Experimental Methods Flashcards
What are polyclonal antibodies?
a mixture of different antibodies directed toward a variety of epitopes on immunizing antigen
How are polyclonal antibodies generated?
- inject host animal with antigen
- the antigen results in a B cell response and plasma cells will secrete antibody
- collect serum from host and isolate the antibodies
What are monoclonal antibodies?
antibodies that are generated from a single B cell clone
How are monoclonal antibodies (mAb) generated?
- isolate a single B cell and fuse the B cell with myeloma cell, creating a hybridoma
- collect antibodies produced from hybridomas
How are monoclonal antibodies modified?
conjugating them to other compounds, but leave antigen binding sites available to bind antigen
What can monoclonal antibodies be conjugated to?
- biotin
- HRP
- fluorophore
- magnetic bead
- agarose
What are the uses for polyclonal antibodies?
- immunoprecipitation
- hemagglutination
What is immunoprecipitation?
antibody-antigen complexes are precipitated from solution
What is immunoprecipitation used for?
isolation of proteins
What is hemagglutination?
clumping of antibody-RBC complexes
What is hemagglutination used for?
blood typing
What are the uses for monoclonal antibodies?
- enzyme-linked immunosorbent assays (ELISAs)
- western blotting
- immunohistochemistry
What do ELISAs utilize?
monoclonal antibodies conjugated to enzymes that produce color changes
What are the types of ELISAs?
- indirect
- sandwich
What are indirect ELISAs used for?
to detect presence of antibodies in a sample
How are indirect ELISAs performed?
- coat plate with antigen
- add sample that contains antibodies (typically serum)
- add antibody-enzyme complex
- add enzyme substrate
- measure color change
What are sandwich ELISAs used for?
to detect presence of an antigen
How are sandwich ELISAs performed?
- coat plate with mAbs specific to antigen
- add mAb conjugated to enzyme that is specific for a different epitope on antigen
- add enzyme substrate
- detect color change
What is western blotting used for?
used to quantify specific proteins in a sample
How is western blotting performed?
- separate proteins in a mixture by molecular weight using gel electrophoresis
- transfer bands to a membrane using electrophoresis
- incubate membrane with enzyme conjugated to mAb
- add enzyme substrate
- use imaging software to view bands
What is immunohistochemistry used for?
to detect presence of antigen within tissues
How is immunohistochemistry performed?
- add enzymes conjugated mAbs to tissue slices on microscope slide
- add enzyme substrate
- view presence of antigen with microscope
