibody concentration or activity measured

Answer 24

antibody concentration or activity is measured by the interaction with the antigen

Question 25

what happens when you combine antibodies with antigen in certain proportions

Answer 25

you get a lattice structure:
- if antigen originally soluble = precipitation
- if antigen cellular/particular = agglutination

Question 26

what happens when optimal proportions of antibody and antigen are not used

Answer 26

if optimal proportions of antibody and antigen are not used then you get:
- excess antibody which causes cell lysis
- excess antibody inactivates the virus

Question 27

what is immunoprecipitation (single protein)

Answer 27

IP: affinity purification of antigens
antibodies bound to inert beads, they are free in solution and thus not packed in a column

Question 28

what is the batch process in immunoprecipitation (single protein)

Answer 28

Batch (not continuous) process:
- each incubation or wash step followed by a centrifugation or magnetic step
agarose beads are used
porous = large surface area for antibody binding
used with centrifugation

Question 29

what magnetic metal is used in immunoprecipitation (single protein)

Answer 29

it has a smaller surface area/bead
but more beads= larger surface area for antibody binding
used with magnets:
-moves beads to the side/bottom of the tube
- the preferred material for beads

Question 30

what antibody-binding proteins are used in immunoprecipitation (antibody immobilisation)

Answer 30

antibody binding proteins are:
- proteins A, G, AIG, L
- affinity with heavy chains

protein AIG beads + antibody (IgG) = affinity-bound
immobilised antibody

Question 31

what is covalent antibody immobilisation

Answer 31

it is a chemical group of beads that reacts with the amine group on the antibody.
bonding is permanent; beads reused

surface-activated beads + amine ligand (antibody) = covalently
immobilised
antibody

Question 32

where is co-immunoprecipitation used

Answer 32

it is used to study interactions of primary antigen proteins with other proteins. thus protein-protein interactions

Question 33

where is CHIP: chromatin immunoprecipitation used

Answer 33

it is used to:
-identify regions of the genome with which DNA-binding proteins associate.
e.g:
- histones
- transcriptional factors
- target proteins are immunoprecipitated with cross-linked nucleotide sequences
- DNA removed for analysis

RIP - RNA immunoprecipitation
similar to CHIP, but nucleic acid is RNA

Question 34

what is tagged-protein (IP)

Answer 34

-if no antibody is available for target proteins
-antibodies are engineered with epitope for antibody binding
- Tags:
~short peptide sequence, e.g.
> FLAf amino acid sequence: DYKDDDDK

Question 35

what is the direct antibody binding method

Answer 35

its when 1 antibody with conjugate is involved
advantages:
-saves time-less steps in the protocol
disadvantage:
- expensive

Question 36

what is the indirect antibody binding method

Answer 36

its when primary and secondary antibodies are involved
a secondary antibody with conjugate
advantage:
- signal amplification = good staining
disadvantage:
- non specific binding = background staining

Question 37

what conjugates and enzymes are used in the direct and indirect binding methods

Answer 37

-conjugates:
fluorescent dyes
e.g. fluorescein
-enzymes:
e.g. horseradish peroxidase

Question 38

how is immunoblotting (Dot Blots) carried out

Answer 38

1- sample is spotted onto a nitrocellulose/PVDR membrane and dried
2- membrane incubated in blocking buffer
3- primary/secondary or conjugated antibodies added and incubated
4- dots visualised with chemiluminescence (enzymes)
larger, darker dots = more antigen present

Question 39

what is western blotting and where is it used

Answer 39

it is a qualitative/semi-quantitative assay for single proteins in a mixture
it is used as a test for:
- HIV
- Hepatitis B
- BSE/CJD
- Lyme disease

Question 40

who developed immunoassays and what does it measure

Answer 40

it was developed by Rosalyn Yalow and Solomon Berson in 1960
is used anti-insulin antibodies to measure levels of insulin in plasma

Question 41

what do immunoassays quantify and what are the different types of immunoassays

Answer 41

immunoassays quantify the target protein/antigen in the sample.

most immunoassays are variations of ELISA
there are 4 basic types:
- direct ELISA
- indirect ELISA
- sandwich ELISA
- competitive ELISA

Question 42

what happens when you add horseradish peroxidase to OPD (substrate)

Answer 42

horseradish peroxidase + OPD (substrate) –> product
colourless –> yellow product

you can measure the colour change of the product using a spectrometer

Question 43

what is Direct ELISA used to test and its basic principle to carry out the test

Answer 43

Direct ELISA is used to test for an antigen in a patient’s serum
basic principle:
1- microtitre plate incubated with patient serum
if present, the antigen will attach
2- antibody to target antigen added
the antibody is labelled with an enzyme (conjugated antibody)
3- substrate to the enzyme added
it is a signal (colour change) to indicate if the target antigen is present
4- wash step in buffer
you need to wash between each stage to remove any unbound reagent

Question 44

what is indirect ELISA/ sandwich ELISA used to test
what are its basic principle to carry out the test

Answer 44

it is used to test for an antigen in the patient’s serum
basic principle:
(this step only occurs in sandwich ELISA) 1- microtitre plate pretreated with antibody to target antigen
(step 2 for sandwich ELISA/ step 1 for indirect ELISA) 1/2- the microtitre plate is incubated with patient’s serum
- if present the target antigen will attach
2/3 - antibody to target antigen added
3/4 - secondary antibody (labelled with enzyme) added
4/5 - substrate to an enzyme added
- signal (colour change) indicates the presentence of the target antigen
5/6 - wash step in buffer
- wash between each stage to remove any unbound reagent

Question 45

what is competitive ELISA used to test
what are its basic principle to carry out the test

Answer 45

it is used to test for an antigen in the patients serum
basic principle:
1- microtitre plate pre-treated with antibody to target antigen
2- known quantity of enzyme-conjugated antigen + patients serum added to plate
3- artificial and natural antigens compete for binding to the antibody
more natural antigen = less artificial antigen bound to plate
4- substrate of the enzyme added
the lower the signal colour change is, the greater the quantity of natural antigen present
5- signal is compared with the signal from assay wells containing labelled antigens only
6- wash step in buffer
wash between each stage to remove any unbound reagent

Question 46

what are the advantages and disadvantages of the 4 basic types of ELISA

Answer 46

Direct ELISA
advantage: disadvantage:
- fast technique - poor sensitivity

Indirect ELISA (antigen and antibody detection
advantage: disadvantage:
- greater sensitivity - a slower technique
(in comparison to direct ELISA) (in comparison to Direct ELISA)

Sandwich ELISA
advantage: disadvantage:
- very sensitive - very expensive
(up to 6x more sensitive than direct and indirect ELISA)

competitive ELISA
advantage: disadvantage:
-ideal for small antigens that cannot - time-consuming
bind 2 antibodies (as in sandwich ELISA)

Question 47

what are the applications of ELISA and why

Answer 47

> Diagnostic/clinical testing
it detects antigens of infectious agents or antibodies against them in body fluids, e.g:
- HIV
- HBV (Hepatitis B virus)
- thyroxine ( to diagnose hypo-or-hyperthyroidism)
- Lyme disease
- TB

> Food Science
to detect contamination by food allergens

> Toxicology
to detect human growth hormone in athlete’s blood samples