Immunological Methods 1 & 2 Flashcards
What is an epitope?
An anibody binding site on an antigen
What is the difference between monoclonal and polyclonal antibody production?
Polyclonal antibody production
- Contains many antibodies that recognizes many determinants on an antigen.
- Many different classes of antibodies
- Can make a specific antibody using only a highly purified antigen
- difficult reproducing
Monoclonal antibody production
- contains a single antibody recognizing a single determinant
- 1 class
- Can make a specific antibody using an impure antigen
- reproducible
Name 3 ways the specificity of the antibody can be used as a tool to identify, isolate or even quantify a certain antigen or antibody.
- Cell populations can be identified and characterized by immunofluorescence or
immunohistochemistry. - Isolating cell populations based on their expression of surface markers
(receptors). - Specific antigen/antibodies can be quantified by e.g. ELISA or RIA (radioimmunoassay)
What are some qualitative immunological techniques?
- Precipitation techniques
- Immunodiffusion
- Agglutination techniques
- Direct agglutination
- Indirect/passive agglutination
- Immunofluorescense
- Immunohistochemistry
- Flow cytometry
- Immunoblotting
What are some quantitative immunological techniques?
- ELISA
- Blotting
- (RIA)
- Flow cytometry
What is percipitation?
A process where soluble antigens bind with their specific antibody at an optimum temperature and pH, resulting in the formation of an insoluble precipitate
A perciptitation technique involves immunodiffusion, describe a technique.
Double-diffusion according to Ouchterlony.
It refers to the double diffusion of antigens and antibodies in a gel. Once an insoluble complex is formed, a precipitation line is created at the place of equivalence. This result is visible to the human eye and exhibits three possible reactions; reaction of identity, reaction of non-identity or partial identity.
Describe the 2 agglutination techniques.
(Hint: Hemagglutination a type of agglutination that occurs when antibodies bind to specific binding sites on antigens expressed on red blood cells, good for classing blood types.)
Direct agglutination, happens via antibodies that binds to its corresponding antigen. Detects specific antibodies or other serum proteins that bind to a patient’s RBC.
Indirect agglutination, is when the antibodies bind to surface particles of an antigen coated carrier ex blood cells with LPS. Used to detect the presence of either antibodies or specific antigens.
What is western blot? (quantitative) HInt: you did this in a lab in ettan
It is used in research to separate and identify proteins.
In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein
Name the ELISA purpose and principle
ELISA is used to determine the amount of antibodies, antigens, proteins and glycoproteins in biological samples.
The basic principle involves adding an anti-A antibody covalently linked to an enzyme. Then washing out the unbinded antibodies. Then an enzyme is added to makes the colorless substrate colored.
What is the difference between direct ELISA and indirect?
Direct ELISAs use a conjugated primary antibody, while indirect ELISAs include an additional amplification step.
What is competetive ELISA?
Competitive ELISA is used to detect and quantify the presence of specific antigens (proteins, peptides, or other molecules) in a sample.
It relies on the competition between a labeled antigen and an unlabeled antigen in the sample for binding to a limited number of antibody binding sites on a solid support, typically a microtiter plate.
What is RIA(Radioimmunoassay)? (Quantitative)
Similar to ELISA, but dependent on radioisotopes instead of enzymes.
This is used in detection of serum proteins of low concentrations, e.g.
growth hormones, insulin..
ELISpot
It is used to measure the frequency of cytokine positive individual cells, typically lymphocytes, that produce a particular cytokine or secrete antibodies in response to an antigen.
ELISpot assesses the immune response to pathogens, allergens, and other antigens.
The technique is highly sensitive and allows for the quantification of individual immune cells’ responses.
Cytokine-specific antibodies are bound to the surface of a plastic well
What is Flow cytometry? (Qualitative)
One can distinguish cells from other cells in the same sample using Flow cytometry.
Method
Cells in solution bind to antibodies that are linked to fluorescent substance and therefore become stained. The cells are sent through a tube one by one and hit by a laser beam. The laser beam causes the fluorescent substance to be excited and the light flashed can be drawn up by a sensor behind it.
There will also be light that deflects. This can be measured with receptors that can draw up and provide insight into the cell’s properties.
