Antibodies Flashcards
Differences between an innate and adaptive response?
Innate is fast, non specific. Uses phagocytes and natural killer cells, and humoral complement system and cytokines
Adaptive; slow response, highly specific, one term memory and involves T helped cells, cytotoxic T cels and humoral B cells
What is the structure of antibodies?
2 heavy and 2 light chains. Each has a variable region with 2 antigen binding sites. and a constant domain which remains the same. 4 polypeptide chains joined covalently.
Wha is the complementarity determining region?
Region within the antigen binding site which is hyper variable.
What is an epitope?
Area of an antigen that is detected by the immune system and binds to the antigen binding site of antibodies.
How do antibodies and antigens bind?
antigen binding site binds non covalently to epitope: H bonds, electrostatic attractions, VdWs.
What are the key features of a polyclonal immune response?
Diverse and specific.
Why are antigens good reagents? (3)
They have a high specificity, a good avidity (able to bind to any molecule, even if synthetic) and don’t require a carrier molecule.
What should you consider when creating polyclonal antisera? (4)
Cost, amount of antigen available, amount of antibody produced, phylogenetic relationship (greater phylogenetic distance means increased immune response - more product)
How are polyclonal antisera produced?
immunisation activated B cells and a booster injection several weeks later increases antibody production and allows class switching to IgG. after 4-8 weeks the blood is collected and serum containing IgG is collected.
Why can overproduction of monoclonal antibodies be dangerous?
can cause uncontrolled growth of a single B lymphocyte - tumours form.
How are monoclonal antibody reagents grown?
isolation of a B cell and growth in vitro - only one antibody grown.
Why are monoclonal antibodies useful as a reagent? (4)
High speciality, low cross receptivity with unrelated Ag, inexhaustible supply, commercial applications in diagnostics and vaccinations.
How are monoclonal antibodies produced?
an animal is immunised with the antigen of choice, after the booster shot, the B cells producing the antibodies are collected.
B cells from a tumour are also collected - these cells do not produce Ab’s.The cancerous B cells and normal cells are fused in a gel, and immortalised.
The cells are then treated with a drug that will kill the cancerous cells - killing off any unfused cells. the fused cells are immortal from both drug treatment and native B cells as they are both cancerous and native.
These fused hybridomas are grown in cultures and tested for specific antibody by testing against Ag.
Thus creating a monoclonal antibody as derived from a single B cell.
What is huminisation?
Where the antibody derived from the animal is combined with the constant region from a human antibody - making it useable in humans.
What is biopharming in relationship to antibodies?
Where plants are genetically modified to express antibodies.
What are the advantages of plantibodies?
More efficient expression, cheaper purification, stable storage in seeds, can be post translationally modified- improve activity.
Outline the process of immunoblotting.
Proteins are denatured and separated on a polyacrlyamide gel using a denaturing agent and electric current. This separates proteins by length and charge.
The proteins are then stained in a gel to visualise them, they are transferred to a nitrocellulose sheet by an electric current- either wet or semidry transfer. Known marker proteins can show the length of proteins.
On the nitrocellulose sheet the proteins are hybridised to a specific labelled antibody, washed and hybridised to a secondary antibody specific to the primary. the secondary antibody is conjugated to a marker which allows you to visualise the protein.
What markers are used on the secondary antibody to visualise the proteins?
Fluorescence, cheti luminescent or a dye.
Uses of immunoblotting? (3)
Determines size of protein.
semi quantitive - quantity of protein.
Screen samples for antibody presence.
Uses of immunocapture?
Pregnancy tests - specific antibody on plane will capture antigen as it is washed over it, in a pregnancy tests detects ß HCG .
Rapid ELISA for FluA antigens
what is ELISA?
Enzyme linked immunosorbent assay. plate based assay technique that captures substances as they move through the plate.
What is immunoprecipitation?
Separating an antigen protein out of a solution by adding a complimentary antigen and washing out the rest of the sample. An Fc binding protein aids binding of the Ag/Ab complex.
Uses of immunoprecipitation?
detect proteins of interest,
characterise weight of protein.
study structure of proteins
Detect antigen in a sample.
What is affinity chromatography?
Purification of a soluble molecule from a solution, requires specific and reversible binding.
Ligand antibody is coupled to a solid matrix in a chromatography column.
Solution washed through, allowing binding to ligand, and solution is washed away.
Bound molecule is eluted off at a later point - allowing you to study composition of the elution.
