Lecture 18 Flashcards
When do the largest precipitates form when antigen and antibody are mixed?
The largest precipates form at the zone of equivalence where there is roughly equal concentration of antigen and antibody as some antigen will be bound by more than one antibody allowing a large precipitate to form
While if there is an excess of antibody then not that many antibodys will be required so there is a low amount of crosslinking
If there is an excess of antigen then all of the antibody binding sites will be filled so there will be no crosslinking
What is immunodiffusion?
This is when antigen and antibody is loaded in wells and they are allowed to diffuse out in the gel, at the zone of equivalence a line will form due to the larger precipate
How can countercurrent electrophoresis be used in immunology?
The gel is placed at a pH of 8 as this causes typical antibody molecules to be charged
This can speed up the process of immune diffusion where a line will form between loaded antigen and antibody dependent on both antibody and antigen concentration, charge, affinity of the antigen-antibody interaction and antigen size
What is rocket gel electrophoresis?
This is when wells are loaded with antigen and then subjected to an electric current this will produce a rocket with a height which is proportional to the antigen concentration
What is ouchterlony electrophoresis?
This is when serum from a healthy person is loaded adjacent to that of someone one with recurrent infections in the middle of a gel
The different forms of antibody are then separated through applying an electric current
Rabbit antihuman antiserum is then placed in a central trough and allowed to diffuse to form precipitation lines
If the person being investigated has zones missing then they must be deficient in that type of antibody
What is nephelometry?
This is where patient serum is mixed with antigen in a clear tube
The ability of light to pass through this tube will be impeded by the complexes that form and can be measured by placing the tube into a spectrophotometer
The technique is used to quantitate antibodies against DNA in SLE patients and has the advantage of being faster than diffusion techniques
What are the features of conventional antisera?
There are multiple immunisations
Recognition of multiple epiptopes
A range of affinities
Required specificity is small fraction of total antibodies
This causes an extensive need for purification and batch-batch variation
How can monoclonal antibodies be generated?
Spleen cells are mixed with myeloma cells in the presence of substances like ethylene glycol to from hybrid myelomas (hybridomas)
However in order to prevent over growth of these cells from the typical (useless) myeloma cells amniopterin is used to prevent all cultured cells from being able to synthesize amino acids forcing them to use their salvage pathways which are first knocked out in the myeloma cells so only hybridomas will grow in the medium
What are the properties of monoclonal antibodies?
They are homogenous with respect to specificity and antibody class
They recognize a single determinate on the antigen of interest
Not necessarily mono-specific
Very high titres can be obtained
Produed with little or no contamination by irrelevant antibodies
Little or no purification is usually required
Invariant reagent that is availbel in essentially infinite supply
Ready use as standardised reagents
What are the different forms of label used antibodies?
They may be labelled with radioactivity, enzymes or fluorochromes
What are the different forms of ELISA?
Direct elisa where the antigen is bound on a solid phase substrate, used to determine if the patient responds to a specific antigen
Sanwich ELISA where there are antibodies bound to the substrate and is used to detect the presence of a specific antigen in a patients sera
Microparticle ELISA where the antibody is bound to beads which has the advantage of greater sensitivity so less patient sera is needed
How can antibodies be used to examine cell biology?
If the cells are treated with fixing agents then the antigens inside them will be preserved and the membrane will become permeable allowing fluorescently labelled antibodies to detect cell structures
What are complement fixation tests?
These are tests often done to diagnose recent infections with difficult to identify organisms
The patients serum is incubated with relevant antigens if the patient contains antibodies to these then an immune complex will form
Complement is then added which will be used up by the immune complexes if present
To determine if this has occurred sheep red blood cells that have been incubated with anti sheep red blood cell antibody are added
If complement has been used then these cells will not lyse
To diagnose a recent infection the lab needs to show a four fold rise in titre between acute and covalescent phases
What is the role of microlymphocytoxicity in HLA tissue typing?
This is a test where peripheral blood lymphocytes are incubated with a panel of a range of antibodies directed against all the main HLA antigens and complement is added and the lymphocytes are examined microscopically if the cells have lysed then that HLA must be present
What is the role of polymerase chain reaction in HLA tissue typing?
These methods depend on whether or not DNA in the patients HLA genes correctly matches the PCR primers
A panel of PCR primers is used each of which precisely match a different HLA allele
The DNA will only amplify if the match is exact
