immunoaffinity Flashcards
define epitope
the targeted molecular structure of a specific antibody
Describe the structure of antibodies
.Heavy chains inside, light chains .Biological activity in base .2 Fab fragments for binding-terminated by N terminals called variable regions .A disulfide bond hinge region . Divalent ( can bind two antigens)
effect of papain and pepsin on antibody
.papain cleaves hinge region to leave two detached fab fragments and constant region
. pepsin cleave below hinge region leaving attached fab fragments. Fragments the constant region
How is diversity in antibodies produced?
.Somatic recombination of genes coding for VDJ region.
. RNa splicing
Polyclonal antibody
a collection of antibodies from different B cells that recognise multiple epitopes on the same antigen
Monoclonal antibody
derived from a single antibody producing B call- only bind to one unique epitope
Considerations of using polyclonal antibodies
.cheap to produce
.tolerant of small changes in protein structure
Considerations of using monoclonal antibodies
.Expensive to produce
.Highly specific for protein form
What is the purpose of excess antibody with respect to antigens in immuno affinity chromatography?
Favors monovalent binding-why is this good? Monovalent bonding means that majority of antigen is bound.
characteristics of good binding matrix
.Provide mechanical/physical/chemical stability
.Acceptable pressure drop
. Exhibit minimal non-specific binding
.Provide surface area of Ab-An interactions.
Classes:
.natural polymers
.Synthetic polymers
.inorganic materials
Describe mechanism of low pH elution strategy for IAC
Disrupts ionic bonds
comment: proteins have to be pH stable
Describe mechanism of chaotropic salts elution strategy for IAC
interferes with stabilizing intra-molecular interactions. High salt concentration disrupts water molecules around the affinity interaction
Describe mechanism of organic solvent elution strategy for IAC
Hydrophobic effects- signifcant elution of smaller molecules, efficient column regeneration
Describe mechanism of organic solvent elution strategy for IAC
Hydrophobic effects- significant elution of smaller molecules, efficient column regeneration
Describe mechanism of displacer elution strategy for IAC
competition- exploits high affinity interactions. Can promote bio-specific interactions because of mild conditions. disadvantage: longer elution time.
Describe mechanism of displacer elution strategy for IAC
competition- exploits high affinity interactions. Can promote bio-specific interactions because of mild conditions. disadvantage: longer elution time.
What does ELISA stand for
Enzyme linked immunosorbent Assay
examples of foreign antigens
foreign proteins, viruses, environmental pollutants ( tattoo ink, smoke..), bacteria and parasites, foreign transplanted tissue
Why did we stop using radio immuno assay
.safety
.produces radioactive waste
.short shelf life
indirect ELISA method
- Bind known antigen to wells of microtiter plate
- Block unnocupied are bound by solution of non-interacting proteins.
- Continue…
Difference between direct and indirect ELISA
in direct elisa, the antibody that binds to the target antigen in enzyme linked- whilst in indirect elisa, a secondary antibody binds to the primary antibody, and the secondary enzyme is the one that is enzyme linked
advantage of indirect elisa over primary elisa
.to identify an antigen, the primary antibody must be specific, hence monoclonal antibodies are used, which are expensive to manufacture. in indirect antibody, these monoclonal antibodies can be reused, as the “single-use” enzyme reaction is done by a secondary antibody.
.Because multiple secondary antibodies can bind to one antibody, this leads to signal amplification. Hence, indirect is more sensitive than direct.
purpose and method of sandwich ELISA
Sandwich ELISAs capture and detect antigens. Antigen sample is loaded into antibody coated well. enzyme-bound antibody is then added.
Advantages of sandwich ELISA
sample doesn’t require prior purification, 2-5 times more sensitve than direct/indirect ELISA
structure of enzyme-bound Ab
biotin bound to enzyme label via avidin or streptavidin in a tetravalent manner. enzyme labels:
.Horse radish peroxidase
. Alkline phosphatase
List 4 uses of antibodies in biotech
- ELISA
- Immunofluorescence microscopy
- western blotting
- Immunoprecipitation
What does immunofluorescence microscopy allow us to visualize?
The abundance and distribution of antigens. The use of different colours can identify different antigens in a biological system
What does western blotting allow us to do?
- Verify the expression of a protein
- determine the relative amount of a protein present in different samples
- Analyze protein-protein interactions.
Western blot method
- separate proteins via SDS or native PAGE electrophoresis
- Transfer proteins to the membrane
- block non-specific sites on membrane
- incubate with primary Ab, wash\
- incubate with secondary antibody, wash
- detect secondary antibody
Reaction with enzyme to produce light
HRP catalyses breakdown of H202 to H20 and O2
luminol is oxidized and emits light
captured on film
Main purpose of protein complex immunoprecipitation
identification of protein interaction partners ( ie other proteins, ligands, co-factors…) to a protein of interes. Non-ionic denaturation is required as to not disrupt these interactions
Purpose and functioning of chromatin immunoprecipitation
Used to determine the location of specific protein-DNA binding sites on the genome.
uses a bead bound to an IgG specific to a putative DNA binding protein, can precipitate out Dna segment of interest
