Immunology Techniques Flashcards
What is immunoassay?
- a biochemical test that measures the presence or conc of a specific molecule through the use of an antibody or antigen.
- the molecule detected by the immunoassay is the ‘analyte’ and is in many cases a protein
What is polyclonal antibody generation?
- antibodies produced by several clones of B cell recognising different parts of the same antigen
What is monoclonal antibody generation?
- single clone- recognises the same epitope within the same antigen
What can be added to antibodies in vaccines in order to elicit a stronger response?
- adjuvants (alum, CFA, poly I:C)
- mix to stimulate the innate IC
What are the adv and disadv of bleeding subject and generating polyclonal antibodies?
- adv:
- a mixture of ab directed toward a variety of epitopes are formed, good for agglutination and immunoprecipitation
- disadv:
- may produce cross-reactivites against other ag
- different bleed-points may yield different degress of affinity
How do you produce mass quantities of single-specificity Ab?
- fuse the B cells producing this antibody with myeloma (tumour) cells
- the myeloma fuse and can then proliferate continuously
- all the B cells will produce the same antibody from the same clone
How can monoclonal antibodies be modified for use in lab etc?
- binding sites of mAb retained, Fc region replaced with other molecule
- conjugation of Fc region with other molecules
- biotin/enzymes used in ELISA assays
- abzymes- mAb that mimic enzymes by binding and stabilizing transition states of chemical reactions
What are the ways in which immunoprecipitation can be performed?
- in solution
- in gel matrices
How is immunoprecipitation performed in solution?
- formation of such large immune comlexes that they fall out of solution (precipitate)
- only occurs when Ag/Ab concs are roughly equal
- used to purify antigen molecules/ remove antigens from solution
How is immunoprecipitation of soluble antigens performed in gel matrices?
- Ouchterlony method
- Ag placed in center well, serum samples placed in outer wells
- visible ‘precipitin line’ forms where large immune complexes form
What are Hemagglutination reactions used for?
- used to detct any Ag conjugated to the surface of rbcs
- used for blood typing
- easy, sentitive, cheap
What are hemagglutination inhibition reactions used for?
- used to detect the presence of viruses and of antiviral Ab
What is bacterial agglutination used for?
- used to detect antibodies to bacteria
- can provide quantitative info about serum antibacterial Ab concs
- can be diagnostic
What are Radioimmunoassays used to measure?
- concs of biologically relevant proteins/hormones in body fluids
Describe the process of Indirect ELISAs
- detects presence and concs of Ab in a sample
- method of choice to detect presence of serum Ab against a wide array of pathogens
Describe Sandwich ELISA
- measures Ab presence and levels
- useful for measurement of soluble cytokine concs
Describe the process of Competetive ELISA
- measures amount of Ag in a sample
- more Ag in original sample, lower the final signal
- can also be used to measure specific Ab
- competition between serum antibodies and an enzyme-conjugated Ag-specific mAb
What are the available enzyme systems for ELISA assays?
- Alkaline phosphatase
- Horseradish peroxidase
What are the various substrates used in an ELISA?
- chromogenic = coloured product, quick yes/no response
- fluorogenic = gives off fluorescent light that can be detected and measured
- chemiluminogenic = chrmical reaction that gives off light that can be detected and measured
How are ELISAs modified using biotin-streptavidin?
- bind biotin to antibody Fc region
- bind strepavidin to enzyme molecule
- allows yse of a variety of primary biotin-tagged Ab in different reactions with just 1 enzyme-conjugated stock of strepavidin
- saves money and provides flexibility in experimental design
What do ELISPOT assays measure?
- molecules secreted by individual cells
How can Western blotting be used to identify a specific protein?
- IDs and quantifies (roughly) specific protein in a complex protein mixture
- sample prepared in a loading buffer
- proteins separated on polyacrtlamide gel by electrophoresis
- proteins transferred from gel to membrane
- identification by enzyme-tagged specific Ab binding to Ag, followed by substrate exposure
- expensive and high level of skill
What is the different between immunocytochemistry and immunohistochemistry?
- immunocytochemistry = performed on isolated cells
- immunohistochemistry = performed on tissue sections
