Week 4 - Immunological methods Flashcards
what is the immune system
- protective systems in mammals
- reacts to infective challenges
what are B cells
- 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
what are antibodies used for
antibodies are used to:
- purify proteins
- locate proteins within a cell
- quantify protein levels
why are antibodies labelled with fluorescent molecules
antibodies can be labelled with fluorescent molecules to locate proteins within:
- tissues ( immunohistochemistry)
- cells (immunocytochemistry)
what is the definition of an antibody immunoglobulin
antibody immunoglobulin definition:
protein synthesised by an animal in response to an antigen
how specific are antibodies and how do antibodies recognise antigens
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
what is the definition of antigen
antigen definition:
a substance producing an immune response that produces specific antibodies to it
what type of cells are antigens and what usually attacks it
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
what is the structure of an antibody
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
how do antibody-antigen interact
antigens bind to Fab domain end on antibody
antibody and antigen have complementary shapes, this allows large surface area interaction
What are the four types of bonding types between antigens and antibody
- hydrogen bonds
- electrostatic interactions
- Van Der Waals forces
- hydrophobic interactions
what is the definition of affinity and specificity
affinity: strength of interaction between antibody and antigen
specificity: degree of complementation between antibody and antigen
what is the affinity of antibodies to antigen
the affinity of antibody (Ab) to antigen (Ag)
1 epitope to 1 paratope
Ag + Ab ⇌ AgAb
what is the equilibrium constant K equation
K = [AgAb]/ [Ag] x [Ab]
what is the definition of auidity/functional affinity
the sum of individual affinities within an antibody
how is antibody produced
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
what are polyclonal antibodies
polyclonal antibodies are when B lymphocytes produce many different antibodies for the same antigen, meaning each antibody binds to a different epitope
what are monoclonal antibodies
monoclonal antibodies are identical antibodies to the antigen
they are produced by a clone of cells all derived from a single B lymphocyte
who developed monoclonal antibody production
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
what is PEG
what is a fused cell
PEG: A fusogen
Fused cell: Hybrifoma
where are cells that produce target antibodies purified and grown
cells producing target antibodies are purified and grown in vivo or in vitro
what is the step to step process on how antibodies are made
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
where are monoclonal antibodies used
- 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
how is antibody concentration or activity measured
antibody concentration or activity is measured by the interaction with the antigen
what happens when you combine antibodies with antigen in certain proportions
you get a lattice structure:
- if antigen originally soluble = precipitation
- if antigen cellular/particular = agglutination
what happens when optimal proportions of antibody and antigen are not used
if optimal proportions of antibody and antigen are not used then you get:
- excess antibody which causes cell lysis
- excess antibody inactivates the virus
what is immunoprecipitation (single protein)
IP: affinity purification of antigens
antibodies bound to inert beads, they are free in solution and thus not packed in a column
what is the batch process in immunoprecipitation (single protein)
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
what magnetic metal is used in immunoprecipitation (single protein)
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
what antibody-binding proteins are used in immunoprecipitation (antibody immobilisation)
antibody binding proteins are:
- proteins A, G, AIG, L
- affinity with heavy chains
protein AIG beads + antibody (IgG) = affinity-bound
immobilised antibody
what is covalent antibody immobilisation
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
where is co-immunoprecipitation used
it is used to study interactions of primary antigen proteins with other proteins. thus protein-protein interactions
where is CHIP: chromatin immunoprecipitation used
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
what is tagged-protein (IP)
-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
what is the direct antibody binding method
its when 1 antibody with conjugate is involved
advantages:
-saves time-less steps in the protocol
disadvantage:
- expensive
what is the indirect antibody binding method
its when primary and secondary antibodies are involved
a secondary antibody with conjugate
advantage:
- signal amplification = good staining
disadvantage:
- non specific binding = background staining
what conjugates and enzymes are used in the direct and indirect binding methods
-conjugates:
fluorescent dyes
e.g. fluorescein
-enzymes:
e.g. horseradish peroxidase
how is immunoblotting (Dot Blots) carried out
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
what is western blotting and where is it used
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
who developed immunoassays and what does it measure
it was developed by Rosalyn Yalow and Solomon Berson in 1960
is used anti-insulin antibodies to measure levels of insulin in plasma
what do immunoassays quantify and what are the different types of immunoassays
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
what happens when you add horseradish peroxidase to OPD (substrate)
horseradish peroxidase + OPD (substrate) –> product
colourless –> yellow product
you can measure the colour change of the product using a spectrometer
what is Direct ELISA used to test and its basic principle to carry out the test
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
what is indirect ELISA/ sandwich ELISA used to test
what are its basic principle to carry out the test
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
what is competitive ELISA used to test
what are its basic principle to carry out the test
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
what are the advantages and disadvantages of the 4 basic types of ELISA
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)
what are the applications of ELISA and why
> 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
